Tegument protein UL21 of alpha-herpesvirus inhibits the innate immunity by triggering CGAS degradation through TOLLIP-mediated selective autophagy

Zicheng Ma; Juan Bai; Chenlong Jiang; Huixin Zhu; Depeng Liu; Mengjiao Pan; Xianwei Wang; Jiang Pi; Ping Jiang; Xing Liu

doi:10.1080/15548627.2022.2139921

. 2022 Nov 7;19(5):1512–1532. doi: 10.1080/15548627.2022.2139921

Tegument protein UL21 of alpha-herpesvirus inhibits the innate immunity by triggering CGAS degradation through TOLLIP-mediated selective autophagy

Zicheng Ma ^a, Juan Bai ^a, Chenlong Jiang ^a, Huixin Zhu ^a, Depeng Liu ^a, Mengjiao Pan ^a, Xianwei Wang ^a, Jiang Pi ^b, Ping Jiang ^a,^c,^✉, Xing Liu ^a,^✉

PMCID: PMC10241001 PMID: 36343628

ABSTRACT

Alpha-herpesvirus causes lifelong infections and serious diseases in a wide range of hosts and has developed multiple strategies to counteract the host defense. Here, we demonstrate that the tegument protein UL21 (unique long region 21) in pseudorabies virus (PRV) dampens type I interferon signaling by triggering the degradation of CGAS (cyclic GMP-AMP synthase) through the macroautophagy/autophagy-lysosome pathway. Mechanistically, the UL21 protein scaffolds the E3 ligase UBE3C (ubiquitin protein ligase E3C) to catalyze the K27-linked ubiquitination of CGAS at Lys384, which is recognized by the cargo receptor TOLLIP (toll interacting protein) and degraded in the lysosome. Additionally, we show that the N terminus of UL21 in PRV is dominant in destabilizing CGAS-mediated innate immunity. Moreover, viral tegument protein UL21 in herpes simplex virus type 1 (HSV-1) also displays the conserved inhibitory mechanisms. Furthermore, by using PRV, we demonstrate the roles of UL21 in degrading CGAS to promote viral infection in vivo. Altogether, these findings describe a distinct pathway where alpha-herpesvirus exploits TOLLIP-mediated selective autophagy to evade host antiviral immunity, highlighting a new interface of interplay between the host and DNA virus.

Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AHV-1: anatid herpesvirus 1; ATG7: autophagy related 7; ATG13: autophagy related 13; ATG101: autophagy related 101; BHV-1: bovine alphaherpesvirus 1; BNIP3L/Nix: BCL2 interacting protein 3 like; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCDC50: coiled-coil domain containing 50; CCT2: chaperonin containing TCP1 subunit 2; CGAS: cyclic GMP-AMP synthase; CHV-2: cercopithecine herpesvirus 2; co-IP: co-immunoprecipitation; CQ: chloroquine; CRISPR: clustered regulatory interspaced short palindromic repeat; Cas9: CRISPR-associated system 9; CTD: C-terminal domain; Ctrl: control; DAPI: 4’,6-diamidino-2-phenylindole; DBD: N-terminal DNA binding domain; DMSO: dimethyl sulfoxide; DYNLRB1: dynein light chain roadblock-type 1; EHV-1: equine herpesvirus 1; gB: glycoprotein B; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HSV-1: herpes simplex virus 1; HSV-2: herpes simplex virus 2; IB: immunoblotting; IRF3: interferon regulatory factor 3; lenti: lentivirus; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARCHF9: membrane associated ring-CH-type finger 9; MG132: cbz-leu-leu-leucinal; NBR1: NBR1 autophagy cargo receptor; NC: negative control; NEDD4L: NEDD4 like E3 ubiquitin protein ligase; NH₄Cl: ammonium chloride; OPTN: optineurin; p-: phosphorylated; PFU: plaque-forming unit; Poly(dA:dT): Poly(deoxyadenylic-deoxythymidylic) acid; PPP1: protein phosphatase 1; PRV: pseudorabies virus; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RNF126: ring finger protein 126; RT-PCR: real-time polymerase chain reaction; sgRNA: single guide RNA; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TOLLIP: toll interacting protein; TRIM33: tripartite motif containing 33; UL16: unique long region 16; UL21: unique long region 21; UL54: unique long region 54; Ub: ubiquitin; UBE3C: ubiquitin protein ligase E3C; ULK1: unc-51 like autophagy activating kinase 1; Vec: vector; VSV: vesicular stomatitis virus; VZV: varicella-zoster virus; WCL: whole-cell lysate; WT: wild-type; Z-VAD: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone.

KEYWORDS: Alpha-herpesvirus, CGAS, selective autophagy, type I interferon signaling, UL21 protein

Introduction

Herpesviruses are enveloped, double-stranded, DNA viruses that can be grouped into three subfamilies – alpha-, beta-, and gamma-herpesviruses – according to their distinct biological characteristics [1,2]. Pseudorabies virus (PRV) is the causative agent of pseudorabies, a disease of causing great economic losses in swine, belonging to the alpha-herpesvirus subfamily together with herpes simplex virus type 1 (HSV-1), and herpes simplex type 2 (HSV-2) [3]. Alpha-herpesviruses encode approximately 8 capsid proteins, 23 tegument proteins, and 14 envelope proteins [4,5]. The tegument is located between the capsid and envelope, and plays critical roles in viral entry, secondary envelopment, and viral capsid nuclear transportation during infection. UL21 (unique long region 21) is a conserved tegument protein of alpha-herpesviruses [6] and has been shown to play an important role in viral replication and pathogenesis. In HSV-1, UL21 facilitates viral replication by its involvement in cell-to-cell spread [7], and deletion of UL21 causes a delay in the early stage of viral replication cycle [8]. It has also been demonstrated that UL21 can promote HSV replication and dissemination as a virus-encoded adaptor of PPP1 (protein phosphatase 1) [9]. HSV-2 UL21 has an early function that facilitates viral gene expression, as well as a late essential function that promotes the egress of capsids from the nucleus by regulating Us3 kinase and nuclear egress complex (NEC) activity [6,10,11]. In PRV, the UL21 gene is a major neurovirulence determinant, and point mutations affecting the UL21 gene of live-vaccine strain Bartha contributes to its attenuated phenotype [12,13]. It has also been suggested that UL21 contributes to PRV neuroinvasion through interacting with DYNLRB1 (dynein light chain roadblock-type 1) [14]. Despite these observations, the underlying mechanism of how UL21 regulates the replication and pathogenesis of alpha-herpesviruses is not readily reconciled by the currently known functions.

Pattern-recognition receptors (PRRs) initiate the antiviral innate immune defense in host cells by sensing invading viruses, in which DNA sensors play vital roles, such as TLR9 (toll like receptor 9), IFI16 (interferon gamma inducible protein 16), AIM2 (absent in melanoma 2), ZBP1/DAI (Z-DNA binding protein 1), DDX41 (DEAD-box helicases 41), and CGAS (cyclic GMP-AMP synthase) [15–20]. Canonically, the CGAS pathway is activated when it binds virus or cell-associated DNA in the cytoplasm of cells, leading to the production of the secondary messenger molecule, 2ʹ3’-cyclic GMP-AMP (2ʹ3’-cGAMP or cGAMP). Subsequently, cGAMP serves as a ligand to activate STING1 (stimulator of interferon response cGAMP interactor 1) and the downstream TBK1 (TANK binding kinase 1) and transcription factor IRF3 (interferon regulatory factor 3) pathways [20]. Hence, CGAS, as a DNA sensor, is essential in type I IFN (interferon) induction and responsible for regulating innate immune responses against DNA viruses, especially herpesviruses [21,22].

Macroautophagy, also referred to as autophagy, is a highly conserved homeostatic process in eukaryotic cells that disassembles and recycles dysfunctional cellular components [23]. The relationship between PRV replication and autophagy has been reported. Sun et al. found that PRV could induce autophagy in the early stage of infection, however, US3 protein could reduce the autophagy level by activating the AKT/mTOR pathway upon PRV replication [24]. Xu et al. showed that PRV infection could induce autophagy in vitro through the classical BECN1-ATG7-ATG5 pathway, resulting in increased PRV replication in mouse neuron 2A (N2a) cells [25]. Also, recent studies have uncovered autophagy as a selective degradation system when delivering substrates, such as damaged organelles or aggregated proteins, to autophagosomes for lysosomal degradation via cargo receptors, including SQSTM1/p62 (sequestosome 1), NBR1 (NBR1 autophagy cargo receptor), OPTN (optineurin), CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2), BNIP3L/Nix (BCL2 interacting protein 3 like), and TOLLIP (toll interacting protein) [26,27]. Accumulating evidence has demonstrated that autophagy plays a vital role in the regulation of immune responses [28–30]. In this study, we identified that the tegument protein UL21 of alpha-herpesviruses dampens CGAS-mediated antiviral signaling by degrading CGAS through autophagic signaling. Mechanistically, we demonstrated that UL21 triggered CGAS degradation through TOLLIP-mediated selective autophagy, where CGAS underwent K27-linked ubiquitination at residue K384 by the E3 ligase UBE3C (ubiquitin protein ligase E3C). Importantly, we demonstrated the contribution of UL21 in antagonizing CGAS signaling in vivo. Our findings revealed an important mechanism underlying evasion of the host antiviral immune response by alpha-herpesviruses.

Results

PRV and HSV-1 UL21 inhibits CGAS-STING1-mediated innate immunity

UL21 is a conserved tegument protein in the alpha-herpesviruses and has multiple important albeit poorly understood functions in viral replication and pathogenesis [6–14]. Here, we chose PRV as a virus model and constructed an UL21 null mutant (ΔUL21) and its revertant virus (ΔUL21R) by using CRISPR (the clustered regulatory interspaced short palindromic repeat)-Cas9 (CRISPR-associated system 9) system (Figure S1A-C) to determine the role of UL21 in viral replication and the pathogenesis of alpha-herpesvirus. The results of viral characterization showed that plaque size of the ΔUL21 were significantly smaller than those of wild-type virus (PRV-WT) and ΔUL21R (Figure 1A). Next, the viral titers were determined in PK-15, HEK-293 T, MEF, and N2a cells. With the low multiplicity of infection (MOI; 0.01 and 0.05), the titers of the ΔUL21 were found to decrease approximately 1 log as compared with those of PRV-WT or ΔUL21R in these cell lines. Also, the same phenotype was seen with the high MOI (Figure 1B). Moreover, a mouse infection experiment showed that the pathogenesis of the ΔUL21 was significantly lower than that of PRV-WT and ΔUL21R (Figure 1C). Further, PRV DNA loads in the brain, lung, and brainstem tissues of mice in the ΔUL21 group were significantly lower than those in the PRV-WT and ΔUL21R infection groups (Figure 1D). Consistently, the neuropathological lesions in the brains, inflammatory cell infiltrate, and congestion in the lungs of the mice in the ΔUL21 group were obviously slightly less than those in the PRV-WT and ΔUL21R groups (Figure 1E). These data collectively indicate that UL21 positively contributes to the replication and pathogenesis of PRV.

Figure 1. — PRV UL21 promotes viral replication and pathogenicity. (A) Plaque morphology of PRV-WT, ΔUL21, and ΔUL21R in Vero cells. Plaque sizes of PRV-WT, ΔUL21, and ΔUL21R were plotted as a percentage of the average PRV-WT plaque sizes. (B) Viral replication in PK-15, HEK-293 T, MEF, and N2a cells. Cells were infected with PRV-WT, ΔUL21, or ΔUL21R at 0.01, 0.05 or 3 plaque forming unit (PFU)/cell. At 48 (0.01, 0.05 PFU/cell) or 24 (3 PFU/cell) hpi, cells were harvested, and the total virus yields were determined in Vero cells by using a plaque assay. (C) Mouse survival was recorded and shown as a percentage over time. Mice housed in 4 groups (10 mice/group) were mock-infected or infected with PRV-WT, ΔUL21, or ΔUL21R (1 × 10⁴ pfu/mice) through intraperitoneal injection. (D) Four groups of mice (4 mice/group) were treated as in (C) and sacrificed at 3 dpi. The viral DNA loads in the brain, lung, and brainstem were quantified by RT-PCR. (E) hematoxylin and eosin (H&E) stained brain and lung sections from the indicated mice 3 dpi (1 × 10⁴ pfu/mice) (scale bar: 100 μm). The data are representative of at least three independent experiments with similar results (mean ± SD of n = 3 biological replicates in B, n = 4 biological replicates in D). * p < 0.05, ** p < 0.01 (two-tailed unpaired t test).

It is known that PRV, as a double-stranded DNA virus, has developed multiple strategies to antagonize DNA sensor signaling pathways to facilitate its replication and pathogenesis [31]. Therefore, we hypothesized that UL21 in alpha-herpesvirus might promote viral infection by overcoming the restriction of DNA sensor mediated innate immunity. As shown in Figure 2A, PRV UL21 (UL21) was found to inhibit the ligand of cytolytic DNA sensor poly(dA:dT)-mediated IFNB/IFN-β promoter activity in HEK-293 T cells. qPCR analysis indicated that ectopic expression of UL21 depressed Poly(deoxyadenylic-deoxythymidylic) acid [poly(dA:dT)] induced transcription of IFNB/IFN-β, ISG15, IFIT2/ISG54, and IFIT1/ISG56 (Figure 2B). In addition, supernatants from UL21 and poly(dA:dT) co-treated HEK-293 T cells did not completely inhibit the replication of vesicular stomatitis virus-green fluorescent protein (VSV-GFP) as measured by the GFP signaling (Figure 2C), suggesting that UL21 dampened the secretion of antiviral factors induced by poly(dA:dT). Consistently, ectopic expression of UL21 could inhibit the phosphorylation of TBK1 and IRF3 stimulated by poly(dA:dT) (Figure 2D).

Figure 2. — PRV UL21 inhibits poly(dA:dT) and CGAS-STING1-mediated type I interferon pathway. (A and E) PRV UL21 inhibits poly(dA:dT) or CGAS-STING1 stimulated *IFNB* promoter activity. (A) HEK-293 T cells seeded in 24-well plates were co-transfected with pIFNB-Luc (50 ng) and pRL-TK (10 ng) along with increasing doses of Flag-UL21 (100, 300, or 500 ng). At 24 hpt, cells were stimulated with poly(dA:dT) at a concentration of 200 ng/mL. After another 24 h, cells were harvested for a luciferase assay. (E) HEK-293 T cells seeded in 24-well plates were co-transfected with pIFNB-Luc (50 ng) and pRL-TK (10 ng), HA-CGAS (500 ng), or MYC-STING1 (50 ng) alone with the along with increasing doses of Flag-UL21 (100, 300, or 500 ng). At 48 hpt, the cells were harvested for a luciferase assay. (B and F) PRV UL21 inhibits poly(dA:dT) or HA-CGAS + MYC-STING1 stimulated *IFNB, ISG15, IFIT2*, and *IFIT1* transcripts. HEK-293 T cells were treated similar to (A) and (E), but without pIFNB-Luc and pRL-TK transfection. The cells were harvested for RNA extraction and RT-PCR detection of *IFNB, ISG15, IFIT2*, and *IFIT1*, and *RNA18S* rRNA mRNA levels. (C and G) HEK-293 T cells were treated as in (B) and (F). The supernatants were harvested and incubated with fresh confluent HEK-293 T cells. After 24 h, the cells were infected with VSV-GFP at an MOI of 0.01. At 24 hpi, GFP signaling was detected by microscopy (scale bar: 100 μm). (D and H) HEK-293 T cells were treated the same as in (B) and (F). The cell lysates were harvested for western blot analysis with antibodies against Flag-tag, IRF3, p-IRF3 (phosphorylated interferon regulatory factor 3), p-TBK1 (TBK1, phosphorylated TANK binding kinase 1), and ACTB/β-actin. Data are representative of at least three independent experiments with similar results (mean ± SD of n = 3 biological replicates in A, B, E, and F). * p < 0.05, ** p < 0.01 (two-tailed unpaired t test).

Among the cytolytic DNA sensors, the CGAS-STING1 pathway plays a central role in the innate immune defense against viral infections [20]. As such, we reasoned that UL21 might negatively regulate the CGAS-STING1-mediated innate immune response. As shown in Figure 2E, ectopic expression of UL21 in HEK-293 T cells inhibited IFNB promoter activity stimulated by CGAS-STING1 overexpression. The transcripts of IFNB, ISG15, IFIT2, and IFIT1 induced by overexpression of CGAS-STING1 were downregulated in UL21 transfected cells (Figure 2F). Furthermore, supernatants from HEK-293 T cells co-transfected with UL21 and CGAS-STING1 could not completely block the proliferation of VSV-GFP as reflected by the GFP signaling (Figure 2G). To verify the effect of UL21 on CGAS-STING1 signaling, we examined TBK1 and IRF3 phosphorylation, a hallmark of innate immune activation [20]. Ectopic expression of UL21 blocked phosphorylation of TBK1 and IRF3 stimulated by CGAS-STING1 (Figure 2H). Notably, HSV-1 UL21 was also found to inhibit the CGAS-STING1-mediated interferon pathway (Figure S2). Interestingly, we found that CGAS expression was decreased when cells were transfected with alpha-herpesvirus UL21 (Figure 2E and Figure S2A), implying that alpha-herpesvirus UL21 inhibits CGAS-STING1-mediated innate immunity by inducing CGAS degradation.

PRV and HSV-1 UL21 degrades CGAS through the autophagy-lysosome pathway

To further dissect the effect of UL21 on CGAS expression, we determined the CGAS protein level in cells transiently transfected with CGAS, along with UL21, GFP, or an empty vector. As shown in Figure 3A, CGAS expression was decreased in cells co-transfected with UL21, but not GFP or the vector plasmid. Importantly, the protein level of CGAS was negatively correlated with the transfected dose of UL21 (Figure 3B). In contrast, STING1 expression was unchanged with elevated levels of ectopically expressed UL21 protein (Figure S3A, B). Similarly, CGAS, but not STING1 expression, was also downregulated in HSV-1 UL21 transfected cells (Figure S3C, D). These data suggested that alpha-herpesvirus UL21 specifically degrades CGAS.

Figure 3. — The autophagy-lysosome pathway is associated with CGAS degradation induced by PRV UL21. (A) Ectopic expression of PRV UL21 decreases the CGAS protein level. HEK-293 T cells seeded in 6-well plates were transfected with HA-CGAS (2 μg) along with indicated plasmids (empty vector, Flag-GFP, or Flag-UL21) (2 μg). At 24 hpt, the cells were harvested for western blot analysis with antibodies against HA-tag, Flag-tag, and ACTB. (B) PRV UL21 degrades CGAS in a dose-dependent manner. HEK-293 T cells seeded in 6-well plates were transfected with HA-CGAS (2 μg) together with empty vector or increasing doses of Flag-UL21 (0.5, 1, or 2 μg). At 24 hpt, the cells were harvested for western blot analysis with antibodies against HA-tag, Flag-tag, and ACTB. (C) Effects of Z-VAD, MG132, and 3-MA on CGAS degradation induced by PRV UL21. HEK-293 T cells seeded in 6-well plates were transfected with HA-CGAS (2 μg) together with empty vector or Flag-UL21 (2 μg). At 12 hpt, cells were treated with DMSO or Z-VAD (20 μM), MG132 (10 μM), or 3-MA (5 mM). After another 12 h, cells were harvested for western blot analysis with antibodies against HA-tag, Flag-tag, and ACTB. (D) Effects of NH₄Cl, 3-MA, and CQ on CGAS degradation induced by PRV UL21. HEK-293 T cells seeded in 6-well plates were transfected with HA-CGAS (2 μg) together with empty vector or Flag-UL21 (2 μg). At 12 hpt, cells were treated with DMSO or NH₄Cl (20 mM), 3-MA (5 mM), or CQ (50 μM). After another 12 h, cells were harvested for western blot analysis with antibodies against HA-tag, Flag-tag, and ACTB. Data are representative of at least three independent experiments with similar results.

The caspase, ubiquitin-proteasome, and autophagy-lysosome pathways are three major systems that control protein degradation in eukaryotic cells [32–34]. To determine which pathway is involved in the CGAS degradation initiated by UL21, HEK-293 T cells were ectopically expressed together with CGAS and UL21, and then treated cells by dimethyl sulfoxide (DMSO, control), carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD, pan-caspase inhibitor), cbz-leu-leu-leucinal (MG132, proteasome inhibitor), or 3-methyladenine (3-MA, autophagy inhibitor). As shown in Figure 3C, the expression of CGAS was rescued in 3-MA treated cells, but not in DMSO, Z-VAD, or MG132 treated cells. The same phenotype was seen in HSV-1 UL21 transfected cells, in which 3-MA treatment restored the CGAS expression (Figure S3E). These results implied that alpha-herpesvirus UL21 degrades CGAS expression through the autophagy-lysosome pathway. To further evaluate the underlying mechanism, we monitored the CGAS protein levels in cells treated with lysosomal inhibitors chloroquine (CQ) and ammonium chloride (NH₄Cl). As illustrated in Figure 3D, the expression of CGAS was reversed in CQ and NH₄Cl treated groups, similar with the results upon 3-MA treatment. Taken together, these data suggested that alpha-herpesvirus UL21 decreases CGAS expression by degradation via the autophagy-lysosome pathway.

PRV and HSV-1 UL21 induces the autophagic process and degrades CGAS

As illustrated above, UL21 degrades CGAS expression through the autophagy-lysosome pathway. We further speculated that UL21 may affect autophagosome formation. As shown in Figure 4A, ectopic expression of UL21 significantly increased the endogenous LC3-I to LC3-II conversion, accompanied by CGAS degradation, and the same phenotype was also observed for HSV-1 UL21 (Figure S3F). In addition, the increased LC3-GFP puncta formation was obviously detected in cells overexpressing UL21 (Figure 4B). Furthermore, MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) conversion and CGAS degradation were also confirmed in cells during PRV infection. As illustrated in Figure 4C and 4D, with the high MOI (3 MOI), at 12 h post infection, the endogenous LC3-I to LC3-II conversion was significantly decreased in HEK-293 T and PK-15 cells infected with ΔUL21 as compared to those in cells infected with PRV-WT and ΔUL21R. Additionally, the degradation of CGAS in cells infected with the ΔUL21 was decreased to some extent as compared to those in the PRV-WT and ΔUL21R groups. However, the differences in CGAS expression among ΔUL21, PRV-WT and ΔUL21R groups were not detected at 12 h post infection with the low MOI (0.05 MOI) (Figure S3G, H), despite the autophagy formation. To explain the discrepancy, we determined the kinetics of UL21 expression infected with PRV with high and low MOIs (3 and 0.05). The high MOI infection resulted in UL21 expression starting from 4 h post infection. In contrast, UL21 expression was only slightly detected at 12 h post infection under the low MOI condition (Figure S3I-L). In concurrence with the UL21 expression kinetics, CGAS degradation was only detectable at 4 and 8 h post infection under the high MOI (Figure S3M-P) but not the low MOI (Figure S3Q-T) infection condition.

Figure 4. — PRV UL21 degrades CGAS through the autophagy pathway. (A) Ectopic expression of PRV UL21 induces autophagy and decreases the endogenous level CGAS protein. HEK-293 T cells seeded in 6-well plates were transfected with Flag-UL21 (2 μg). At 24 hpt, the cells were harvested for western blot analysis with antibodies against LC3, CGAS, Flag-tag, and ACTB. (B) PRV UL21 induces GFP-LC3 puncta formation. HeLa cells seeded in 6-well plates were transfected with GFP-LC3 (2 μg) along with empty vector or Flag-UL21 (2 μg). At 24 hpt, cells were stained with anti-Flag (red) and subjected to analysis by confocal microscopy. Nuclei were stained with DAPI (blue) (scale bar: 10 μm). (C and D) PRV UL21 is important in virus-induced autophagy and CGAS degradation. HEK-293 T (C) or PK-15 (D) cells were mock-infected or infected with PRV-WT, ΔUL21, or ΔUL21R at 3 PFU/cell. At 12 hpi, the cells were harvested for western blot analysis with antibodies against LC3, CGAS, STING1, UL21, UL54, and ACTB. Densitometric quantification of LC3-II:I. The protein bands shown in panel C and D were quantified using NIH ImageJ software. The data are presented as the relative amount of LC3-II: I normalized to the total level of ACTB in each sample. (E) ATG7 deficiency inhibits PRV UL21-mediated CGAS degradation. HEK-293 T cells were transfected with siRNA targeting to *ATG7* (siATG7) (10 mM) or control siRNA (siNC) (10 mM). After 12 h, cells were transfected with the HA-CGAS (2 μg) together with empty vector (2 μg) or Flag-UL21 (2 μg). After another 24 h, the cells were harvested for western blot analysis with antibodies against HA-tag, ATG7, Flag-tag, and ACTB. (F and G) ATG7 deficiency inhibits PRV UL21 induced CGAS degradation in viral infection. HEK-293 T (F) or PK-15 (G) cells were transfected with siATG7 (10 mM) or siNC (10 mM). After 12 h, cells were mock-infected or infected with PRV, ΔUL21, or ΔUL21R at 3 PFU/cell. At 12 hpi, the cells were harvested for western blot analysis with antibodies against CGAS, ATG7, UL21, UL54, and ACTB. Densitometric quantification of CGAS. The protein bands shown in panel F and G were quantified using NIH ImageJ software. The data are presented as the relative amount of CGAS normalized to the total level of ACTB in each sample. (H and I) The effects of CGAS on viral replication. Wild-type (WT), *CGAS*^−/−, or *CGAS*^−/–rescued HEK-293 T cells (H), and WT, *CGAS*^−/−, or *CGAS*^−/–rescued PK-15 cells (I) were mock-infected or infected with PRV-WT, ΔUL21, or ΔUL21R at 0.01 PFU/cell. At 48 hpi, cells and supernatants were harvested, and the total virus yields were determined in Vero cells by a plaque assay. Data are representative of at least three independent experiments with similar results (mean ± SD of n = 3 biological replicates in H and I). * p < 0.05, ** p < 0.01 (two-tailed unpaired t test).

To further clarify the relationship between autophagy induction and CGAS degradation induced by UL21, we firstly determined the expression of CGAS in HEK-293 T cells transfected with UL21 when ATG7 (autophagy related 7), the key adaptor of autophagy [35], was knocked down. As predicted, in cells with ectopic expression of UL21, the CGAS protein level was restored after small interfering RNA (siRNA) targeting to ATG7 (siATG7) treatment compared to that after control siRNA (siNC) treatment (Figure 4E). Consistently, during PRV infection, the decreased expression of CGAS observed after PRV-WT and ΔUL21R infection was restored to the level observed during ΔUL21 infection when ATG7 was knocked down in HEK-293 T and PK-15 cells (Figure 4F,). Notably, the degradation of CGAS is important for PRV replication, especially the ΔUL21. In CGAS knockout cell lines, the replication of the ΔUL21 was significantly increased, and the differences between the ΔUL21 and PRV-WT or ΔUL21R were obviously different (Figure 4H, and Figure S4A, B). To exclude the possibility that CGAS degradation was indirectly caused by another viral protein, we assessed the expression of UL16 (unique long region 16), which can form a complex with UL21 in alpha-herpesviruses. The deletion of UL21 did not alter the expression profile of UL16 (Figure S4C), and UL16 had no effect on the protein expression of CGAS (Figure S4D, E). Altogether, these results demonstrated that alpha-herpesvirus UL21 specifically mediates CGAS degradation, which is relevant to the autophagy pathway.

TOLLIP-mediated autophagy is responsible for CGAS degradation induced by PRV and HSV-1 UL21

Next, we determined the mechanism of CGAS degradation induced by UL21-mediated autophagy. We detected the interactions between UL21 and CGAS. Figure 5A shows that CGAS, but not the vector or GFP, co-immunoprecipitated with UL21. The immunostaining assay showed that CGAS was colocalized with UL21 (Figure 5B). Also, HSV-1 UL21 was shown to interact with CGAS (Figure S5A).

Figure 5. — TOLLIP is the selective autophagic cargo mediating UL21 induced CGAS degradation. (A) PRV UL21 interacts with CGAS. HEK-293 T cells were transfected with Flag-UL21 (3 μg) together with indicated plasmids (empty vector [3 μg], HA-CGAS [3 μg], or HA-GFP [3 μg]). At 24 hpt, cells were processed for immunoprecipitation (IP) with anti-Flag magnetic beads. Whole-cell lysates (WCLs) and precipitated proteins were probed with antibodies against Flag-tag, HA-tag, and ACTB. (B) PRV UL21 colocalizes with CGAS in the cytoplasm. HeLa cells were transfected with HA-CGAS (2 μg) along with empty vector or Flag-UL21 (2 μg). At 24 hpt, cells were stained with anti-HA (red) and anti-Flag (green) and subjected to analysis by confocal microscopy. Nuclei were stained with DAPI (blue) (scale bar: 10 μm). (C) PRV UL21 interacts with SQSTM1 and TOLLIP. HEK-293 T cells were transfected with MYC-UL21 (3 μg) together with indicated plasmids (empty vector [3 μg], Flag-NBR1 [3 μg], Flag-OPTN [3 μg], Flag-SQSTM1 [3 μg], Flag-CALCOCO2 [3 μg], Flag-BNIP3L [3 μg], or Flag-TOLLIP [3 μg]). At 24 hpt, cells were processed for IP with anti-MYC magnetic beads. WCLs and precipitated proteins were probed with antibodies against Flag-tag, MYC-tag, and ACTB. (D) CGAS interacts with SQSTM1 and TOLLIP. HEK-293 T cells were transfected with HA-CGAS (3 μg) together with indicated plasmids (empty vector [3 μg], Flag-NBR1 [3 μg], Flag-OPTN [3 μg], Flag-SQSTM1 [3 μg], Flag-CALCOCO2 [3 μg], Flag-BNIP3L [3 μg], or Flag-TOLLIP [3 μg]). At 24 hpt, the cells were processed for IP with anti-HA magnetic beads. WCLs and precipitated proteins were probed with antibodies against Flag-tag, HA-tag, and ACTB. (E) UL21 strengthen the interaction between TOLLIP and CGAS. HEK-293 T cells were transfected with HA-CGAS (3 μg) together with indicated plasmids (Flag-SQSTM1 [3 μg] with MYC-UL21 [3 μg], or empty vector [3 μg]) or (Flag-TOLLIP [3 μg] with MYC-UL21 [3 μg] or empty vector [3 μg]). At 24 hpt, cells were processed for IP with anti-HA magnetic beads. WCLs and precipitated proteins were probed with antibodies against Flag-tag, MYC-tag, HA-tag, and ACTB. (F) SQSTM1 is not associated with UL21 induced CGAS degradation. Wild type (WT) or *SQSTM1*^−/− HEK-293 T cells seeded in 6-well plates were transfected with HA-CGAS (2 μg) together with or without MYC-UL21 (2 μg) and with or without Flag-SQSTM1 (2 μg). At 24 hpt, cells were harvested for western blot analysis with antibodies against HA-tag, MYC-tag, SQSTM1, and ACTB. (G and H) *TOLLIP* deficiency inhibits UL21 induced CGAS degradation. Wild-type (WT) or *TOLLIP*^−/− HEK-293 T (G) and PK-15 (H) cells seeded in 6-well plates were transfected with HA-CGAS (2 μg) together with or without MYC-UL21 (2 μg) and with or without Flag-TOLLIP (2 μg). At 24 hpt, cells were harvested for western blot analysis with antibodies against HA-tag, MYC-tag, TOLLIP, and ACTB. (I) *TOLLIP* deficiency inhibits UL21-mediated CGAS degradation in PRV infection. WT and *TOLLIP*^−/− HEK-293 T cells were mock-infected or infected with PRV-WT, ΔUL21, or the ΔUL21R at 3 PFU/cell. At 12 hpi, cells were harvested for western blot analysis with antibodies against CGAS, TOLLIP, UL21, UL54 and ACTB. Data are representative of at least three independent experiments with similar results.

The canonical or the selective autophagic pathways are different routes the cell uses to degrade intracellular proteins and organelles [36]. In the canonical autophagic pathway, ULK1 (unc-51 like autophagy activating kinase 1), ATG13 (autophagy related 13), RB1CC1/FIP200 (RB1 inducible coiled-coil 1), and ATG101 (autophagy related 101) are key players [26]. However, knockdown of ULK1, ATG13, RB1CC1, or ATG101 had no effects on the UL21-mediated degradation of CGAS (Figure S5B, C), suggesting that the UL21-mediated degradation of CGAS may not occur via the canonical autophagic, but the selective autophagic pathway.

In the selective autophagic pathway, the cargo receptors deliver substrates to autophagosomes for selective degradation [37]. To explore which cargo receptor mediates UL21 induced CGAS degradation, we assessed the interactions among CGAS, the cargo receptors, and UL21. As shown in Figure 5C, SQSTM1 and TOLLIP were found to bind to UL21. In addition, CGAS strongly interacted with SQSTM1, but weakly bound to TOLLIP (Figure 5D), indicating that SQSTM1 and TOLLIP might be associated with CGAS and UL21. Intriguingly, the interaction between TOLLIP and CGAS was strengthened in the presence of UL21 (Figure 5E). Based on these results, we speculated that SQSTM1 or TOLLIP may be the cargo receptor mediating CGAS degradation. Thus, HEK-293 T cells with SQSTM1 or TOLLIP knocked out (SQSTM1^−/− or TOLLIP^−/−) were constructed using the CRISPR-Cas9 system (Figure S6A–D). In SQSTM1^−/− HEK-293 T cells, CGAS degradation induced by UL21 was maintained at levels similar to WT HEK-293 T cells (Figure 5F). Notably, the CGAS degradation induced by UL21 was decreased in TOLLIP^−/− HEK-293 T cells as compared with that in the WT HEK-293 T cells (Figure 5G). The phenotype was also seen in the PK-15 cells by using the lentivirus CRISPR-Cas9 system (Figure 5H and Figure S6E). Also, CGAS degradation mediated by HSV-1 UL21 was found to be decreased in TOLLIP^−/− HEK-293 T cells (Figure S6F). In PRV infection, the decreased expression of CGAS in PRV-WT and ΔUL21R was restored to a level similar to that seen in the ΔUL21 in TOLLIP^−/− HEK-293 T cells (Figure 5I). These data demonstrated that TOLLIP is the cargo receptor that mediates the degradation of CGAS induced by alpha-herpesvirus UL21.

PRV and HSV-1 UL21 induces K27-linked ubiquitination of CGAS at site of K384

In the selective autophagic pathway, the cargo receptors usually recognize ubiquitinated substrates, which are then delivered to autophagosomes for selective degradation [37]. Therefore, we determined whether UL21 could induce CGAS ubiquitination. As shown in Figure 6A, after co-transfection of wild-type ubiquitin and CGAS into HEK-293 T cells, UL21 expression, but not empty vector expression, readily induced CGAS ubiquitination. Biochemical analysis of cells expressing UL21 together with different ubiquitin mutants in which specific lysine residues were substituted showed that only K27, and not K6, K11, K29, K33, K48, or K63, linked the ubiquitination for CGAS (Figure 6B).

Figure 6. — UL21 induces K27-linked ubiquitination of CGAS at K384 site. (A) UL21 induces the ubiquitination of CGAS. HEK-293 T cells were transfected with Flag-UL21 (3 μg) and HA-Ub (3 μg) together with empty vector (3 μg) or MYC-UL21 (3 μg). At 24 hpt, cells were processed for immunoprecipitation (IP) with anti-Flag magnetic beads. Whole-cell lysates (WCLs) and precipitated proteins were probed with antibodies against HA-tag, MYC-tag, Flag-tag, and ACTB. (B) UL21 catalyzes K27-linked ubiquitination of CGAS. HEK-293 T cells were transfected with Flag-CGAS (3 μg), MYC-UL21 (3 μg), empty vector (3 μg), ubiquitination-HA (3 μg) or its mutants (HA-K6 [3 μg], HA-K11 [3 μg], HA-K27 [3 μg], HA-K29 [3 μg], HA-K33 [3 μg], HA-K48 [3 μg], or HA-K63 [3 μg]). At 24 hpt, cells were processed for IP with anti-Flag magnetic beads. WCLs and precipitated proteins were probed with antibodies against HA-tag, MYC-tag, Flag-tag, and ACTB. (C) Schematic representation of wild-type CGAS and its deletion mutants. HEK-293 T cells were transfected with the empty vector (2 μg), Flag-CGAS (2 μg) or its truncated mutants (Flag-CGAS [1–160 aa; 2 μg], Flag-CGAS [160–522 aa; 2 μg], Flag-CGAS [1–330 aa; 2 μg], Flag-CGAS [160–330 aa; 2 μg], Flag-CGAS [330–522 aa; 2 μg], and Flag-CGAS [290–400 aa; 2 μg]). At 24 hpt, cells were harvested for western blot analysis with antibodies against Flag-tag and ACTB. (D) UL21 interacts with CGAS at the internal domain 290–400 aa. HEK-293 T cells were transfected with the MYC-UL21 (3 μg) together with the indicated plasmids (empty vector [3 μg], Flag-CGAS [3 μg], Flag-CGAS [1–160 aa; 3 μg], Flag-CGAS [160–522 aa; 3 μg], Flag-CGAS [1–330 aa; 3 μg], Flag-CGAS [160–330 aa; 3 μg], Flag-CGAS [330–522 aa; 3 μg], or Flag-CGAS [290–400 aa; 3 μg]). At 24 hpt, cells were processed for IP with anti-Flag magnetic beads. WCLs and precipitated proteins were probed with antibodies against MYC-tag, Flag-tag, and ACTB. (E) UL21 catalyzes K27-linked ubiquitination of CGAS at K384 site. HEK-293 T cells were transfected with MYC-UL21 (3 μg) and HA-Ub-K27 (3 μg) together with the indicated plasmids (empty vector [3 μg], Flag-CGAS-WT [3 μg], Flag-CGAS-K301R [3 μg], Flag-CGAS-K327 R [3 μg], Flag-CGAS-K347R [3 μg], Flag-CGAS-K362R [3 μg], Flag-CGAS-K365R [3 μg], Flag-CGAS-K384R [3 μg], or Flag-CGAS-K394R [3 μg]). At 24 hpt, cells were processed for IP with anti-Flag magnetic beads. WCLs and precipitated proteins were probed with antibodies against HA-tag, Flag-tag, MYC-tag, and ACTB. (F and G) Effects of UL21 on human and porcine CGAS mutant expression. HEK-293 T (F) or PK-15 (G) cells seeded in 6-well plates were transfected with MYC-UL21 (2 μg) together with Flag-CGAS/Flag-CGAS [P] or its mutants (2 μg). At 24 hpt, cells were harvested for western blot analysis with antibodies against MYC-tag, Flag-tag, and ACTB. Data are representative of at least three independent experiments with similar results.

Next, we aimed to identify the potential ubiquitination sites on CGAS. We firstly explored the domain of CGAS that interacts with UL21 by developing a series of CGAS deletion mutants (Figure 6C). The results of an immunoprecipitation assay showed that only the truncated Flag-CGAS (160–522 aa) and Flag-CGAS (290–400 aa) were strongly bound to UL21, indicating that the internal domain of CGAS (amino acids 290–400) was required for the CGAS-UL21 interaction (Figure 6D). As UL21 could degrade both human and porcine CGAS, we compared the conserved lysines (K) located within amino acids 290–400, which contained seven lysines (Figure S7A). Further, a systematic lysine (K) to arginine (R) mutation scanning was carried out to map the ubiquitination sites on CGAS. When K384 in human CGAS was mutated to arginine, the K27-linked ubiquitination of CGAS was almost completely abolished (Figure 6E). As expected, ectopic expression of UL21 could not degrade CGAS with a mutation at K384R (human CGAS) or K359R (porcine CGAS) (Figure 6 F–). Additionally, HSV-1 UL21 could also not degrade the mutated human CGAS K384R (Figure S7B). Collectively, these results suggested that K384 in human or K359 in porcine is the key site responsible for CGAS ubiquitination.

UBE3C is the E3 ligase that mediates CGAS ubiquitination induced by PRV and HSV-1 UL21

Because alpha-herpesvirus UL21 has no E3 ubiquitin ligase activity, we reasoned that UL21 might function as a scaffold to link CGAS to its E3 ligase for ubiquitination and then degradation. To elucidate this, we first sought to identify the potential E3 ligases from the UL21 interactome through MS analysis of cellular factors that co-purified with Flag-UL21 transfected HEK-293 T cells (Table S1A). Additionally, we sought to find CGAS or TOLLIP associated E3 ligases through the UbiBrowser database (http://ubibrowser.ncpsb.org/). Twenty E3 ligases were found in the UL21 interactome (Table S1B and Figure S8A). Furthermore, 20 and 132 E3 ligases were separately predicted to link to CGAS or TOLLIP, respectively, with 9 E3 ligases shared among both proteins (Table S1C, D and Figure S8A). Among them, we found that ubiquitin protein ligase E3C (UBE3C) was unique. To further define the role of UBE3C in the process of CGAS degradation induced by UL21, we detected CGAS expression after knocking down UBE3C by siRNA. As expected, CGAS expression was restored only in the UBE3C reduced group, but not in other E3 ligase reduced groups (Figure S8B, C). Importantly, in UBE3C^−/− HEK-293 T and PK-15 cells, CGAS degradation induced by UL21 was reduced (Figure 7A,, and Figure S9A-C). The effects of HSV-1 UL21 on CGAS degradation was also decreased in UBE3C^−/− HEK-293 T cells (Figure S9D). We then determined the interaction between UL21 and UBE3C by co-immunoprecipitation (co-IP) and confocal assays. As shown in Figure 7C and 7D, UL21 interacted and colocalized with UBE3C, which was also confirmed by the interaction between HSV-1 UL21 and UBE3C (Figure S9E). Importantly, overexpression of UL21 could not induce K27-linked ubiquitination of CGAS in UBE3C^−/− HEK-293 T cells, which only occurred in WT HEK-293 T cells (Figure 7E). Collectively, these data indicated that UBE3C is the specific E3 ligase responsible for mediating CGAS ubiquitination and degradation induced by alpha-herpesvirus UL21.

Figure 7. — UBE3C is the E3 ligase that mediates CGAS ubiquitination induced by UL21. (A and B) UBE3C mediates CGAS degradation induced by UL21. Wild-type (WT) or *UBE3C*^−/− HEK-293 T (A) and PK-15 (B) cells seeded in 6-well plates were transfected with HA-CGAS (2 μg) together with or without MYC-UL21 (2 μg), and with or without Flag-UBE3C (2 μg). At 24 hpt, cells were harvested for western blot analysis with antibodies against HA-tag, MYC-tag, UBE3C, and ACTB. (C) UL21 interacts with UBE3C. HEK-293 T cells were transfected with MYC-UL21 (3 μg) together with indicated plasmids (empty vector [3 μg], Flag-NBR1 [as the negative control; 3 μg], or Flag-UBE3C [3 μg]). At 24 hpt, cells were processed for immunoprecipitation (IP) with anti-MYC magnetic beads. Whole-cell lysates (WCLs) and precipitated proteins were probed with antibodies against Flag-tag, MYC-tag, and ACTB. (D) UL21 colocalizes with UBE3C. HeLa cells were transfected with Flag-UBE3C (2 μg) along with empty vector (2 μg) or MYC-UL21 (2 μg). At 24 hpt, cells were stained with anti-Flag (red) and anti-MYC (green) subjected to analysis by confocal microscopy. Nuclei were stained with DAPI (blue) (scale bar: 10 μm). (E) UBE3C is necessary for the K27-linked ubiquitination of CGAS induced by UL21. Wild-type (WT) or *UBE3C*^−/− HEK-293 T cells were transfected with MYC-UL21 (2 μg), Flag-CGAS (2 μg), HA-Ub (K27 only) (2 μg), or HA-Ub (K27R) (K27 lysine residues mutated to arginine) (2 μg). At 24 hpt, cells were processed for IP with anti-Flag magnetic beads. WCLs and precipitated proteins were probed with antibodies against Flag-tag, MYC-tag, HA-tag, and ACTB. Data are representative of at least three independent experiments with similar results.

The N terminus of PRV and HSV-1 UL21 interacts with CGAS and mediates its degradation

UL21 is a conserved tegument protein in alpha-herpesvirus [6]. To determine the domain of UL21 responsible for CGAS degradation, we constructed four truncates according to the amino acid (aa) sequence conservation of UL21 across the alpha-herpesviridae family (Figure 8A and Figure S10A). The results of the co-IP showed that the N-terminal (1–200 aa) of UL21 was essential for the interaction with CGAS (Figure 8B). Furthermore, UL21 (1–200 aa) was found to colocalize with CGAS in the cytoplasm (Figure 8C). Interestingly, the predicted structure of the N terminus of UL21 at 1–200 aa in alpha-herpesviruses (PRV and HSV-1) is very similar (Figure S10B). These results indicated that the N-terminal region of UL21 at 1–200 aa is located within the cytoplasm and mediates the interaction with CGAS and may function in CGAS degradation.

Next, we determined the roles of the truncates on the CGAS-mediated innate immune response. As shown in Figure 8D, UL21 (1–200 aa), but not UL21 (200–532 aa) was found to inhibit CGAS-mediated IFNB promoter activity. Further, we evaluated the effects of UL21 (1–200 aa) and UL21 (200–532 aa), separately defined as N- or C-terminal, on CGAS degradation. The results showed that the N-terminal region of UL21 induced CGAS degradation mediated by autophagy (Figure 8E,). Consistently, CGAS degradation was induced by the N-terminal region of HSV-1 UL21 (Figure S10C). In addition, as expected, the N terminus of UL21 interacted with TOLLIP and UBE3C, and further strengthened the combination of TOLLIP and CGAS, the same as full-length (FL) of UL21 (Figure S10D–F). Consistently, we found that CGAS degradation mediated by the UL21 N-terminal 1–200 aa or full-length (FL) was restored in TOLLIP^−/− and UBE3C^−/− HEK-293 T cells (Figure 8G,). Finally, to define CGAS degradation induced by the N-terminal region of UL21 during PRV infection, we constructed recombinant viruses with deletions at 200–532 aa or 1–200 aa, named as ΔUL21 (1–200 aa) or ΔUL21 (200–532 aa), respectively (Figure S10G, H). As shown in Figure 8I and 8J, CGAS degradation was seen in WT HEK-293 T cells infected with PRV wild-type (PRV-WT), ΔUL21 (1–200 aa), and ΔUL21R, but not in ΔUL21 or ΔUL21 (200–532 aa). In contrast, this phenotype was dismissed in TOLLIP^−/− or UBE3C^−/− HEK-293 T cells. The phenotype was also seen in WT and TOLLIP^−/− or UBE3C^−/− PK-15 cells (Figure S10I, J). Altogether, these results demonstrated that the N-terminal region of UL21 at 1–200 aa is responsible for inducing CGAS degradation during alpha-herpesvirus infection.

Cgas is essential in controlling PRV infection in mice

Finally, cgas knockout mice were exploited to investigate the significance of Cgas in PRV infection in vivo. Bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) and cgas knockout (cgas^−/−) mice were infected with PRV and then harvested for detecting the induction of interferon stimulated genes. Real-time polymerase chain reaction (RT-PCR) experiments indicated that the transcription of Ifnb, Isg15, Ifit2, and Ifit1 in WT BMDMs induced by infection with ΔUL21 or ΔUL21 (200–532 aa) was significantly higher than those infected with PRV-WT, ΔUL21 (1–200 aa), and ΔUL21R. In contrast, transcription of Ifnb, Isg15, Ifit2, and Ifit1 induced by PRV was significantly dampened in cgas^−/− BMDMs as compared with WT BMDMs (Figure 9A). As predicted, in WT BMDMs, the phosphorylation of TBK1 and IRF3 were obviously increased after infection with ΔUL21 (200–532 aa), but slightly increased after infection with PRV-WT, ΔUL21 (1–200 aa), and ΔUL21R. Otherwise, the levels of phosphorylated TBK1 and IRF3 were dramatically impaired in cgas^−/− BMDMs (Figure 9B).

Figure 9. — CGAS is essential for controlling PRV infection. (A) RT-PCR analysis of *Ifnb, Isg15, Ifit2, Ifit1* in wild-type (WT) and *cgas*^−/− BMDMs after PRV infection. WT and *cgas*^−/− BMDMs cells were mock-infected or infected with PRV-WT, ΔUL21, ΔUL21 (1–200 aa), ΔUL21 (200–532 aa), or ΔUL21R at 3 PFU/cell. At 12 hpi, the cells were harvested for RNA extraction and RT-PCR detection of *Ifnb, Isg15, Ifit2*, and *Ifit1* mRNA, and *Rna18s* rRNA levels. (B) Immunoblot analysis of lysates of WT or *cgas*^−/− BMDMs after PRV infection. WT and *cgas*^−/− BMDMs cells were mock infected or infected with PRV-WT, ΔUL21, ΔUL21 (1–200 aa), ΔUL21 (200–532 aa), or ΔUL21R at 3 PFU/cell. At 12 hpi, cells were harvested for western blot analysis with antibodies against CGAS, STING1, p-TBK1, TBK1, p-IRF3, IRF3, UL21, UL54, and ACTB. (C) Mouse survival was recorded and shown as a percentage over time. WT and *cgas*^−/− mice separately housed in 6 groups (n = 8 mice/group) were mock-infected or infected with PRV-WT, ΔUL21, ΔUL21 (1–200 aa), ΔUL21 (200–532 aa), or ΔUL21R (1 × 10⁴ pfu/mice) through intraperitoneal injection. (D) Viral DNA loads in the brain, lung, or brainstem were quantified by RT-PCR. WT and *cgas*^−/− mice separately housed in 6 groups (n = 4 mice/group) were mock-infected or infected with PRV-WT, ΔUL21, ΔUL21 (1–200 aa), ΔUL21 (200–532 aa), or ΔUL21R (1 × 10⁴ pfu/mice) through intraperitoneal injection. Three days post-infection, the brain, lung or brainstem tissues were harvested for RT-PCR detection of viral loads. (E) The Isg expression in lung tissues from the mice in (D). Total RNA was extracted from the lung tissues and used for RT-PCR detection of *Ifnb, Isg15, Ifit2*, and *Ifit1* mRNA, and *Rna18s* rRNA levels. (F) H&E-stained brain and lung sections from the mice in (D) (scale bar: 100 μm). The data are representative of at least three independent experiments with similar results (mean ± SD of n = 3 biological replicates in A, n = 4 biological replicates in D, E and F). * p < 0.05, ** p < 0.01, ns: not significant (two-tailed unpaired t test).

Next, WT or cgas^−/− mice were mock-infected (a control used in infection experiments treated with the same volume of phosphate-buffered saline [PBS] without the virus) or infected with PRV-WT, ΔUL21, ΔUL21 (1–200 aa), ΔUL21 (200–532 aa), or ΔUL21R (diluted in the same volume of PBS) at 1 × 10⁴ plaque forming units/mice, and their survival was monitored. As shown in Figure 9C, there was a 0% survival rate in WT or cgas^−/− mice inoculated with PRV-WT, ΔUL21 (1–200 aa), and ΔUL21R. As expected, all mice survived in the mock-infected group. However, there were different patterns in WT or cgas^−/− mice infected with ΔUL21 and ΔUL21 (200–532 aa). Of the WT mice, only 5/8 mice infected with ΔUL21 survived and 4/8 infected with ΔUL21 (200–532 aa) survived. Only 1/8 cgas^−/− mice survived in the ΔUL21 and ΔUL21 (200–532 aa) groups. Further, we collected the brain, lung, and brainstem tissues at 3 dpi and measured the PRV loads. As illustrated in Figure 9D, in WT mice, PRV DNA loads in the lung, brain, and brainstem tissues collected from the ΔUL21 and ΔUL21 (200–532 aa) groups were significantly lower than those in PRV-WT, ΔUL21 (1–200 aa), and ΔUL21R groups. In contrast, in cgas^−/− mice, the differences were reduced. The expression of Isg in lung tissues was also determined by RT-PCR, which showed that the transcripts of Ifnb, Isg15, Ifit2, and Ifit1 in lung tissues of WT mice infected with ΔUL21 and ΔUL21 (200–532 aa) were significantly higher than those in PRV-WT, ΔUL21 (1–200 aa), and ΔUL21R. In contrast, in cgas^−/− mice, the transcripts of Ifnb, Isg15, Ifit2, and Ifit1 were sharply decreased, and the differences induced by PRV were almost diminished (Figure 9E). Consistently, in general, the neuropathological lesions in the brain and the inflammatory cell infiltrate and congestion in the lungs of WT mice were obviously less than those in cgas^−/− mice (Figure 9F). The neuropathological lesions of brain tissue from WT mice infected with ΔUL21 and ΔUL21 (200–532 aa) were milder than those in PRV-WT, ΔUL21 (1–200 aa), and ΔUL21R groups. In contrast, the neuropathological lesions of brain tissues in the cgas^−/− mice infected with ΔUL21 and ΔUL21 (200–532 aa) were similar to the lesions in the PRV-WT, ΔUL21 (1–200 aa), and ΔUL21R groups. The same phenotypes were also seen in the lung injury. Altogether, these results suggested that Cgas plays a critical role in controlling PRV infection in vivo.

Discussion

The cytoplasmic DNA sensor CGAS mediates innate immune responses against alpha-herpesviruses. Conversely, alpha-herpesviruses typically attempt to subvert the host immune response to benefit their life cycle. The tegument protein UL37 of HSV-1 has been shown to deamidate CGAS, impairing the ability of CGAS to catalyze cGAMP synthesis and inhibiting CGAS-induced antiviral responses [38]. HSV-1 tegument protein UL41, an mRNA-specific endonuclease, has been shown to downregulate the mRNA and protein level of CGAS to abrogate CGAS-STING1-mediated signaling [39]. Another tegument protein, VP22, was demonstrated to interact with CGAS and directly inhibit its enzymatic activity [40]. Additionally, a recent report demonstrated that VP22 can also restrict CGAS-DNA phase separation to mediate immune evasion [41]. In this study, we propose a model whereby the tegument protein UL21 in alpha-herpesviruses antagonize CGAS-mediated innate signaling pathway (Figure 10). Briefly, to counteract the host antiviral immune response, we showed that the UL21 protein induces K27-linked ubiquitination of CGAS mediated by the E3 ligase UBE3C. Subsequently, the UL21 protein preferentially recruits and interacts with the selective autophagic receptor TOLLIP to recognize ubiquitinated CGAS, which finally fuses with the lysosome for CGAS degradation. Thus, alpha-herpesvirus UL21 inactivates CGAS, leading to type I interferon inhibition and a higher viral replication efficiency.

Figure 10. — A working model of the role of the UL21 protein of PRV in the regulation of CGAS mediated signaling. Black arrows indicate the CGAS-STING1-mediated type I IFN signaling pathway. Blue arrows indicate expression of the UL21 protein. Red arrows indicate the process of UL21-mediated autophagic degradation of CGAS.

UL21 is a conserved tegument protein of alpha-herpesviruses that consists of about 530 aa [6]. Several studies have demonstrated that UL21 influences the neuroinvasion and neurovirulence of PRV proteins in mice – phenotypes that are both associated with the carboxyl terminus of UL21 [12,14,42]. In HSV-1, UL21 promotes viral replication and spread by directly binding with PPP1, which is dependent upon the N-terminal domain and flexible linker (1–281 aa) [9]. It has been demonstrated that targeting of the host phosphatase PPP1CA/PPP1α (protein phosphatase 1 catalytic subunit alpha), a member of the PPP1 family, by the HSV-1 viral protein ICP34.5 contributes to HSV induced encephalitis [43]. Coincidently, our mass spectrometry data showed that PPP1 was also an interactor of PRV UL21 (Blue marked in Table S1A). These results implied that UL21 in alpha-herpesviruses is a conserved viral phosphatase adaptor. Here, we confirmed that UL21 is a pathogenic factor in PRV infection. We identified a previously unrecognized mechanism, whereby the N-terminal domain (1–200 aa) of UL21 plays a dominant role in inactivating CGAS signaling via TOLLIP-mediated selective autophagy. Sequence alignment revealed that the homology of the N-terminal domain of UL21 is highly conserved among alpha-herpesviruses, suggesting the CGAS inactivating function is shared by UL21 protein from other alpha-herpesviruses. In this study, we showed that HSV-1 UL21 is capable of inducing CGAS degradation as well. Whether the function of UL21 on CGAS degradation is conserved in other alpha-herpesvirus orthologs needs to be further explored in the future. In addition, research regarding the relationship between the PPP1-binding activity, CGAS inhibition, and viral pathogenicity of alpha-herpesvirus UL21 is warranted.

As mentioned above, most work on UL21 has been performed using HSV-1 and pseudorabies virus. Baines et al. showed that UL21 of HSV-1 is expressed as a late (gamma 1) gene [44]. However, Yoshifumi Muto et al. found that HSV-1 UL21 could be expressed at 6 h post infection at an MOI of 3 [45]. Under the high MOI condition (MOI = 3), PRV UL21 expression started from 4 h post infection, whereas the UL21 was not detectable until 12 h post-infection with the low MOI (0.05 MOI). Consistent with the UL21 expression patterns, CGAS degradation only occurred at the early time points in cells infected with the high MOI PRV, but not in cells with the low MOI. Due to the complexity of viral gene expressions at late timepoints, it is difficult to explore whether CGAS degradation is solely induced by UL21. Besides, we found that the LC3-I expression was almost stable at 4, 8, and 12 h under the low MOI infection condition but decreased in cells with the high MOI infection. We suspected that PRV infection with the high MOI may suppress LC3-I expression, which might be attributed to two reasons. One possible reason is that UL21 expression promotes LC3-I transferring to LC3-II; the other one is that some other viral genes may downregulate LC3-I expression. The detailed mechanisms need to be explored further in the future.

Selective autophagy, a bulk degradation process in eukaryotes, is extremely discerning, serving as a rheostat for immune reactions to maintain cell homeostasis with the help of cargo receptors [46], including SQSTM1/p62, NBR1, OPTN, CALCOCO2/NDP52, BNIP3L/Nix, TOLLIP, and newly identified CCDC50 (coiled-coil domain containing 50) and CCT2 (chaperonin containing TCP1 subunit 2) [47–54]. Upon viral infection, selective autophagy can exploit the cargo receptors to deliver the viruses or viral components to the autophagosome for autophagy-lysosomal degradation, which is helpful to the host clearance of viral infection [23]. In turn, selective autophagy can also target and degrade the critical components of immune factors to abrogate antiviral immunity, favoring viral replication. For instance, CALCOCO2/NDP52 has been shown to promote the degradation of IRF3 in response to Sendai virus (SeV) infection [55], and NBR1 has been shown to mediate selective autophagy to degrade MAVS induced by the PB1 protein of influenza A virus [56]. CCDC50 was demonstrated to play important roles in RIGI-IFIH1/MDA5 and STING1 degradation during viral infection [53,57]. TOLLIP, the Cue5 homolog in yeast, has been reported to be a selective autophagy receptor to mediate the clearance of Huntington disease-linked polyQ proteins, implicating the critical role of TOLLIP in innate immunity and human health and disease [52,58]. Recently, TOLLIP was shown to have regulatory functions in endosomal microautophagy [59]. Our data showed that alpha-herpesvirus UL21 induced CGAS degradation via TOLLIP-mediated autophagy, which negatively modulated innate immunity and promoted viral replication. This study is the first to report a connection between CGAS and TOLLIP, providing evidence that TOLLIP plays a role in the innate immune response during viral infection.

Selective autophagy receptors are equipped with a ubiquitin-binding domain, which is used for the recognition of ubiquitinated cargo. Ubiquitination has also been shown to be equally important for CGAS signaling. Upon DNA virus infection, USP14 (ubiquitin specific peptidase 14) is recruited by IFN-inducible TRIM14 (tripartite motif containing 14) to cleave the K48-linked polyubiquitin chains of CGAS, thereby inhibiting SQSTM1-dependent degradation of CGAS [60]. The deubiquitinases USP27X (ubiquitin specific peptidase 27 X-linked) and USP29 (ubiquitin-specific peptidase 29) directly interact with CGAS and remove K48-linked polyubiquitination chains on CGAS, resulting in the stabilization of CGAS [61,62]. Additionally, the E3 ligase TRIM56 (tripartite motif containing 56) induces the mono-ubiquitination of CGAS at K347 site, which is important for efficient cytosolic DNA sensing in response to DNA virus infection [63]. RNF185 (ring finger protein 185) catalyzes the K27-linked polyubiquitination of CGAS at K173 and K384 sites, both of which enhance CGAS dimerization and promote its enzymatic activity upon detecting cytosolic DNAs [64]. We observed that CGAS underwent UL21 dependent K27-linked ubiquitination at K384 site mediated by the E3 ligase UBE3C. UBE3C, a member of the HECT family, has been reported to catalyze K48-, and to a lesser extent K29- and K11-linked chains, which are also involved in the proteasomal degradation pathway [65]. For example, UBE3C inhibits type I interferon by mediating K48-linked ubiquitination of IRF3 and IRF7, leading to their proteasomal degradation [66]. Intriguingly, our work revealed that UBE3C is the E3 ligase that mediates the K27-linked ubiquitination of CGAS, which is then recognized and degraded by the TOLLIP-mediated selective autophagy. However, further work is required to clarify the detailed mechanism on how UBE3C ubiquitinates CGAS.

In summary, we described the ubiquitination and autophagic degradation of CGAS-mediated by the conserved tegument protein UL21 of alpha-herpesviruses. This work introduces the UL21-UBE3C-CGAS-TOLLIP axis in DNA virus infections, which plays important roles in the immune escape of herpesviruses. Furthermore, this axis has the potential to serve as a novel target for the development of antiviral therapeutics that regulate the host immune response.

Materials and methods

Cells, viruses and plasmids

Human embryonic kidney (HEK)-293 T (CRL-11268), HeLa (CCL-2), Porcine kidney cell 15 (PK-15; CCL-33), Vero (CCL-81), and Neuro-2a (N2a; CCL-131) cells were obtained from the American Type Culture Collection (ATCC). Mouse embryonic fibroblasts (MEFs) were kindly gifted from Dr. Rong Zhang (Fudan University, China). CGAS^−/−, SQSTM1^−/−, TOLLIP^−/−, and UBE3C^−/− HEK-293 T cells were obtained by using the CRISPR-Cas9 system with specific small guide RNAs (sgRNAs) inserted into plasmid PX458 (Addgene, 48,138; deposited by Feng Zhang). CGAS^−/− PK-15 cells were a gift from Beibei Chu (Henan Agricultural University, China) as previously described [67]. TOLLIP^−/− and UBE3C^−/− PK-15 cells were generated by using the LentiCRISPRV2 system (Addgene, 52,961; deposited by Feng Zhang) and selected with puromycin at a concentration of 5 μg/mL (Santa Cruz Biotechnology, sc-108,071). All cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, 11,995,065) containing 10% fetal bovine serum (FBS; Gibco, 10,099,141) in a humidified 5% CO₂ incubator at 37°C.

PRV ZJ01 (GenBank: KM061380.1) is a prototype PRV strain used in this study as wild-type PRV (PRV-WT) [68]. The recombinant viruses with UL21 deletion (ΔUL21) or its revertant (ΔUL21R), or with UL21 specific domain mutations (1–200 or 200–532 aa) named as ΔUL21 (1–200 aa) or ΔUL21 (200–532 aa), were all generated by the CRISPR-Cas9 system as described below. Preparation of viral stock and titration of infectivity were carried out in Vero cells.

Flag-UL21 was obtained by inserting the PRV UL21 gene into the EcoRI and XhoI sites of pCMV-N-Flag (Beyotime, D2722). Flag-UL21 was used as a PCR template to generate the truncation mutants, also inserting into the EcoRI and XhoI sites of pCMV-N-Flag, specifically Flag-UL21 (1–300 aa), Flag-UL21 (200–532 aa), Flag-UL21 (1–200 aa), and Flag-UL21 (300–532 aa). Flag-UL21 (HSV-1) was obtained by inserting the HSV-1 UL21 gene into the EcoRI and XbaI sites of pCMV-N-Flag, which was then used as a PCR template to generate the Flag-UL21 (1–200 aa) (HSV-1) and Flag-UL21 (200–535 aa) (HSV-1) truncation mutants. Flag-UL16 and Flag-UL16 (HSV-1) were obtained by separately inserting the PRV UL16 or HSV-1 UL16 gene into the BamHI and EcoRI sites of pCMV-N-Flag. HA-CGAS was obtained by inserting the human CGAS gene into the SalI and XhoI sites of pCMV-HA (Clontech, 635,690). The human CGAS gene was also cloned into pCMV-N-Flag) using the HindIII and XhoI sites on Flag-CGAS, which was used as the template to construct truncation mutants named as Flag-CGAS (1–160 aa), Flag-CGAS (160–522 aa), Flag-CGAS (1–330 aa), Flag-CGAS (160–330 aa), Flag-CGAS (330–522 aa), and Flag-CGAS (290–400 aa). Subsequently, Flag-CGAS was also used as the template to generate other mutations (i.e., Flag-CGAS-K301R, Flag-CGAS-K327R, Flag-CGAS-K347R, Flag-CGAS-K362R, Flag-CGAS-K365R, Flag-CGAS-K384R, and Flag-CGAS-K394R) by site-directed mutagenesis with QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, 200,523). MYC-STING1, MYC-UL21, and MYC-UL21 (HSV-1) were obtained by inserting human STING1, PRV UL21, or HSV-1 UL21 genes into the HindIII and EcoRI sites of pCMV-N-MYC (Beyotime, D2756). The porcine CGAS gene was gifted by Beibei Chu (Henan Agricultural University, China) and cloned into pCMV-N-Flag by using the BamHI and HindIII sites named as Flag-CGAS [P], which was subsequently used as the template to generate other mutations (i.e., Flag-CGAS [P]-K274R, Flag-CGAS [P]-K302R, Flag-CGAS [P]-K322R, Flag-CGAS [P]-K337R, Flag-CGAS [P]-K340R, Flag-CGAS [P]-K359R, and Flag-CGAS [P]-K369R). The GFP cassette was separately inserted into pCMV-HA or pCMV-N-Flag by using the EcoRI and XhoI sites to generate HA-GFP or Flag-GFP. Flag-UBE3C (RC215110), Flag-NBR1 (RC215397), Flag-OPTN (RC202470), Flag-SQSTM1 (RC210581), Flag-CALCOCO2 (RC203843), Flag-BNIP3L (RC203315), and Flag-TOLLIP (RC200227) were purchased from Origene. MYC-UBE3C and MYC-TOLLIP were obtained by cloning the UBE3C and TOLLIP genes into the SalI and SpeI sites of pCMV-N-MYC. pCMV HA-Ub (wild type [WT]) (17,608, deposited by Ted Dawson) and the mutants HA-Ub (K6 only) (22,900, deposited by Sandra Weller), HA-Ub (K11 only) (22,901, deposited by Sandra Weller), HA-Ub (K27 only) (22,902, deposited by Sandra Weller), HA-Ub (K29 only) (22,903, deposited by Sandra Weller), HA-Ub (K33 only) (17,607, deposited by Ted Dawson), HA-Ub (K48 only) (17,605, deposited by Ted Dawson), HA-Ub (K63 only) (17,606, deposited by Ted Dawson), GFP-LC3 (11,546, deposited by Karla Kirkegaard), PX335 (42,335, deposited by Feng Zhang), and lentivirus package plasmids pVSV-G (8454, deposited by Bob Weinberg) and psPAX2 (12,260, deposited by Didier Trono) were purchased from Addgene. HA-Ub (K27R) was generated by site-directed mutagenesis with QuikChange II Site-Directed Mutagenesis Kit using HA-Ub (WT) as the template. IFNB-Luc and pRL-TK were described elsewhere [69]. The primers used for plasmid construction are listed in Table S2. All the constructs were confirmed by Sanger sequencing (Sangon Biotech).

Generation of recombinant PRV by CRISPR-Cas9 system

To construct the plasmid pX335-UL21 single guide RNA (sgRNA)1 and PX335-UL21 sgRNA2, we obtained sgRNA1 and sgRNA2 targeting PRV UL21 from the annealing product of sgRNA1-Fwd and sgRNA1-Rev and sgRNA2-Fwd and sgRNA2-Rev oligos, respectively, and inserted them into the pX335 vector at the BbsI site to produce the recombinant plasmid pX335-UL21 sgRNA1 and pX335-UL21 sgRNA2, respectively. The plasmids pX335-GFP sgRNA1 and PX335-GFP sgRNA2 were obtained as described above. To obtain the donor plasmids pUC19-UL22-EGFP-UL20, pUC19-UL22-UL20, pUC19-UL22-UL21-UL20, pUC19-UL22-UL21(1–200 aa)-UL20, or pUC19-UL22-UL21(200–532 aa)-UL20, we cloned the indicated DNA fragments into pUC19 using the HindIII, BamHI, KpnI, and EcoRI sites. All oligo sequences are listed in Table S2. All the plasmids were confirmed by Sanger sequencing.

We firstly rescued the recombinant viruses with UL21 deletion replaced by GFP named as ΔUL21 (GFP). To do this, we first transfected donor plasmid pUC19-UL22-EGFP-UL20 together with pX335-UL21 sgRNA1 and PX335-UL21 sgRNA2 into the HEK-293 T cells. Twenty-four hours post-transfection (hpt), the cells were infected with wild-type PRV (PRV-WT) at an MOI of 0.01. Forty-eight hours post-infection (hpi), the cell supernatants were harvested, which were used for selection of the recombinant virus. Briefly, the harvested cell supernatants were serially diluted and incubated with the Vero cells to allow for infection. Afterward, the plaques carrying GFP signaling were picked. After 5 rounds of purification, the virus was confirmed by PCR, western blot, and sequencing. The resultant virus was named ΔUL21 (GFP). Based on the recombinant virus ΔUL21 (GFP), we obtained recombinant ΔUL21, ΔUL21R, ΔUL21 (1–200 aa), or ΔUL21 (200–532 aa) viruses by using the same methods, but picking the plaques without GFP signaling.

Generation of knockout cell lines by CRISPR-Cas9 system

Single clonal knockout HEK-293 T cells were obtained using the PX458 vector that expresses Cas9 and sgRNA targeting SQSTM1, TOLLIP, UBE3C, and CGAS. The sequences of the sgRNA are listed in Table S2. Green fluorescent protein (GFP) positive single cells were sorted at 48 h post-transfection and serially diluted to a single cell into 96-well plates and screened for gene knockout based on western blot and Sanger sequencing. Pooled knockout PK-15 cells were obtained by lentiviral transduction. Briefly, the LentiCRISPRV2 vector that expressing Cas9 and TOLLIP, UBE3C sgRNA together with package plasmids (pVSV-G and psPAX2) were transfected in HEK-293 T cells. The produced lentivirus was collected after 48 hpt. PK-15 cells were then infected with the lentivirus. At 16 h after infection, the cells were overlaid with fresh medium. At 3 d post-infection, the cells were selected using 5 μg/mL puromycin (Santa Cruz Biotechnology). All experiments were performed within 2 weeks after lentiviral transduction.

Antibodies and chemical reagents

The antibodies and chemical reagents used are listed as follows: anti-FLAG (HRP conjugate, M2; Sigma-Aldrich, A8592), anti-HA (HRP conjugate, 6E2; Cell Signaling Technology, 2999), anti-MYC (HRP conjugate, 9B11; Cell Signaling Technology, 2040), anti-ACTB/β-actin (AC-15; Sigma-Aldrich, A5441), anti-CGAS (human-specific, D1D3G; Cell Signaling Technology, 15,102), anti-CGAS (porcine-specific; Proteintech, 26,416-1-AP), anti-STING1 (Cell Signaling Technology, 13,647), anti-STING1 (Proteintech, 19,851-1-AP), anti-phospho-TBK1 (Ser172, D52C2; Cell Signaling Technology, 5483), anti-TBK1 (D1B; Cell Signaling Technology, 3504), anti-phospho-IRF3 (Ser396, D6O1M; Cell Signaling Technology, 29,047), anti-IRF3 (D83B9; Cell Signaling Technology, 4302S), anti-ATG7 (D12B11; Cell Signaling Technology, 8558), anti-LC3 (Proteintech, 14,600-1-AP), anti-SQSTM1/p62 (Proteintech, 18,420-1-AP), anti-TOLLIP (Proteintech, 11,315-1-AP), anti-UBE3C (Abcam, ab226173), anti-unique long region 54 (UL54) (a gift from Dr. Chunfu Zheng, Fujian Medical University, China) [70], mouse anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2357), goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, sc-2005). 3-MA (Sigma-Aldrich, M9281), NH₄Cl (Sigma-Aldrich, A9434), CQ (Sigma-Aldrich, C6628), MG-132 (Sigma-Aldrich, 474,787), and Z-VAD (Sigma-Aldrich, 627,610). The anti-UL21 and anti-UL16 antibodies were prepared in our lab.

Plasmids and siRNA transfections

In HEK-293 T cells, the plasmid transfections were conducted with Lipofectamine 3000 (Thermo Fisher Scientific, L3000015). In PK-15 cells, the plasmid transfection was performed with Lipofectamine LTX & Plus Reagent (Thermo Fisher Scientific, 15,338,100). For siRNA transfection, the siRNA was transfected into cells HEK-293 T or PK-15 cells by using Lipofectamine™ RNAiMAX (Thermo Fisher Scientific, 13,778,075). All the protocols were followed according to the manufacturer’s instruction. The siRNA sequences are listed in Table S2.

Dual luciferase assay

For the luciferase reporter assays, HEK-293 T cells grown in 24-well plates were transfected with the luciferase reporter plasmid IFNB-Luc and pRL-TK, together with HA-CGAS, MYC-STING1, Flag-UL21, or an empty vector using Lipofectamine 3000 according to the manufactural instruction. In poly(dA:dT) (InvivoGen, tlrl-patc) treatment assay, HEK-293 T cells grown in 24-well plates were firstly transfected with a luciferase reporter plasmid IFNB-Luc and pRL-TK together with Flag-UL21 or an empty vector. Twenty-four hpt, cells were then stimulated with poly(dA:dT) at 200 ng/mL for another 24 h. At 48 hpt, cells were harvested for a dual-luciferase reporter assay as previously described [69].

VSV-GFP infection inhibition assay

The GFP signaling of VSV-GFP was used to evaluate the antiviral cytokine secretion. The supernatants harvested from the dual-luciferase assay were added to fresh confluent HEK-293 T cells and incubated for 24 h. The cells were then infected at an MOI of 0.01 with VSV-GFP. At 12 h post-infection, VSV-GFP replication was visualized by monitoring the GFP expression level by fluorescence microscopy (Zeiss, Axio vert.A1).

Viral infection

Cells were infected with viruses at the indicated multiplicity of infection. After adsorption for 2 h, the monolayers were overlaid with DMEM supplemental with 1% FBS and incubated at 37°C. For viral titer determination, samples were harvested at 24 or 48 hpi and viruses, released by three cycles of freezing and thawing, were titrated on Vero cells by using plaque assay as previously described [69]. For western blot assay, the cell lysates were harvested at indicated timepoints for determination.

Real-time qPCR

Total RNA was harvested from cells by using an RNeasy Plus mini kit (Qiagen, 74,136) according to the manufacturer’s instruction. Aliquots of RNA (about 200 ng) were used for cDNA synthesis with a high-capacity cDNA reverse transcription kit with RNase inhibitor (Applied Biosystems, 4,374,966). Quantitative real-time PCR (qPCR) was performed on Applied Biosystems ABI Prism 7900 H T instrument with SYBR green master mix (Applied Biosystems, 4,385,616). For antiviral genes detection, the gene expression levels were normalized to that of endogenous control RNA18S rRNA. Relative gene expression was determined as described previously [71]. Primer sequences used for human/mouse IFNB, human/mouse IFIT1, human/mouse IFIT2, human/mouse ISG15, and RNA18S rRNA are listed in Table S2. For siRNA knockdown efficiency evaluation, the gene expression levels were also normalized to that of endogenous control RNA18S rRNA. Primer sequences used for human ATG7, ULK1, ATG13, RB1CC1, ATG101, UBE3C, RNF126 (ring finger protein 126), TRIM33 (tripartite motif containing 33), NEDD4L (NEDD4 like E3 ubiquitin protein ligase), and MARCHF9 (membrane associated ring-CH-type finger 9) are listed in Table S2. The mRNA expressions of these genes were determined by RT-PCR. The primers are also listed in Table S2. The viral loads in cells or the brain, lung, and brainstem samples of the PRV-infected mice were determined by absolute RT-PCR for the gB (PRV glycoprotein B) gene as previously described [68]. The primers are also listed in Table S2.

Mass spectrometry analysis

HEK-293 T cells were transfected with Flag-GFP or Flag-UL21. At 48 hpt, the cells were harvested and lysed with Nonidet P-40 (NP-40) buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40 [Beyotime, P0013 F], 150 mM NaCl, 1 mM EDTA, 1: 400 protease inhibitor mixture [Beyotime, ST506]). After centrifugation at 12,000 g, 10 min, supernatants were collected and mixed with 50-μL anti-Flag M2 magnetic beads, and then incubated for 4 h at 4°C. Precipitates were washed extensively with lysis buffer and separately analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) using the Q Exactive (Thermo Fisher Scientific, 0726055) and Easy-nLC 1000 (Thermo Fisher Scientific, LC120) instruments. MS data were analyzed using MASCOT. The experiment was conducted at the Beijing Genomics Institute (BGI).

Sequence alignment

A sequence alignment was used for comparing the conservation of UL21 across alpha-herpesviruses. PRV ZJ01 (GenBank: KM061380.1) is a prototype PRV strain used in this study. The following sequences were aligned using ClustalW [72] and conservation calculated using Jalview [73] with PRV UL21 (abbreviation and Uniprot ID are shown in parentheses): HSV-1 (Herpes simplex virus 1, P10205), HSV-2 (Herpes simplex virus 2, G9I242), CHV-2 (cercopithecine herpesvirus 2, Q5Y0T2), SHV-1 (saimiriine herpesvirus 1, E2IUE9), BHV-1 (bovine alpha-herpesvirus 1, Q65563), EHV-1 (equine herpesvirus 1, P28972), AHV-1 (anatid herpesvirus 1, A4GRJ2), and VZV (varicella-zoster virus, Q6QCT9). Alignment results are shown with conserved residues highlighted.

Confocal fluorescence microscopy

Hela cells seeded in 6-well plates (5 × 10⁵ cells/well) on coverslips were transfected with indicated plasmids. Twenty-four hpt, the cells were fixed with 4% paraformaldehyde (Beyotime, P0099) for 20 min at room temperature and then washed three times with phosphate-buffered saline (PBS; Beyotime, C0221A). Cells were permeabilized with 0.1% Triton X-100 (Beyotime, P0096) in PBS for 10 min and blocked with 5% bovine serum albumin (BSA; Beyotime, ST2254) for 1 h. Afterward, the cells were incubated with primary antibodies overnight at 4°C. After washing with PBS, a fluorescein-conjugated secondary antibody (Proteintech, SA00013) was incubated with the cells for 1 h. Finally, the cells were washed with PBS and stained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542). The stained cells were observed with a Nikon microscope (Nikon, Nikon A1 confocal microscope) with a 60× oil objective.

Western blot

Protein from cell lysates were subjected to electrophoresis on 8%, 10%, or 12% Bis-Tris sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels (pH 6.4). Then, the protein gels were transferred to polyvinylidene difluoride membranes and blocked in 3% (wt:vol) BSA in phosphate-buffered saline with Tween-20 (Beyotime, ST825; PBST) or Tris-buffered saline with Tween-20 (TBST) (for phosphorylated protein antibodies) for 2 h. The blocked membranes were incubated with the indicated primary antibodies at 4°C overnight. After washing with PBST or TBST, membranes were incubated with HRP-conjugated secondary antibodies diluted in 3% (wt:vol) BSA in PBST or TBST for 1 h at room temperature. The images were developed using Chemistar High-sig ECL western blot substrate (Tanon, 4600) through Tanon 5200 system (Tanon).

Immunoprecipitation and ubiquitination assays

For immunoprecipitation, the cells were harvested and lysed with NP-40 buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1:400 protease inhibitor mixture). After centrifugation, supernatants were collected and mixed with 50 μL of anti-Flag M2 magnetic beads (Sigma-Aldrich, M8823), anti-MYC magnetic beads (Santa Cruz Biotechnology, sc-500,772), or anti-HA magnetic beads (Cell Signaling Technology, 11,846), which were incubated at 4°C for 6 h. Afterward, the beads were washed three times with wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, and protease inhibitor mixture) and collected. The precipitated proteins were analyzed by western blot using the indicated antibodies. For detection of CGAS ubiquitination, cells were lysed with lysis buffer (1% SDS, 150 mM NaCl, and 10 mM Tris-HCl, pH 8.0) with 2 mM sodium orthovanadate, 50 mM sodium fluoride, and protease inhibitors. The samples were processed the same as immunoprecipitation, but washed with wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, and protease inhibitor mixture) containing 2 M urea to remove the nonspecific binding of other ubiquitinated proteins.

Mice

cgas^−/− mice and wild-type (WT) mice were purchased from Nanjing Biomedical Research Institute of Nanjing University. All the mice were housed in specific pathogen-free barrier facilities in Nanjing Agricultural University. For the generation of BMDMs, bone marrow cells were isolated from the tibia and femur of cgas^−/− mice and WT mice (6–8 weeks) and plated in dishes at a density of 1 × 10⁶ cells/ml, which were then cultured with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 20% fetal bovine serum (FBS) and 30% L929 supernatant for 4 days before medium replacement. The cells were further cultured for another 4 days and harvested as adherent BMDMs, which was used for PRV infection. For mouse infection challenge, cgas^−/− mice and WT mice (6–8 weeks) were separately housed in 6 groups: 1) mock-infected, 2) PRV-WT-infected, 3) ΔUL21-infected, 4) ΔUL21 (1–200 aa)-infected, 5) ΔUL21 (200–532 aa)-infected, and 6) ΔUL21R-infected.

Ethics statement

All mouse experiments were performed according to the National Guidelines for Housing and Care of Laboratory Animals (China) and Institutional Animal Care and Ethics Committee of Nanjing Agricultural University (permit no. IACECNAU20210602). All mice were housed in the animal facility of Nanjing Agricultural University (Nanjing, Jiangsu, China).

Statistical analysis

All data were presented as means ± SD and analyzed using GraphPad Prism 7.0 software. An one-way ANOVA with Dunnett’s multiple comparisons or an unpaired two-tailed Student’s t test was used as indicated in the legends. The P values were calculated from three biological replicates unless otherwise indicated in the legends. Data were reproduced in independent experiments as indicated in the figure legends.

Supplementary Material

Supplemental Material

Click here for additional data file.^{(8.7MB, zip)}

Acknowledgments

We would like to thank Chunfu Zheng (Fujian Medical University, China), Rong Zhang (Fudan University, China), and Beibei Chu (Henan Agricultural University, China) for providing valuable reagents. We thank Bin He (University of Illinois at Chicago, USA) and Zhiwei Wu (Nanjing University, China) for valuable suggestions. Also, we thank for the technical support by the instrument platform of Institute of Immunology, College of Veterinary Medicine, Nanjing Agricultural University.

Funding Statement

This work was supported by the National Natural Science Foundation of China (32272985), the Earmarked Fund for CARS-35, and the startup funding form Nanjing Agricultural University (090-804125).

Data availability

All study data are included in the manuscript and its Supporting Information files.

Disclosure statement

The authors declare that there are no conflicts of interest.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2022.2139921

References

[1].Sehrawat S, Kumar D, Rouse BT.. Herpesviruses: harmonious pathogens but relevant cofactors in other diseases? Front Cell Infect Microbiol. 2018;8:177. [DOI] [PMC free article] [PubMed] [Google Scholar]
[2].Sharma V, Mobeen F, Prakash T. Comparative genomics of herpesviridae family to look for potential signatures of human infecting strains. Int J Genomics. 2016;2016:9543274. [DOI] [PMC free article] [PubMed] [Google Scholar]
[3].Pomeranz LE, Reynolds AE, Hengartner CJ. Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol Mol Biol Rev. 2005. Sep;69(3):462–500. [DOI] [PMC free article] [PubMed] [Google Scholar]
[4].Loret S, Guay G, Lippe R. Comprehensive characterization of extracellular herpes simplex virus type 1 virions. J Virol. 2008. Sep;82(17):8605–8618. [DOI] [PMC free article] [PubMed] [Google Scholar]
[5].Karasneh GA, Shukla D. Herpes simplex virus infects most cell types in vitro: clues to its success. Virol J. 2011. Oct 26;8(1):481. [DOI] [PMC free article] [PubMed] [Google Scholar]
[6].Le Sage V, Jung M, Alter JD, et al. The herpes simplex virus 2 UL21 protein is essential for virus propagation. J Virol. 2013. May;87(10):5904–5915. [DOI] [PMC free article] [PubMed] [Google Scholar]
[7].Sarfo A, Starkey J, Mellinger E, et al. The UL21 tegument protein of herpes simplex virus 1 is differentially required for the syncytial phenotype. J Virol. 2017. Nov 1;91(21). DOI: 10.1128/JVI.01161-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
[8].Mbong EF, Woodley L, Frost E, et al. Deletion of UL21 causes a delay in the early stages of the herpes simplex virus 1 replication cycle. J Virol. 2012. Jun;86(12):7003–7007. [DOI] [PMC free article] [PubMed] [Google Scholar]
[9].Benedyk TH, Muenzner J, Connor V, et al. pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread. PLoS Pathog. 2021. Aug;17(8):e1009824. [DOI] [PMC free article] [PubMed] [Google Scholar]
[10].Muradov JH, Finnen RL, Gulak MA, et al. pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex. PLoS Pathog. 2021. Aug;17(8):e1009679. [DOI] [PMC free article] [PubMed] [Google Scholar]
[11].Gao J, Finnen RL, Sherry MR, et al. Differentiating the roles of UL16, UL21, and Us3 in the nuclear egress of herpes simplex virus capsids. J Virol. 2020. Jun 16;94(13). DOI: 10.1128/JVI.00738-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
[12].Curanovic D, Lyman MG, Bou-Abboud C, et al. Repair of the UL21 locus in pseudorabies virus Bartha enhances the kinetics of retrograde, transneuronal infection in vitro and in vivo. J Virol. 2009. Feb;83(3):1173–1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
[13].Klupp BG, Lomniczi B, Visser N, et al. Mutations affecting the UL21 gene contribute to avirulence of pseudorabies virus vaccine strain Bartha. Virology. 1995. Oct 1;212(2):466–473. [DOI] [PubMed] [Google Scholar]
[14].Yan K, Liu J, Guan X, et al. The Carboxyl terminus of tegument protein pUL21 contributes to pseudorabies virus neuroinvasion. J Virol. 2019. Apr 1;93(7). DOI: 10.1128/JVI.02052-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
[15].Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor recognizes bacterial DNA. Nature. 2000. Dec 7;408(6813):740–745. [DOI] [PubMed] [Google Scholar]
[16].Unterholzner L, Keating SE, Baran M, et al. IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol. 2010. Nov;11(11):997–1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
[17].Hornung V, Ablasser A, Charrel-Dennis M, et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature. 2009. Mar 26;458(7237):514–518. [DOI] [PMC free article] [PubMed] [Google Scholar]
[18].Takaoka A, Wang Z, Choi MK, et al. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature. 2007. Jul 26;448(7152):501–505. [DOI] [PubMed] [Google Scholar]
[19].Zhang Z, Yuan B, Bao M, et al. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat Immunol. 2011. Sep 4;12(10):959–965. [DOI] [PMC free article] [PubMed] [Google Scholar]
[20].Sun L, Wu J, Du F, et al. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 2013. Feb 15;339(6121):786–791. [DOI] [PMC free article] [PubMed] [Google Scholar]
[21].Zhao J, Qin C, Liu Y, et al. Herpes simplex virus and pattern recognition receptors: An arms race. Front Immunol. 2020;11:613799. [DOI] [PMC free article] [PubMed] [Google Scholar]
[22].Li XD, Wu J, Gao D, et al. Pivotal roles of CGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science. 2013. Sep 20;341(6152):1390–1394. [DOI] [PMC free article] [PubMed] [Google Scholar]
[23].Choi Y, Bowman JW, Jung JU. Autophagy during viral infection - a double-edged sword. Nat Rev Microbiol. 2018. Jun;16(6):341–354. [DOI] [PMC free article] [PubMed] [Google Scholar]
[24].Sun M, Hou L, Tang YD, et al. Pseudorabies virus infection inhibits autophagy in permissive cells in vitro. Sci Rep. 2017. Jan 6;7(1):39964. [DOI] [PMC free article] [PubMed] [Google Scholar]
[25].Xu C, Wang M, Song Z, et al. Pseudorabies virus induces autophagy to enhance viral replication in mouse neuro-2a cells in vitro. Virus Res. 2018. Mar 15;248:44–52. [DOI] [PubMed] [Google Scholar]
[26].Boyle KB, Randow F. The role of ‘eat-me’ signals and autophagy cargo receptors in innate immunity. Curr Opin Microbiol. 2013. Jun;16(3):339–348. [DOI] [PubMed] [Google Scholar]
[27].Novak I, Kirkin V, McEwan DG, et al. Nix is a selective autophagy receptor for mitochondrial clearance. Embo Rep. 2010. Jan;11(1):45–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
[28].Ma Y, Galluzzi L, Zitvogel L, et al. Autophagy and cellular immune responses. Immunity. 2013. Aug 22;39(2):211–227. [DOI] [PubMed] [Google Scholar]
[29].Du Y, Duan T, Feng Y, et al. LRRC25 inhibits type I IFN signaling by targeting ISG15-associated RIG-I for autophagic degradation. EMBO J. 2018. Feb 1;37(3):351–366. [DOI] [PMC free article] [PubMed] [Google Scholar]
[30].Jin S, Tian S, Luo M, et al. Tetherin suppresses type I interferon signaling by targeting MAVS for NDP52-Mediated selective autophagic degradation in human cells. Mol Cell. 2017. Oct 19;68(2):308–322 e4. [DOI] [PubMed] [Google Scholar]
[31].Li S, Cao L, Zhang Z, et al. Cytosolic and nuclear recognition of virus and viral evasion. Mol Biomed. 2021. Oct 10;2(1):30. [DOI] [PMC free article] [PubMed] [Google Scholar]
[32].Murphy BM, O’Neill AJ, Adrain C, et al. The apoptosome pathway to caspase activation in primary human neutrophils exhibits dramatically reduced requirements for cytochrome C. J Exp Med. 2003. Mar 3;197(5):625–632. [DOI] [PMC free article] [PubMed] [Google Scholar]
[33].Schwartz AL, Ciechanover A. Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol. 2009;49(1):73–96. [DOI] [PubMed] [Google Scholar]
[34].Marshall RS, Li F, Gemperline DC, et al. Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/Ubiquitin receptor RPN10 in arabidopsis. Mol Cell. 2015. Jun 18;58(6):1053–1066. [DOI] [PMC free article] [PubMed] [Google Scholar]
[35].Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol. 2011;27(1):107–132. [DOI] [PubMed] [Google Scholar]
[36].Kwon YT, Ciechanover A. The ubiquitin code in the ubiquitin-Proteasome system and Autophagy. Trends Biochem Sci. 2017. Nov;42(11):873–886. [DOI] [PubMed] [Google Scholar]
[37].Cohen-Kaplan V, Livneh I, Avni N, et al. The ubiquitin-proteasome system and autophagy: coordinated and independent activities. Int J Biochem Cell Biol. 2016. Oct;79:403–418. [DOI] [PubMed] [Google Scholar]
[38].Zhang J, Zhao J, Xu S, et al. Species-Specific deamidation of CGAS by herpes simplex virus UL37 protein facilitates viral replication. Cell Host Microbe. 2018. Aug 8;24(2):234–248 e5. [DOI] [PMC free article] [PubMed] [Google Scholar]
[39].Su C, Zheng C. Herpes simplex virus 1 Abrogates the CGAS/STING-Mediated cytosolic DNA-Sensing pathway via its virion host shutoff protein, UL41. J Virol. 2017. Mar 15;91(6). DOI: 10.1128/JVI.02414-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
[40].Huang J, You H, Su C, et al. Herpes simplex virus 1 tegument protein VP22 abrogates CGAS/STING-Mediated antiviral innate immunity. J Virol. 2018. Aug 1;92(15). DOI: 10.1128/JVI.00841-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
[41].Xu G, Liu C, Zhou S, et al. Viral tegument proteins restrict CGAS-DNA phase separation to mediate immune evasion. Mol Cell. 2021. Jul 1;81(13):2823–2837 e9. [DOI] [PubMed] [Google Scholar]
[42].Klopfleisch R, Klupp BG, Fuchs W, et al. Influence of pseudorabies virus proteins on neuroinvasion and neurovirulence in mice. J Virol. 2006. Jun;80(11):5571–5576. [DOI] [PMC free article] [PubMed] [Google Scholar]
[43].Wilcox DR, Muller WJ, Longnecker R. HSV targeting of the host phosphatase PP1alpha is required for disseminated disease in the neonate and contributes to pathogenesis in the brain. Proc Natl Acad Sci U S A. 2015. Dec 15;112(50):E6937–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
[44].Baines JD, Koyama AH, Huang T, et al. The UL21 gene products of herpes simplex virus 1 are dispensable for growth in cultured cells. J Virol. 1994. May;68(5):2929–2936. [DOI] [PMC free article] [PubMed] [Google Scholar]
[45].Muto Y, Goshima F, Ushijima Y, et al. Generation and characterization of UL21-Null herpes simplex virus type 1. Front Microbiol. 2012;3:394. [DOI] [PMC free article] [PubMed] [Google Scholar]
[46].Goodall EA, Kraus F, Harper JW. Mechanisms underlying ubiquitin-driven selective mitochondrial and bacterial autophagy. Mol Cell. 2022. Mar 29;82(8):1501–1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
[47].Geisler S, Holmstrom KM, Skujat D, et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol. 2010. Feb;12(2):119–131. [DOI] [PubMed] [Google Scholar]
[48].Deosaran E, Larsen KB, Hua R, et al. NBR1 acts as an autophagy receptor for peroxisomes. J Cell Sci. 2013. Feb 15;126(Pt 4):939–952. [DOI] [PubMed] [Google Scholar]
[49].Wild P, Farhan H, McEwan DG, et al. Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science. 2011. Jul 8;333(6039):228–233. [DOI] [PMC free article] [PubMed] [Google Scholar]
[50].Xie X, Li F, Wang Y, et al. Molecular basis of ubiquitin recognition by the autophagy receptor CALCOCO2. Autophagy. 2015;11(10):1775–1789. [DOI] [PMC free article] [PubMed] [Google Scholar]
[51].Yazdankhah M, Ghosh S, Shang P, et al. BNIP3L-mediated mitophagy is required for mitochondrial remodeling during the differentiation of optic nerve oligodendrocytes. Autophagy. 2021. Oct;17(10):3140–3159. [DOI] [PMC free article] [PubMed] [Google Scholar]
[52].Lu K, Psakhye I, Jentsch S. Autophagic clearance of polyQ proteins mediated by ubiquitin-Atg8 adaptors of the conserved CUET protein family. Cell. 2014. Jul 31;158(3):549–563. [DOI] [PubMed] [Google Scholar]
[53].Hou P, Yang K, Jia P, et al. A novel selective autophagy receptor, CCDC50, delivers K63 polyubiquitination-activated RIG-I/MDA5 for degradation during viral infection. Cell Res. 2021. Jan;31(1):62–79. [DOI] [PMC free article] [PubMed] [Google Scholar]
[54].Ma X, Lu C, Chen Y, et al. CCT2 is an aggrephagy receptor for clearance of solid protein aggregates. Cell. 2022. Mar 27;185(8):1325–1345.e22. [DOI] [PubMed] [Google Scholar]
[55].Wu Y, Jin S, Liu Q, et al. Selective autophagy controls the stability of transcription factor IRF3 to balance type I interferon production and immune suppression. Autophagy. 2021. Jun;17(6):1379–1392. [DOI] [PMC free article] [PubMed] [Google Scholar]
[56].Zeng Y, Xu S, Wei Y, et al. The PB1 protein of influenza A virus inhibits the innate immune response by targeting MAVS for NBR1-mediated selective autophagic degradation. PLoS Pathog. 2021. Feb;17(2):e1009300. [DOI] [PMC free article] [PubMed] [Google Scholar]
[57].Hou P, Lin Y, Li Z, et al. Autophagy receptor CCDC50 tunes the STING-mediated interferon response in viral infections and autoimmune diseases. Cell Mol Immunol. 2021. Oct;18(10):2358–2371. [DOI] [PMC free article] [PubMed] [Google Scholar]
[58].Lu K, Psakhye I, Jentsch S. A new class of ubiquitin-Atg8 receptors involved in selective autophagy and polyQ protein clearance. Autophagy. 2014;10(12):2381–2382. [DOI] [PMC free article] [PubMed] [Google Scholar]
[59].Zellner S, Schifferer M, Behrends C. Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling. Mol Cell. 2021. Mar 18;81(6):1337–1354 e8. [DOI] [PubMed] [Google Scholar]
[60].Chen M, Meng Q, Qin Y, et al. TRIM14 inhibits CGAS degradation mediated by selective autophagy receptor p62 to promote innate immune responses. Mol Cell. 2016. Oct 6;64(1):105–119. [DOI] [PubMed] [Google Scholar]
[61].Guo Y, Jiang F, Kong L, et al. Cutting Edge: USP27X deubiquitinates and stabilizes the DNA sensor CGAS to regulate cytosolic DNA-Mediated signaling. J Immunol. 2019. Oct 15;203(8):2049–2054. [DOI] [PubMed] [Google Scholar]
[62].Zhang Q, Tang Z, An R, et al. USP29 maintains the stability of CGAS and promotes cellular antiviral responses and autoimmunity. Cell Res. 2020. Oct;30(10):914–927. [DOI] [PMC free article] [PubMed] [Google Scholar]
[63].Seo GJ, Kim C, Shin WJ, et al. TRIM56-mediated monoubiquitination of CGAS for cytosolic DNA sensing. Nat Commun. 2018. Feb 9;9(1):613. [DOI] [PMC free article] [PubMed] [Google Scholar]
[64].Wang Q, Huang L, Hong Z, et al. The E3 ubiquitin ligase RNF185 facilitates the CGAS-mediated innate immune response. PLoS Pathog. 2017. Mar;13(3):e1006264. [DOI] [PMC free article] [PubMed] [Google Scholar]
[65].Ambrozkiewicz MC, Cuthill KJ, Harnett D, et al. Molecular evolution, neurodevelopmental roles and clinical significance of HECT-Type UBE3 E3 ubiquitin ligases. Cells. 2020. Nov 10;9(11):2455. [DOI] [PMC free article] [PubMed] [Google Scholar]
[66].Yu Y, Hayward GS. The ubiquitin E3 ligase RAUL negatively regulates type i interferon through ubiquitination of the transcription factors IRF7 and IRF3. Immunity. 2010. Dec 14;33(6):863–877. [DOI] [PMC free article] [PubMed] [Google Scholar]
[67].Wang J, Wang CF, Ming SL, et al. Porcine IFITM1 is a host restriction factor that inhibits pseudorabies virus infection. Int J Biol Macromol. 2020. May 15;151:1181–1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
[68].Gu Z, Hou C, Sun H, et al. Emergence of highly virulent pseudorabies virus in southern China. Can J Vet Res. 2015. Jul;79(3):221–228. [PMC free article] [PubMed] [Google Scholar]
[69].Lv L, Bai J, Gao Y, et al. Peroxiredoxin 1 interacts with TBK1/IKKepsilon and negatively regulates pseudorabies virus propagation by promoting innate immunity. J Virol. 2021. Sep 9;95(19):e0092321. [DOI] [PMC free article] [PubMed] [Google Scholar]
[70].Li M, Wang S, Cai M, et al. Identification of nuclear and nucleolar localization signals of pseudorabies virus (PRV) early protein UL54 reveals that its nuclear targeting is required for efficient production of PRV. J Virol. 2011. Oct;85(19):10239–10251. [DOI] [PMC free article] [PubMed] [Google Scholar]
[71].Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3(6):1101–1108. [DOI] [PubMed] [Google Scholar]
[72].Larkin MA, Blackshields G, Brown NP, et al. Clustal W and clustal X version 2.0. Bioinformatics. 2007. Nov 1;23(21):2947–2948. [DOI] [PubMed] [Google Scholar]
[73].Waterhouse AM, Procter JB, Martin DM, et al. Jalview version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009. May 1;25(9):1189–1191. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material

Click here for additional data file.^{(8.7MB, zip)}

Data Availability Statement

All study data are included in the manuscript and its Supporting Information files.

[cit0001] [1].Sehrawat S, Kumar D, Rouse BT.. Herpesviruses: harmonious pathogens but relevant cofactors in other diseases? Front Cell Infect Microbiol. 2018;8:177. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0002] [2].Sharma V, Mobeen F, Prakash T. Comparative genomics of herpesviridae family to look for potential signatures of human infecting strains. Int J Genomics. 2016;2016:9543274. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0003] [3].Pomeranz LE, Reynolds AE, Hengartner CJ. Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol Mol Biol Rev. 2005. Sep;69(3):462–500. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0004] [4].Loret S, Guay G, Lippe R. Comprehensive characterization of extracellular herpes simplex virus type 1 virions. J Virol. 2008. Sep;82(17):8605–8618. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0005] [5].Karasneh GA, Shukla D. Herpes simplex virus infects most cell types in vitro: clues to its success. Virol J. 2011. Oct 26;8(1):481. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0006] [6].Le Sage V, Jung M, Alter JD, et al. The herpes simplex virus 2 UL21 protein is essential for virus propagation. J Virol. 2013. May;87(10):5904–5915. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0007] [7].Sarfo A, Starkey J, Mellinger E, et al. The UL21 tegument protein of herpes simplex virus 1 is differentially required for the syncytial phenotype. J Virol. 2017. Nov 1;91(21). DOI: 10.1128/JVI.01161-17. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0008] [8].Mbong EF, Woodley L, Frost E, et al. Deletion of UL21 causes a delay in the early stages of the herpes simplex virus 1 replication cycle. J Virol. 2012. Jun;86(12):7003–7007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0009] [9].Benedyk TH, Muenzner J, Connor V, et al. pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread. PLoS Pathog. 2021. Aug;17(8):e1009824. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0010] [10].Muradov JH, Finnen RL, Gulak MA, et al. pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex. PLoS Pathog. 2021. Aug;17(8):e1009679. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0011] [11].Gao J, Finnen RL, Sherry MR, et al. Differentiating the roles of UL16, UL21, and Us3 in the nuclear egress of herpes simplex virus capsids. J Virol. 2020. Jun 16;94(13). DOI: 10.1128/JVI.00738-20. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0012] [12].Curanovic D, Lyman MG, Bou-Abboud C, et al. Repair of the UL21 locus in pseudorabies virus Bartha enhances the kinetics of retrograde, transneuronal infection in vitro and in vivo. J Virol. 2009. Feb;83(3):1173–1183. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0013] [13].Klupp BG, Lomniczi B, Visser N, et al. Mutations affecting the UL21 gene contribute to avirulence of pseudorabies virus vaccine strain Bartha. Virology. 1995. Oct 1;212(2):466–473. [DOI] [PubMed] [Google Scholar]

[cit0014] [14].Yan K, Liu J, Guan X, et al. The Carboxyl terminus of tegument protein pUL21 contributes to pseudorabies virus neuroinvasion. J Virol. 2019. Apr 1;93(7). DOI: 10.1128/JVI.02052-18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0015] [15].Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor recognizes bacterial DNA. Nature. 2000. Dec 7;408(6813):740–745. [DOI] [PubMed] [Google Scholar]

[cit0016] [16].Unterholzner L, Keating SE, Baran M, et al. IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol. 2010. Nov;11(11):997–1004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0017] [17].Hornung V, Ablasser A, Charrel-Dennis M, et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature. 2009. Mar 26;458(7237):514–518. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0018] [18].Takaoka A, Wang Z, Choi MK, et al. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature. 2007. Jul 26;448(7152):501–505. [DOI] [PubMed] [Google Scholar]

[cit0019] [19].Zhang Z, Yuan B, Bao M, et al. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat Immunol. 2011. Sep 4;12(10):959–965. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0020] [20].Sun L, Wu J, Du F, et al. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 2013. Feb 15;339(6121):786–791. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0021] [21].Zhao J, Qin C, Liu Y, et al. Herpes simplex virus and pattern recognition receptors: An arms race. Front Immunol. 2020;11:613799. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0022] [22].Li XD, Wu J, Gao D, et al. Pivotal roles of CGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science. 2013. Sep 20;341(6152):1390–1394. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0023] [23].Choi Y, Bowman JW, Jung JU. Autophagy during viral infection - a double-edged sword. Nat Rev Microbiol. 2018. Jun;16(6):341–354. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0024] [24].Sun M, Hou L, Tang YD, et al. Pseudorabies virus infection inhibits autophagy in permissive cells in vitro. Sci Rep. 2017. Jan 6;7(1):39964. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0025] [25].Xu C, Wang M, Song Z, et al. Pseudorabies virus induces autophagy to enhance viral replication in mouse neuro-2a cells in vitro. Virus Res. 2018. Mar 15;248:44–52. [DOI] [PubMed] [Google Scholar]

[cit0026] [26].Boyle KB, Randow F. The role of ‘eat-me’ signals and autophagy cargo receptors in innate immunity. Curr Opin Microbiol. 2013. Jun;16(3):339–348. [DOI] [PubMed] [Google Scholar]

[cit0027] [27].Novak I, Kirkin V, McEwan DG, et al. Nix is a selective autophagy receptor for mitochondrial clearance. Embo Rep. 2010. Jan;11(1):45–51. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0028] [28].Ma Y, Galluzzi L, Zitvogel L, et al. Autophagy and cellular immune responses. Immunity. 2013. Aug 22;39(2):211–227. [DOI] [PubMed] [Google Scholar]

[cit0029] [29].Du Y, Duan T, Feng Y, et al. LRRC25 inhibits type I IFN signaling by targeting ISG15-associated RIG-I for autophagic degradation. EMBO J. 2018. Feb 1;37(3):351–366. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0030] [30].Jin S, Tian S, Luo M, et al. Tetherin suppresses type I interferon signaling by targeting MAVS for NDP52-Mediated selective autophagic degradation in human cells. Mol Cell. 2017. Oct 19;68(2):308–322 e4. [DOI] [PubMed] [Google Scholar]

[cit0031] [31].Li S, Cao L, Zhang Z, et al. Cytosolic and nuclear recognition of virus and viral evasion. Mol Biomed. 2021. Oct 10;2(1):30. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0032] [32].Murphy BM, O’Neill AJ, Adrain C, et al. The apoptosome pathway to caspase activation in primary human neutrophils exhibits dramatically reduced requirements for cytochrome C. J Exp Med. 2003. Mar 3;197(5):625–632. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0033] [33].Schwartz AL, Ciechanover A. Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol. 2009;49(1):73–96. [DOI] [PubMed] [Google Scholar]

[cit0034] [34].Marshall RS, Li F, Gemperline DC, et al. Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/Ubiquitin receptor RPN10 in arabidopsis. Mol Cell. 2015. Jun 18;58(6):1053–1066. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0035] [35].Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol. 2011;27(1):107–132. [DOI] [PubMed] [Google Scholar]

[cit0036] [36].Kwon YT, Ciechanover A. The ubiquitin code in the ubiquitin-Proteasome system and Autophagy. Trends Biochem Sci. 2017. Nov;42(11):873–886. [DOI] [PubMed] [Google Scholar]

[cit0037] [37].Cohen-Kaplan V, Livneh I, Avni N, et al. The ubiquitin-proteasome system and autophagy: coordinated and independent activities. Int J Biochem Cell Biol. 2016. Oct;79:403–418. [DOI] [PubMed] [Google Scholar]

[cit0038] [38].Zhang J, Zhao J, Xu S, et al. Species-Specific deamidation of CGAS by herpes simplex virus UL37 protein facilitates viral replication. Cell Host Microbe. 2018. Aug 8;24(2):234–248 e5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0039] [39].Su C, Zheng C. Herpes simplex virus 1 Abrogates the CGAS/STING-Mediated cytosolic DNA-Sensing pathway via its virion host shutoff protein, UL41. J Virol. 2017. Mar 15;91(6). DOI: 10.1128/JVI.02414-16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0040] [40].Huang J, You H, Su C, et al. Herpes simplex virus 1 tegument protein VP22 abrogates CGAS/STING-Mediated antiviral innate immunity. J Virol. 2018. Aug 1;92(15). DOI: 10.1128/JVI.00841-18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0041] [41].Xu G, Liu C, Zhou S, et al. Viral tegument proteins restrict CGAS-DNA phase separation to mediate immune evasion. Mol Cell. 2021. Jul 1;81(13):2823–2837 e9. [DOI] [PubMed] [Google Scholar]

[cit0042] [42].Klopfleisch R, Klupp BG, Fuchs W, et al. Influence of pseudorabies virus proteins on neuroinvasion and neurovirulence in mice. J Virol. 2006. Jun;80(11):5571–5576. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0043] [43].Wilcox DR, Muller WJ, Longnecker R. HSV targeting of the host phosphatase PP1alpha is required for disseminated disease in the neonate and contributes to pathogenesis in the brain. Proc Natl Acad Sci U S A. 2015. Dec 15;112(50):E6937–44. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0044] [44].Baines JD, Koyama AH, Huang T, et al. The UL21 gene products of herpes simplex virus 1 are dispensable for growth in cultured cells. J Virol. 1994. May;68(5):2929–2936. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0045] [45].Muto Y, Goshima F, Ushijima Y, et al. Generation and characterization of UL21-Null herpes simplex virus type 1. Front Microbiol. 2012;3:394. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0046] [46].Goodall EA, Kraus F, Harper JW. Mechanisms underlying ubiquitin-driven selective mitochondrial and bacterial autophagy. Mol Cell. 2022. Mar 29;82(8):1501–1513. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0047] [47].Geisler S, Holmstrom KM, Skujat D, et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol. 2010. Feb;12(2):119–131. [DOI] [PubMed] [Google Scholar]

[cit0048] [48].Deosaran E, Larsen KB, Hua R, et al. NBR1 acts as an autophagy receptor for peroxisomes. J Cell Sci. 2013. Feb 15;126(Pt 4):939–952. [DOI] [PubMed] [Google Scholar]

[cit0049] [49].Wild P, Farhan H, McEwan DG, et al. Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science. 2011. Jul 8;333(6039):228–233. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0050] [50].Xie X, Li F, Wang Y, et al. Molecular basis of ubiquitin recognition by the autophagy receptor CALCOCO2. Autophagy. 2015;11(10):1775–1789. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0051] [51].Yazdankhah M, Ghosh S, Shang P, et al. BNIP3L-mediated mitophagy is required for mitochondrial remodeling during the differentiation of optic nerve oligodendrocytes. Autophagy. 2021. Oct;17(10):3140–3159. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0052] [52].Lu K, Psakhye I, Jentsch S. Autophagic clearance of polyQ proteins mediated by ubiquitin-Atg8 adaptors of the conserved CUET protein family. Cell. 2014. Jul 31;158(3):549–563. [DOI] [PubMed] [Google Scholar]

[cit0053] [53].Hou P, Yang K, Jia P, et al. A novel selective autophagy receptor, CCDC50, delivers K63 polyubiquitination-activated RIG-I/MDA5 for degradation during viral infection. Cell Res. 2021. Jan;31(1):62–79. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0054] [54].Ma X, Lu C, Chen Y, et al. CCT2 is an aggrephagy receptor for clearance of solid protein aggregates. Cell. 2022. Mar 27;185(8):1325–1345.e22. [DOI] [PubMed] [Google Scholar]

[cit0055] [55].Wu Y, Jin S, Liu Q, et al. Selective autophagy controls the stability of transcription factor IRF3 to balance type I interferon production and immune suppression. Autophagy. 2021. Jun;17(6):1379–1392. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0056] [56].Zeng Y, Xu S, Wei Y, et al. The PB1 protein of influenza A virus inhibits the innate immune response by targeting MAVS for NBR1-mediated selective autophagic degradation. PLoS Pathog. 2021. Feb;17(2):e1009300. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0057] [57].Hou P, Lin Y, Li Z, et al. Autophagy receptor CCDC50 tunes the STING-mediated interferon response in viral infections and autoimmune diseases. Cell Mol Immunol. 2021. Oct;18(10):2358–2371. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0058] [58].Lu K, Psakhye I, Jentsch S. A new class of ubiquitin-Atg8 receptors involved in selective autophagy and polyQ protein clearance. Autophagy. 2014;10(12):2381–2382. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0059] [59].Zellner S, Schifferer M, Behrends C. Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling. Mol Cell. 2021. Mar 18;81(6):1337–1354 e8. [DOI] [PubMed] [Google Scholar]

[cit0060] [60].Chen M, Meng Q, Qin Y, et al. TRIM14 inhibits CGAS degradation mediated by selective autophagy receptor p62 to promote innate immune responses. Mol Cell. 2016. Oct 6;64(1):105–119. [DOI] [PubMed] [Google Scholar]

[cit0061] [61].Guo Y, Jiang F, Kong L, et al. Cutting Edge: USP27X deubiquitinates and stabilizes the DNA sensor CGAS to regulate cytosolic DNA-Mediated signaling. J Immunol. 2019. Oct 15;203(8):2049–2054. [DOI] [PubMed] [Google Scholar]

[cit0062] [62].Zhang Q, Tang Z, An R, et al. USP29 maintains the stability of CGAS and promotes cellular antiviral responses and autoimmunity. Cell Res. 2020. Oct;30(10):914–927. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0063] [63].Seo GJ, Kim C, Shin WJ, et al. TRIM56-mediated monoubiquitination of CGAS for cytosolic DNA sensing. Nat Commun. 2018. Feb 9;9(1):613. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0064] [64].Wang Q, Huang L, Hong Z, et al. The E3 ubiquitin ligase RNF185 facilitates the CGAS-mediated innate immune response. PLoS Pathog. 2017. Mar;13(3):e1006264. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0065] [65].Ambrozkiewicz MC, Cuthill KJ, Harnett D, et al. Molecular evolution, neurodevelopmental roles and clinical significance of HECT-Type UBE3 E3 ubiquitin ligases. Cells. 2020. Nov 10;9(11):2455. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0066] [66].Yu Y, Hayward GS. The ubiquitin E3 ligase RAUL negatively regulates type i interferon through ubiquitination of the transcription factors IRF7 and IRF3. Immunity. 2010. Dec 14;33(6):863–877. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0067] [67].Wang J, Wang CF, Ming SL, et al. Porcine IFITM1 is a host restriction factor that inhibits pseudorabies virus infection. Int J Biol Macromol. 2020. May 15;151:1181–1193. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0068] [68].Gu Z, Hou C, Sun H, et al. Emergence of highly virulent pseudorabies virus in southern China. Can J Vet Res. 2015. Jul;79(3):221–228. [PMC free article] [PubMed] [Google Scholar]

[cit0069] [69].Lv L, Bai J, Gao Y, et al. Peroxiredoxin 1 interacts with TBK1/IKKepsilon and negatively regulates pseudorabies virus propagation by promoting innate immunity. J Virol. 2021. Sep 9;95(19):e0092321. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0070] [70].Li M, Wang S, Cai M, et al. Identification of nuclear and nucleolar localization signals of pseudorabies virus (PRV) early protein UL54 reveals that its nuclear targeting is required for efficient production of PRV. J Virol. 2011. Oct;85(19):10239–10251. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0071] [71].Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3(6):1101–1108. [DOI] [PubMed] [Google Scholar]

[cit0072] [72].Larkin MA, Blackshields G, Brown NP, et al. Clustal W and clustal X version 2.0. Bioinformatics. 2007. Nov 1;23(21):2947–2948. [DOI] [PubMed] [Google Scholar]

[cit0073] [73].Waterhouse AM, Procter JB, Martin DM, et al. Jalview version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009. May 1;25(9):1189–1191. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Tegument protein UL21 of alpha-herpesvirus inhibits the innate immunity by triggering CGAS degradation through TOLLIP-mediated selective autophagy

Zicheng Ma

Juan Bai

Chenlong Jiang

Huixin Zhu

Depeng Liu

Mengjiao Pan

Xianwei Wang

Jiang Pi

Ping Jiang

Xing Liu

ABSTRACT

Introduction

Results

PRV and HSV-1 UL21 inhibits CGAS-STING1-mediated innate immunity

Figure 1.

Figure 2.

PRV and HSV-1 UL21 degrades CGAS through the autophagy-lysosome pathway

Figure 3.

PRV and HSV-1 UL21 induces the autophagic process and degrades CGAS

Figure 4.

TOLLIP-mediated autophagy is responsible for CGAS degradation induced by PRV and HSV-1 UL21

Figure 5.

PRV and HSV-1 UL21 induces K27-linked ubiquitination of CGAS at site of K384

Figure 6.

UBE3C is the E3 ligase that mediates CGAS ubiquitination induced by PRV and HSV-1 UL21

Figure 7.

The N terminus of PRV and HSV-1 UL21 interacts with CGAS and mediates its degradation

Figure 8.

Cgas is essential in controlling PRV infection in mice

Figure 9.

Discussion

Figure 10.

Materials and methods

Cells, viruses and plasmids

Generation of recombinant PRV by CRISPR-Cas9 system

Generation of knockout cell lines by CRISPR-Cas9 system

Antibodies and chemical reagents

Plasmids and siRNA transfections

Dual luciferase assay

VSV-GFP infection inhibition assay

Viral infection

Real-time qPCR

Mass spectrometry analysis

Sequence alignment

Confocal fluorescence microscopy

Western blot

Immunoprecipitation and ubiquitination assays

Mice

Ethics statement

Statistical analysis

Supplementary Material

Acknowledgments

Funding Statement

Data availability

Disclosure statement

Supplementary material

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases