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Journal of Virology logoLink to Journal of Virology
. 2021 Jan 13;95(3):e02114-20. doi: 10.1128/JVI.02114-20

The Vaccinia Virus B12 Pseudokinase Represses Viral Replication via Interaction with the Cellular Kinase VRK1 and Activation of the Antiviral Effector BAF

Amber B Rico a,b,#, Alexandria C Linville a,c,#, Annabel T Olson a,c, Zhigang Wang a,b, Matthew S Wiebe a,b,
Editor: Joanna L Shislerd
PMCID: PMC7925102  PMID: 33177193

Viruses from diverse families encode both positive and negative regulators of viral replication. While their functions can sometimes be enigmatic, investigation of virus-encoded, negative regulators of viral replication has revealed fascinating aspects of virology.

KEYWORDS: B1, BAF, VRK, poxvirus, protein kinases

ABSTRACT

The poxviral B1 and B12 proteins are a homologous kinase-pseudokinase pair, which modulates a shared host pathway governing viral DNA replication and antiviral defense. While the molecular mechanisms involved are incompletely understood, B1 and B12 seem to intersect with signaling processes mediated by their cellular homologs termed the vaccinia-related kinases (VRKs). In this study, we expand upon our previous characterization of the B1-B12 signaling axis to gain insights into B12 function. We begin our studies by demonstrating that modulation of B12 repressive activity is a conserved function of B1 orthologs from divergent poxviruses. Next, we characterize the protein interactome of B12 using multiple cell lines and expression systems and discover that the cellular kinase VRK1 is a highly enriched B12 interactor. Using complementary VRK1 knockdown and overexpression assays, we first demonstrate that VRK1 is required for the rescue of a B1-deleted virus upon mutation of B12. Second, we find that VRK1 overexpression is sufficient to overcome repressive B12 activity during B1-deleted virus replication. Interestingly, we also evince that B12 interferes with the ability of VRK1 to phosphoinactivate the host defense protein BAF. Thus, B12 restricts vaccinia virus DNA accumulation in part by repressing the ability of VRK1 to inactivate BAF. Finally, these data establish that a B12-VRK1-BAF signaling axis forms during vaccinia virus infection and is modulated via kinases B1 and/or VRK2. These studies provide novel insights into the complex mechanisms that poxviruses use to hijack homologous cellular signaling pathways during infection.

IMPORTANCE Viruses from diverse families encode both positive and negative regulators of viral replication. While their functions can sometimes be enigmatic, investigation of virus-encoded, negative regulators of viral replication has revealed fascinating aspects of virology. Studies of poxvirus-encoded genes have largely concentrated on positive regulators of their replication; however, examples of fitness gains attributed to poxvirus gene loss suggests that negative regulators of poxvirus replication also impact infection dynamics. This study focuses on the vaccinia B12 pseudokinase, a protein capable of inhibiting vaccinia DNA replication. Here, we elucidate the mechanisms by which B12 inhibits vaccinia DNA replication, demonstrating that B12 activates the antiviral protein BAF by inhibiting the activity of VRK1, a cellular modulator of BAF. Combined with previous data, these studies provide evidence that poxviruses govern their replication by employing both positive and negative regulators of viral replication.

INTRODUCTION

Poxviruses, of which vaccinia virus is the prototypical member, are large, enveloped viruses with double-stranded DNA genomes approximately 200 kb in size. Unlike other mammalian DNA viruses, poxviruses exhibit significant independence from host cells and carry out their life cycle in the cytoplasm. The relative autonomy of poxviruses is achieved by a large repertoire of virally encoded proteins including replicative machinery (17) and host defense modulators (810). Characterization of these vaccinia virus proteins has revealed fascinating insights into both virology and cellular signal transduction. Needed to overcome cellular defense mechanisms prohibiting cytoplasmic genome replication, the vaccinia Ser/Thr protein kinase B1 is one such example of a virally encoded protein necessitated by the cytoplasmic life cycle of poxviruses (1115).

Studies of vaccinia B1 function using temperature-sensitive viruses (11, 16, 17) first uncovered its role in modulating cellular signal transduction to allow for cytoplasmic DNA replication. These studies demonstrated that B1 promotes vaccinia infection by phosphorylating and inactivating the cellular host defense protein barrier to autointegration factor (BAF) (1820), which otherwise binds vaccinia genomes and other cytoplasmic DNA, impeding their replication. Other experiments characterizing a group of three conserved cellular protein kinases with homology to B1, referred to as vaccinia virus-related kinases (VRKs) (12, 21, 22), revealed that VRK1 can also phosphorylate BAF and rescues the replication of a temperature-sensitive B1 mutant (12, 21, 2325). Together, these studies revealed that a B1-VRK1-BAF signaling axis exists within poxvirus-infected cells; the molecular underpinnings of which have been further affirmed and elucidated in experiments employing VRK1-knockout cells and a B1-deleted vaccinia virus (ΔB1) (23).

Mutant poxviruses can adapt rapidly to nonpermissive cell culture conditions, uncovering novel functional linkages between viral genes (2630). Recently, Olson et al. (31) used adaptive evolution of ΔB1 vaccinia virus to identify an additional pathway governed by the vaccinia B1 kinase. Specifically, disruption of the vaccinia B12 open reading frame rescued ΔB1 virus replication. This study revealed that B1 also promotes vaccinia virus replication by regulating a previously unappreciated inhibitory activity of B12. Although the mechanism through which B12 represses vaccinia replication is poorly understood, some insights have been gained into its regulation and signaling (32, 33). For example, B12 is a pseudokinase, meaning that it has clear homology to protein kinases but also possesses a few amino acid substitutions that render it catalytically inert (3437). In addition, B12 function can be modulated in a cell-type-specific manner by VRK2, an additional homolog of the B1 and VRK1 kinases (38). Lastly, a correlation between loss of B12 function and increased levels of phosphorylated BAF (31) has been observed, suggesting a regulatory pathway may connect these two proteins.

We describe here the further characterization of the B1-B12 signaling axis during vaccinia infection. We first demonstrate that regulation of B12 and BAF is a conserved function for B1 orthologs from diverse poxviruses. Next, we characterize the protein interactome of B12, discovering that VRK1 is the predominant interacting partner for B12 in cell lines from different species and tissues. Following the identification of VRK1 as a B12 partner, we determine that rescue of ΔB1 virus replication by mutation of B12 occurs via a pathway mediated by VRK1. We additionally present evidence that the increase in BAF phosphorylation observed upon mutation of B12 correlates with VRK1 function. Interestingly, we found that in CV1 cells repression of ΔB1 virus by B12 predominantly results from B12 restricting the ability of VRK1 to promote BAF phosphorylation. In contrast, in haploid VRK1 knockout cells, increased BAF phosphorylation does not account for the rescue in ΔB1 virus replication observed by mutation of B12, indicating that other BAF-independent pathways are also regulated by B12 and perhaps VRK1. Lastly, we show that VRK2 can modulate the B12-VRK1 signaling axis, shedding new light on how VRK2 may control signaling in poxvirus-infected cells. This study synthesizes previous evidence implicating VRK1 involvement in vaccinia replication with new insights into B12 mediated repression of vaccinia replication and reveals that a novel B12-VRK1-BAF signaling axis exists in infected cells.

RESULTS

B1 orthologs from divergent poxviruses promote ΔB1 virus replication.

Serine/threonine protein kinases related to the vaccinia B1 protein are encoded by the vast majority of chordopoxvirinae members, with amino acid identity ranging from 47 to 99% between orthologs. Although the vaccinia B1 protein has been studied extensively, direct functional comparisons between orthologs have not been performed. Thus, it is currently unknown whether serine/threonine protein kinases from other poxviruses regulate both the cellular antiviral factor BAF and the signaling driven by the vaccinia B12 pseudokinase similarly to vaccinia B1. While orthologs of vaccinia B1 are common among poxviruses, orthologs of vaccinia B12 are only found within viruses of the orthopoxvirus genus. For this reason, we hypothesized that the B1 ortholog from ectromelia virus (EMV152, 98% identity to B1), which like vaccinia is an orthopoxvirus and encodes a B12 ortholog, would be able to suppress both BAF and B12 signaling. In contrast, more divergent B1 orthologs from myxomavirus (m142, 47% identity to B1) and goatpox virus (GTPV132, 50% identity to B1), two viruses outside of the orthopoxvirus genus, may lack the ability to suppress B12 signaling. To test this hypothesis, 3×FLAG-tagged B1, EMV152, m142, and GTPV132 were stably expressed in CV1, L929, and VRK2 knockout HAP1 cells (VRK2KO) via lentiviral transduction. These three cell lines were chosen as established examples in which both BAF and B12 can impair viral replication in the absence of B1 or the complementary cellular kinase VRK2 (31, 38). Following transduction and hygromycin selection for transgene incorporation, expression of each ortholog was confirmed by immunoblot analysis with FLAG antibody. In CV1 cells, expression levels of m142 and GTPV132 exceeded that of B1 (Fig. 1A). EMV152 was apparently toxic to CV1 cells in this study, as we were unable to establish stably transduced cells expressing 3×FLAG-EMV152 despite repeated attempts. In L929 and VRK2KO cells expression of m142, GTPV132, and EMV152 all modestly exceeded that of B1 (Fig. 1C and E).

FIG 1.

FIG 1

B1 orthologs from divergent poxviruses complement for vaccinia B1 loss. (A) Protein immunoblotting using anti-tubulin and anti-FLAG in transduced CV1 cells. (B) Plaque-forming assays during ΔB1 infection were carried out on transduced CV1 cells. Cell were fixed and stained at 48 hpi. (C) Protein immunoblotting using anti-tubulin and anti-FLAG in transduced L929 cells. (D) Vaccinia DNA accumulation (left) and viral yield (right) measured for ΔB1 virus at 24 hpi in transduced L929 cell lines infected at an MOI of 3. (E) Protein immunoblotting using anti-tubulin and anti-FLAG in transduced VRK2KO cells. (F) GAPDH-normalized vaccinia DNA accumulation (left) and viral yield (right) measured by qPCR for ΔB1 virus at 24 hpi in transduced VRK2KO cell lines infected at an MOI of 3. The value of ΔB1 virus replication in B1 expressing cells was set to 1 for DNA accumulation assays. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Next, each B1 ortholog was examined for the ability to rescue viral replication of a B1 knockout (ΔB1) vaccinia virus. To assess ΔB1 virus replication and spread in CV1 cells expressing each B1 ortholog, a plaque assay was carried out during ΔB1 infections (Fig. 1B). Although no plaques were visible in control (CTRL) CV1 cells, we observed a robust rescue of ΔB1 virus on CV1 cells expressing B1, m142, and GTPV132 with plaques of equal size for each B1 ortholog.

Next, for L929 cells, DNA accumulation and one-step viral yield were determined upon expression of each B1 ortholog. At 24 h postinfection (hpi), ΔB1 virus replication in L929 cells was 39-, 37-, 45-, and 45-fold higher in B1-, m142-, GTPV132-, and EMV152-expressing cells, respectively, compared to CTRL cells (Fig. 1D, left). Similarly, ΔB1 viral yield mirrored that of DNA accumulation results with B1, m142, GTPV132, and EMV152 expression resulting in 373-, 416-, 250-, and 411-fold increases, respectively, in ΔB1 virus replication compared to CTRL cells (Fig. 1D, right).

Finally, viral DNA accumulation and yield were also determined upon expression of each B1 ortholog in VRK2KO cells. At 24 hpi, ΔB1 virus DNA replication in VRK2KO cells was 13-, 15-, 14-, and 13-fold higher in B1-, m142-, GTPV132-, and EMV152-expressing cells, respectively, compared to CTRL cells (Fig. 1F, left). Similarly, ΔB1 viral yield in the presence of B1, m142, GTPV132, and EMV152 expression resulted in 321-, 371-, 129-, and 308-fold increases, respectively, compared to CTRL cells (Fig. 1F, right). These results suggest that B1 orthologs from ectromelia, myxoma, and goatpox viruses are each capable of suppressing both vaccinia B12 signaling and BAF even though they possess considerable differences in identity to vaccinia B1 in some cases.

Identification of B12-interacting proteins by LC-MS/MS.

Previous evidence has demonstrated that the B1-B12 signaling axis acts as a potent modulator of vaccinia virus DNA replication, although the mechanism(s) of B12-mediated repression remains unclear (31, 38). To begin to elucidate how B12 leads to impairment of vaccinia DNA replication, we immunoprecipitated HA-tagged B12 expressed from either recombinant WRHAB12 vaccinia virus or a lentiviral construct. Hypothesizing that B12 targets evolutionarily conserved pathways, these studies were undertaken in multiple cell lines to allow for comparative analysis between interactome datasets. Following the workflow outlined in Fig. 2A, HA-tagged B12 and cointeracting proteins were eluted from HA antibody-bound Dynabeads, separated 1 cm by electrophoresis on SDS-PAGE gels, and subjected to in-gel trypsin digestion before analysis by label-free nano-LC-MS/MS. Altogether, HA-tagged B12 was immunoprecipitated from three virus-infected cell lines (CV1, HAP1, and L929) and from two uninfected cell lines (CV1 and HAP1) for a total of five independent experiments. To be considered an interactor of B12, immunoprecipitated proteins had to be significantly enriched in HAB12 compared to control immunoprecipitations by a Fisher exact test (P < 0.05) with a Benjamini-Hochberg multiple test correction and be enriched in all infected or uninfected immunoprecipitations performed.

FIG 2.

FIG 2

Experimental scheme and analysis of HAB12 interacting proteins. (A) Experimental workflow for affinity purification and LC-MS/MS analysis. HA-tagged B12 was expressed in CV1, HAP1, or L929 cells by WRHAB12 or lentiviral transduction and immunoprecipitated with anti-HA magnetic beads. (B) Comparison of HAB12 interactomes from CV1, HAP1, and L929 cells infected with WRHAB12 and harvested at 7 hpi. (C) Comparison of HAB12 interactomes from CV1 and HAP1 transduced cells after 24 h of HAB12 expression.

In control and experimental samples, we identified 565, 665, and 873 total proteins from infected CV1, HAP1, and L929 cells, respectively, following HA immunoprecipitation (Fig. 2A). Of these, 85 proteins (15%) met the significance threshold in CV1 cells, 126 proteins (19%) were significantly enriched in HAP1 cells, and 124 proteins (14%) were significantly enriched in L929 cells when comparing WRHAB12- to WR-infected cells (Fig. 2B). Of the enriched proteins, 23 proteins of cellular origin overlapped from all three cell lines with no viral proteins significantly enriched in all three cases (Fig. 2B and Table 1). We did note that, while failing to meet our significance threshold in all three cell lines, the VRK2 kinase was immunoprecipitated by HAB12 from HAP1 cells, confirming that HAB12 interacts with VRK2 as previously described (38).

TABLE 1.

Proteins significantly enriched following HAB12 immunoprecipitation of WRHAB12-infected cell lysates

Protein (human) Gene P and enrichment valuesa
HAP1
L929
CV1
P Enrichment P Enrichment P Enrichment
Serine/threonine-protein kinase B12 HAB12 1.00E–20 670/22 (30) 1.00E–20 976/12 (81) 1.00E–20 945/0 (capped)
Serine/threonine-protein kinase VRK1 VRK1 1.47E–17 65/0 (capped) 3.96E–20 87/0 (capped) 8.67E–11 54/0 (capped)
Heterogenous nuclear ribonucleoprotein H HNRNPH1 1.90E–08 73/12 (6.1) 8.30E–09 14/7 (2) 3.20E–10 58/0 (capped)
40S ribosomal protein S15 RPS15 6.25E–08 27/0 (capped) 3.93E–02 3.8 (5/19) 2.50E–03 14/0 (capped)
Polymerase delta-interacting protein 3 POLDIP3 1.58E–05 18/0 (capped) 7.55E–08 32/0 (capped) 8.10E–05 22/0 (capped)
60S ribosomal protein L26 RPL26 3.10E–05 32/5 (6.4) 7.15E–04 36/7 (5.1) 3.19E–03 22/2 (11)
Nucleolin NCL 8.72E–05 22/2 (11) 2.86E–06 78/17 (4.6) 2.50E–03 14/0 (capped)
Insulin-like growth factor 2 mRNA-binding protein 3 IGF2BP3 9.99E–05 15/0 (capped) 1.55E–03 23/3 (7.7) 4.50E–04 18/0 (CApPED)
Serine/arginine-rich splicing factor 3 SRSF3 1.48E–04 21/2 (11) 3.42E–03 21/3 (7.0) 5.88E–03 12/0 (capped)
Ribosomal protein L19 RPL19 1.85E–04 14/0 (capped) 1.65E–04 17/0 (capped) 9.03E–03 11/0 (capped)
60S ribosomal protein L15 RPL15 4.26E–04 19/2 (9.5) 4.73E–06 33/2 (17) 1.91E–04 4/0 (capped)
40S ribosomal protein S3A RPS3A 5.72E–04 40/12 (3.3) 9.43E–05 62/16 (4.1) 1.07E–05 50/0 (10)
40S ribosomal protein S14 RPS14 7.00E–04 5.8 (4/23) 3.52E–03 36/9 (4.0) 7.92E–03 26/4 (6.5)
40S ribosomal protein S6 RPS6 9.41E–04 20/3 (6.7) 4.18E–06 37/3 (12) 1.46E–05 26/0 (capped)
60S ribosomal protein L8 RPL8 1.17E–03 16/0 (capped) 8.61E–05 30/3 (10) 5.28E–05 23/0 (capped)
tRNA-splicing ligase RTCB homolog RTCB 1.17E–03 11/0 (capped) 2.76E–04 16/0 (capped) 3.83E–03 13/0 (capped)
40S ribosomal protein S16 RPS16 1.34E–03 30/8 (3.8) 1.01E–03 35/7 (5.0) 5.86E–03 27/4 (6.8)
60S ribosomal protein l18A RPL18A 2.00E–03 16/2 (8.0) 2.91E–05 29/2 (15) 2.50E–03 14/0 (capped)
60S ribosomal protein l10A RPL10A 2.81E–03 30/9 (3.3) 1.64E–04 48/10 (4.8) 1.29E–04 35/3 (12)
Heterogenous nuclear ribonucleoprotein M HNRNPM 8.26E–03 27/9 (3.0) 1.31E–04 38/6 (6.3) 4.50E–04 18/0 (capped)
Poly(rC)-binding protein 1 PCBP1 9.89E–03 15/3 (5.0) 9.91E–05 28/0 (capped) 3.83E–03 13/0 (capped)
Elongation factor α1 EEF1A1 1.45E–02 37/16 (2.3) 6.89E–04 46/11 (4.2) 2.99E–04 53/9 (9)
Elongation factor Tu TUFM 1.55E–02 14/3 (4.7) 1.62E–08 35/0 (capped) 7.22E–07 33/0 (capped)
a

P values were determined using the Fisher exact test with a Benjamin-Hochberg multiple-test correction. Enrichment (%) values were calculated as the total spectra identified for HAB12/CTRL. The numerical fold enrichment is indicated in parentheses when calculable; otherwise, it is marked as “capped.”

In uninfected cells expressing HAB12, 571 total proteins were identified from CV1 cells, and 769 were identified from HAP1 cells following HA immunoprecipitation (Fig. 2A). Of these total proteins, 18 (3%) were significantly enriched from CV1 lysates, and 13 (2%) were significantly enriched from HAP1 lysates when comparing HAB12-expressing cells to CTRL cells (Fig. 2C). Three proteins overlapped in both uninfected cell data sets; HAB12 and VRK1, both of which were also present in all of the infected cell datasets, as well as PYGL, which was not significantly enriched in immunoprecipitations of infected L929 cells (Fig. 2C and Table 2). In summary, our orthogonal interaction studies identified VRK1 as the only protein other than HAB12 enriched from all infected and uninfected cell lysates following HAB12 immunoprecipitation. These data suggest VRK1 may have an important role in B12 mediated repression of vaccinia virus DNA replication.

TABLE 2.

Proteins significantly enriched following HAB12 immunoprecipitation of uninfected, HAB12-expressing cell lysates

Protein (human) Gene P and enrichment valuesa
HAP1
CV1
P Enrichment P Enrichment
Serine/threonine-protein kinase B12 HAB12 1.00E–20 121/0 (capped) 1.00E–20 100/18 (5.6)
Glycogen phosphorylase (liver form) PYGL 1.00E–20 126/0 (capped) 1.00E–20 20/0 (capped)
Serine/threonine-protein kinase VRK1 VRK1 1.00E–20 202/0 (capped) 8.67E–11 39/0 (capped)
a

P values were determined using the Fisher exact test with a Benjamin-Hochberg multiple-test correction. Enrichment (%) values were calculated as the total spectra identified for HAB12/CTRL (fold enrichment).

Confirmation of interactions between B12 and the VRKs.

To confirm our IP-MS results, we performed immunoprecipitations using an HA-specific antibody followed by immunoblot analysis to detect VRK1. For these assays, CV1 or HAP1 cells were infected with WRHAB12 for 7 h prior to harvest. Immunoblots of input lysates were performed to verify the expression of HA-tagged B12 and VRK1 by each cell line (Fig. 3A and B, INPUT). The same lysates were then subjected to immunoprecipitation using anti-HA-coated beads and subsequent immunoblot analysis with VRK1 antibody to determine whether HAB12 coprecipitated VRK1. We found that HAB12 coprecipitated VRK1 in both WRHAB12-infected CV1 and HAP1 cell lines (Fig. 3A and B, HA IP) compared to controls.

FIG 3.

FIG 3

B12 coprecipitates with VRK1 and VRK2 in infected cells. (A) CV1 cells were infected with WT(WR) or WRHAB12 virus at an MOI of 5 and HAB12 immunoprecipitated at 7 hpi, followed by VRK1 immunoblotting. (B) HAP1 cells were uninfected or infected with WRHAB12 at an MOI of 5 and HAB12 immunoprecipitated at 7 hpi, followed by VRK1 immunoblotting. (C and D) Control vector, 3×FLAG-tagged VRK1, or VRK2A were overexpressed in CV1 cells by lentiviral transduction and infected with WRHAB12 at an MOI of 5 in panel C or infected with WR or WRHAB12 at an MOI of 5 in panel D. At 7 hpi, cell lysates were subjected to immunoprecipitation using anti-FLAG (C) or anti-HA (D) antibody bound to magnetic beads, followed by protein immunoblotting using anti-HA or anti-FLAG. Input proteins were included to illustrate the presence of the respective proteins in each lysate. The relative quantitation of band intensities is included below some blots.

Our previous study comparing the interaction between B12 with either VRK2A, VRK2B, or B1 demonstrated that B12 preferentially interacts with VRK2A compared to VRK2B and B1 (38). Since VRK1 was one of the most highly enriched B12 interaction partners we detected, we hypothesized that VRK1 may interact preferentially with B12 compared to VRK2A when VRK1 and VRK2A are expressed in cells at similar levels. To test this hypothesis, CV1 cells stably transduced with empty control vector, 3XFLAGVRK1, or 3XFLAGVRK2A were infected with WRHAB12 virus. Lysates were harvested at 7 hpi and subjected to anti-FLAG immunoprecipitation and immunoblot analyses. Immunoblots of input lysates indicated that 3XFLAGVRK2A is expressed 2-fold higher than 3XFLAGVRK1 and that relatively equal amounts of HAB12 were present in the three lysates (Fig. 3C, INPUT). After immunoprecipitation, 3XFLAGVRK1 was pulled down by anti-FLAG beads to levels 1.4-fold higher than 3XFLAGVRK2A (Fig. 3C, FLAG IP and FLAG IB). In order to quantify the amount of HAB12 pulled down by 3XFLAGVRK2A compared to 3XFLAGVRK1 within the linear range of our instrument, two different amounts of immunoprecipitated lysates were loaded for 3XFLAGVRK1 into adjacent lanes representing either 20 or 100% of the volume loaded from the control or VRK2A-containing immunoprecipitations. HA-specific immunoblotting revealed that even when 5-fold less immunoprecipitated lysate was loaded for the 3XFLAGVRK1 containing sample (0.2× lane), 7-fold more HAB12 was detected compared to 3XFLAGVRK2A lysates (Fig. 3C, FLAG IP and HA IB). The amount of HAB12 pulled down by 3XFLAGVRK1 in the “100%” lane exceeded the linear detection range of the chemiluminescence imager and could not be compared to 3XFLAGVRK2A in this experiment.

To supplement these results, reciprocal HA immunoprecipitations were performed. For HA immunoprecipitations, WR infection controls were also included to assess nonspecific binding of the FLAG-tagged proteins to the anti-HA beads. Immunoblots of input lysates show that 3XFLAGVRK1 is present at slightly higher levels than 3XFLAGVRK2A, but that HAB12 detection is similar between 3XFLAGVRK1 and 3XFLAGVRK2A lysates (Fig. 3D, INPUT). Detection of immunoprecipitated HAB12 revealed that relatively equal amounts were purified from each of the lysates (Fig. 3D, HA IP and HA IB). FLAG immunoblotting revealed that HAB12 immunoprecipitated ∼40-fold more 3XFLAGVRK1 than 3XFLAGVRK2A (Fig. 3D, HA IP and FLAG IB). These studies demonstrate that in immunoprecipitations from cellular lysates, 3XFLAGVRK1 is enriched to a greater level than 3XFLAGVRK2, leading us to hypothesize that VRK1 may form a more stable complex with B12 than VRK2A in cells.

VRK1 mediates the rescue of ΔB1 and ΔB1mutB12 viruses.

Our previous studies have demonstrated that B12 impairs the replication of vaccinia virus mutants lacking the B1 kinase via a poorly understood mechanism (31, 38). Interestingly, earlier seminal studies by Boyle et al. (24), revealed that VRK1 can complement for the B1-deficient ts2 mutant vaccinia virus when VRK1 is overexpressed from the viral thymidine kinase locus. Our new discovery of an interaction between B12 and VRK1 led us to theorize that B12 may influence the activity of VRK1 during vaccinia virus infection. To test this hypothesis, we began by investigating the impact of VRK1 overexpression or depletion on the growth of viral plaques after infection.

For overexpression studies, we began by introducing 3XFLAGVRK1 into CV1 cells via lentiviral transduction, successfully overexpressing it at least 30-fold compared to endogenous VRK1 (Fig. 4B). Although wild-type (WT) virus plaques were similar in CTRL and 3XFLAGVRK1-expressing cells, overexpression of VRK1 markedly rescued plaque formation for ΔB1 virus (Fig. 4A). As observed during ΔB1mutB12 infection of CTRL cells, mutation of B12 is sufficient to rescue ΔB1 virus plaque formation in these cells, although ΔB1mutB12 plaques are smaller than WT plaques. Interestingly, upon VRK1 overexpression ΔB1mutB12 plaque size remained unchanged, indicating that 3XFLAGVRK1 overexpression does not further enhance replication of this mutant (Fig. 4A).

FIG 4.

FIG 4

VRK1 levels impact ΔB1 and ΔB1mutB12 virus growth in CV1 cells. (A) Plaque assay of CV1 cells stably expressing 3XFLAGVRK1 or transduced with control vector and infected with 300 PFU of WT, ΔB1, or ΔB1mutB12. Cells were incubated for 72 h at 37°C prior to fixation and staining. (B) Representative immunoblot of CV1 cells stably expressing control vector or 3XFLAGVRK1 using anti-tubulin or anti-VRK1. Arrows at right indicate 3×FLAG-tagged and endogenous VRK1. Cells were infected with WT, ΔB1, ΔB1mutB12, ts24, or ts42 at an MOI of 3 as indicated and harvested for 7 hpi DNA accumulation (C) or 24 hpi viral yield (D). (E) Plaque assay of VRK1-depleted or control CV1 cells infected with 300 PFU of WT or ΔB1mutB12. Cells were incubated for 72 h at 37°C prior to fixation and imaging. (F) Representative immunoblot of VRK1-depleted or control CV1 cells using anti-tubulin or anti-VRK1. The value of CTRL-WT was set to 1 for DNA accumulation assay, and error bars represent standard deviations. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

To determine whether the overexpression of 3XFLAGVRK1 only complemented for a B1-defective virus or could also enhance the replication of other impaired vaccinia virus mutants, the fitness of WT, ΔB1, ΔB1mutB12 virus, a temperature-sensitive D5 primase/helicase mutant (ts24) virus (39), and a temperature-sensitive E9 DNA polymerase mutant (ts42) virus (40) were examined. At 7 hpi, neither WT, ts24, nor ts42 DNA accumulation was impacted by 3XFLAGVRK1 overexpression (Fig. 4C). In comparison, ΔB1 DNA accumulation was increased significantly by 37-fold and ΔB1mutB12 by 2.8-fold (Fig. 4C) upon VRK1 overexpression. Examination of the 24-hpi viral yield revealed similar trends as those seen for 7-hpi DNA accumulation. Specifically, the viral yield for WT, ts24, and ts42 were not impacted by 3XFLAGVRK1 overexpression (Fig. 4D). In contrast, ΔB1 virus increased significantly by 16-fold to levels similar to ΔB1mutB12 virus. While ΔB1mutB12 virus DNA accumulation was increased at 7 hpi (Fig. 4C), the amount of virus produced on CTRL and 3XFLAGVRK1 cells was unchanged, consistent with ΔB1mutB12 plaque size remaining the same between the two cell types. These data demonstrate that 3XFLAGVRK1 overexpression only enhances the fitness of replication deficient B1-deleted viruses.

Next, to assess the impact of VRK1 depletion on these viruses, CV1 cells were transfected with either of two independent siRNAs against VRK1, resulting in depletion of VRK1 to levels that are not detectable compared to a control siRNA (Fig. 4F). While viral replication and spread of WT virus was not impacted by the loss of VRK1, ΔB1mutB12 plaque formation was completely inhibited by VRK1 depletion (Fig. 4E). Together, these results indicate that VRK1 is necessary for the formation of plaques by ΔB1mutB12 virus and that VRK1 overexpression is sufficient to rescue ΔB1 virus in CV1 cells. To complement the above CV1 studies, parental HAP1 and VRK1KO cells were each transduced with CTRL or 3XFLAGVRK1-expressing lentivirus. Immunoblotting of transduced cells confirmed that VRK1KO:CTRL cells do not contain detectable levels of VRK1 and show that 3XFLAGVRK1 effectively reconstituted VRK1 to levels exceeding that in HAP1:CTRL cells (Fig. 5A). Based on the results from Fig. 4, we hypothesized that 3XFLAGVRK1 overexpression would increase viral replication for ΔB1 virus and that the absence of VRK1 would reduce the replication of ΔB1mutB12 virus compared to control cells. At 7 hpi, DNA accumulation for WT virus remained unchanged between all cell types (Fig. 5B). In comparison, 3XFLAGVRK1 overexpression in HAP1 cells resulted in a 2-fold increase in ΔB1 genome replication and a 2.5-fold increase in ΔB1mutB12 DNA compared to HAP1:CTRL cells (Fig. 5B). VRK1 knockout in VRK1KO:CTRL cells decreased ΔB1mutB12 virus DNA replication by 4-fold compared to HAP1:CTRL cells (Fig. 5B). Importantly, reconstitution of VRK1 in VRK1KO:3XFLAGVRK1 cells rescued ΔB1 and ΔB1mutB12 virus replication compared to HAP1:CTRL cells, suggesting that VRK1 is sufficient to enhance viral genome replication at this time point (Fig. 5B).

FIG 5.

FIG 5

VRK1 levels impact ΔB1 and ΔB1mutB12 virus fitness in HAP1 cell lines. (A) Representative immunoblot of HAP1 or HAP1-VRK1KO cells stably expressing control vector or 3XFLAGVRK1 using anti-tubulin or anti-VRK1. Arrows at right indicate 3×FLAG-tagged and endogenous VRK1. Cells were infected with WT, ΔB1, or ΔB1mutB12 at an MOI of 3 as indicated and harvested at 7 hpi DNA accumulation (B), at 24 hpi DNA accumulation (C, left), or at 24 hpi viral yield (C, right). The value of HAP1-CTRL-WT at each respective harvest time was set to 1 for DNA accumulation assays, and error bars represent standard deviations. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

At 24 hpi, WT virus DNA accumulation remained unchanged between all cell types (Fig. 5C, left). VRK1 overexpression in HAP1:3XFLAGVRK1 cells increased ΔB1 DNA accumulation by 2-fold compared to HAP1:CTRL cells, while ΔB1mutB12 DNA levels were identical in these two cell lines. Similar to 7 hpi, knockout of VRK1 in VRK1KO:CTRL cells significantly decreased ΔB1 DNA accumulation by 2-fold and ΔB1mutB12 DNA accumulation by 3-fold, and both were rescued by reconstitution of VRK1 in VRK1KO:3XFLAGVRK1 cells (Fig. 5C, left).

Finally, we examined how the presence or absence of VRK1 impacted viral yield. At 24 hpi, the WT viral yield remained unchanged between all cell types (Fig. 5C, right). In contrast, the ΔB1 viral yield was reduced and exhibited marked sensitivity to VRK1 levels in each cell line. Specifically, during ΔB1 infection a 3-fold decrease was seen between HAP1:CTRL and VRK1KO:CTRL cells, which was subsequently rescued by 3XFLAGVRK1 expression in VRK1KO:3XFLAGVRK1 cells (Fig. 5C, right). Next, we found that ΔB1mutB12 viral yield was not significantly impacted by 3XFLAGVRK1 expression in HAP1:3XFLAGVRK1 cells. However, the ΔB1mutB12 yield decreased substantially (13-fold) between HAP1:CTRL and VRK1KO:CTRL cells and could be rescued by VRK1 expression in VRK1KO:3XFLAGVRK1 cells. Overall, the results in Fig. 5 suggest that in the absence of B1, VRK1 levels are an important determinant of vaccinia DNA replication and viral yield. In addition, since VRK1 knockout prevents the viral rescue of ΔB1mutB12 virus compared to ΔB1, these data support the conclusion that a proviral function of VRK1 is both necessary and sufficient for the rescue of ΔB1mutB12 virus compared to ΔB1 virus.

B12 decreases BAF phosphorylation in a VRK1-dependent manner.

We previously published that mutation of B12 leads to increased BAF phosphorylation during ΔB1mutB12 infection compared to ΔB1 infection (31), which likely ameliorates the DNA binding activity of BAF. In addition, VRK1 has been shown to phosphorylate BAF (25, 41). Therefore, we posited that differential regulation of BAF phosphorylation may partly explain how B12 and VRK1 were impacting viral fitness in CV1 and HAP1 cells. To test our hypothesis, we began by introducing 3XFLAGVRK1 into CV1 cells via lentiviral transduction. These cells were infected with WT, ΔB1, or ΔB1mutB12 virus and BAF phosphorylation assessed at 7 hpi. During WT infection, the phosphorylation status of BAF was similar in CTRL or 3XFLAGVRK1-expressing cells (Fig. 6A, compare lanes 1 and 2). Similar levels of BAF phosphorylation were also observed in cells infected with ts24 and ts42 viruses (data not shown), thus confirming prior studies that viruses expressing B1 can phosphorylate BAF independent of viral DNA replication (19). The ΔB1-infected CTRL cells exhibited less BAF phosphorylation than WT cells due to the loss of the B1 protein kinase (Fig. 6A, compare lanes 1 and 3). However, during ΔB1 infection, 3XFLAGVRK1 expression increased the amount of phosphorylated BAF detected compared to CTRL cells (Fig. 6A, compare lanes 3 and 4). As previously published (31), in CTRL cells ΔB1mutB12 infection resulted in higher levels of phosphorylated BAF than CTRL cells infected with ΔB1 (Fig. 6A, compare lanes 3 and 5), with no apparent additional influence by 3XFLAGVRK1 overexpression during ΔB1mutB12 infection (Fig. 6A, compare lanes 5 and 6). These results demonstrate that 3XFLAGVRK1 overexpression is sufficient to increase the amount of phosphorylated BAF in ΔB1-infected CV1 cells.

FIG 6.

FIG 6

BAF phosphorylation is influenced by B12 and VRK1. (A) Representative immunoblot of CV1 cells stably expressing control vector or 3XFLAGVRK1. Cells were infected with WT, ΔB1, or ΔB1mutB12 at an MOI of 10. (B) Immunoblot of CV1 cells pretreated with control or siVRK1 RNA for 72 h and then infected with WT, ΔB1, or ΔB1mutB12 at an MOI of 10. (C) Immunoblot of HAP1 or HAP1-VRK1KO cells infected with an MOI 10 WT, ΔB1, or ΔB1mutB12. For all immunoblots shown in panels A, B, and C, cells were harvested 7 hpi and subjected to immunoblot analysis for anti-tubulin, anti-VRK1, anti-BAF, and anti-phospho BAF. Open triangles indicate phosphorylated BAF. Closed triangles indicate unphosphorylated BAF.

We next hypothesized that VRK1 was needed for the increase in BAF phosphorylation seen previously during ΔB1mutB12 infection compared to ΔB1. To test this hypothesis, CV1 cells were transfected with siRNA against VRK1, infected with WT, ΔB1, or ΔB1mutB12, and lysates assessed for BAF phosphorylation at 7 hpi. In uninfected lysates, the phosphorylation status of BAF remained similar following VRK1 depletion (Fig. 6B, compare lanes 1 and 2). VRK1 depletion also did not impact BAF phosphorylation during WT infection (Fig. 6B, compare lanes 3 and 4) or ΔB1 infection (Fig. 6B, compare lanes 5 and 6). Interestingly, VRK1 depletion during ΔB1mutB12 infection resulted in decreased detection of phosphorylated BAF compared to CTRL cells (Fig. 6B, compare lanes 7 and 8), indicating that VRK1 was necessary for the increased detection in BAF phosphorylation during ΔB1mutB12 infection compared to ΔB1 in CV1 cells.

BAF phosphorylation during vaccinia virus infection was further investigated utilizing parental HAP1 and VRK1KO cells. First, in uninfected cells VRK1 loss modestly reduced BAF phosphorylation compared to HAP1 cells (Fig. 6C, compare lanes 1 and 2). During WT infection, no difference in BAF modification was observed between HAP1 and VRK1KO cells. In ΔB1-infected cells, a decrease in BAF phosphorylation in both HAP1 and VRK1KO cells was observed compared to WT (Fig. 6C, compare lanes 3 and 4 with 5 and 6). In contrast, during ΔB1mutB12 infection, VRK1 knockout reduced the amount of phosphorylated BAF detected compared to parental HAP1 cells (Fig. 6C, compare lanes 7 and 8). Combined, these results suggest that during ΔB1 infection, B12 redirects or impedes VRK1 activity against BAF, leading to reduced BAF phosphorylation. However, when B12 is mutated, as in ΔB1mutB12-infected cells, VRK1 increases BAF phosphorylation and thereby impairs its antiviral activity against vaccinia virus.

BAF depletion rescues ΔB1 and ΔB1mutB12 replication in VRK1-depleted CV1 cells.

We next hypothesized that BAF was a major determinant through which VRK1 altered ΔB1mutB12 fitness. If this is the case, we would expect that codepletion of both BAF and VRK1 would result in elevated ΔB1mutB12 viral yield compared to depletion of only VRK1. To test this hypothesis, CV1 cells were first transduced with shCTRL or shBAF expressing lentiviruses and then transfected with siVRK1 or a control siRNA. Immunoblot analysis of CV1 shCTRL:siCTRL, shBAF:siCTRL, shCTRL:siVRK1, and shBAF:siVRK1 cells indicated that both VRK1 and BAF depletion were achieved compared to shCTRL:siCTRL cells (Fig. 7A). Next, using one-step growth assays, we found that WT viral yield was not impacted by BAF or VRK1 depletion (Fig. 7B). For ΔB1 virus, VRK1 depletion resulted in a 11-fold decrease in viral yield compared to shCTRL:siCTRL cells, with a 5.5-fold increase in viral yield resulting from BAF depletion when coupled with VRK1 depletion (Fig. 7B). Similar to the phenotype seen in VRK1KO cells, VRK1 depletion resulted in a significant 26-fold decrease in ΔB1mutB12 progeny compared to shCTRL:siCTRL cells that was rescued (9.6-fold) by codepletion of BAF with VRK1 (Fig. 7B). These results lead us to conclude that, in CV1 cells, BAF is the predominant substrate that VRK1 acts upon to promote the fitness of both ΔB1 and ΔB1mutB12 viruses.

FIG 7.

FIG 7

BAF depletion partially rescues ΔB1 and ΔB1mutB12 virus in VRK1-depleted CV1 cells but does not enhance replication in HAP1-VRK1KO cells. (A) Immunoblot using anti-BAF and anti-VRK1 in CV1 cells transduced to express control shCTRL or shBAF and then treated with siVRK1 or a control siRNA. (B) CV1 cells depleted as shown in panel A were infected with WT, ΔB1, or ΔB1mutB12 virus at an MOI of 3 and harvested at 24 hpi for viral yield determination. (C) Protein immunoblotting using anti-tubulin and anti-BAF in shBAF-depleted HAP1 and HAP1-VRK1KO cells. (D) Viral yield measured for WT, ΔB1, and ΔB1mutB12 viruses at 24 hpi in transduced HAP1 and VRK1KO cell lines infected at an MOI of 3. (E) GAPDH-normalized vaccinia DNA accumulation measured for WT, ΔB1, and ΔB1mutB12 viruses at 24 hpi in transduced HAP1 and VRK1KO cell lines infected at an MOI of 3. For all immunoblots, open triangles indicate phosphorylated BAF, and closed triangles indicate unphosphorylated BAF. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

BAF depletion is not sufficient to rescue ΔB1 or ΔB1mutB12 virus in VRK1KO cells.

We next examined whether antiviral activity of BAF was the primary pathway affected by B12-VRK1 signaling in HAP1 cells, as appears to be the case in CV1 cells. Lentiviral expression of BAF-specific shRNA was used to deplete BAF to 5 to 10% of endogenous levels in HAP1 and VRK1KO cells (Fig. 7C). Viral DNA accumulation and yield was then used to assess WT, ΔB1, and ΔB1mutB12 viral rescue following shBAF treatment. Intriguingly, neither viral DNA accumulation nor viral yield of any virus were significantly enhanced by the depletion of BAF in either HAP1 or VRK1KO cells (Fig. 7D and E). These results indicate that in HAP1 cells, B12 may be altering VRK1 signaling via multiple pathways impacting poxvirus infection in additional to the regulation of BAF.

VRK2 can modulate the B12:VRK1 signaling axis.

Data here and in previous studies establish that both VRK1 and VRK2 contribute to vaccinia virus DNA replication. We next directly compared how B1 and B12 affected viral genome replication in the absence of either one or both of these cellular kinases. First, we compared the proviral activity of VRK1 and VRK2 by performing WT, ΔB1, and ΔB1mutB12 virus infections in parental HAP1, VRK1KO, and VRK2KO cells (Fig. 8A). In this experiment, the 24-h viral yield in parental HAP1 cells was reduced 31-fold during ΔB1 compared to WT infection, whereas ΔB1mutB12 virus replication increased 4.3-fold compared to ΔB1 virus. In VRK1KO cells, viral yield was decreased marginally more (100-fold) during ΔB1 virus infection compared to WT. However, in clear contrast to the HAP1 cells, ΔB1mutB12 viral yield was not increased compared to ΔB1 viral yield suggesting that VRK1 is needed to complement ΔB1mutB12 virus yield in HAP1 cells. In VRK2KO cells, we observed the greatest decrease (300-fold) in ΔB1 virus yield compared to WT infection of the three cell lines. Similar to the trend in HAP1 cells, we observed a 36-fold increase in ΔB1mutB12 compared to ΔB1 virus yield in VRK2KO cells, suggesting that VRK2 functions to complement ΔB1 virus yield in HAP1 cells but is not required for the rescue observed upon loss of B12. These results are consistent with a model in which both VRK1 and VRK2 can promote vaccinia virus replication, albeit via distinct pathways. Specifically, VRK1 can function as a proviral factor only in the absence of B12 and is needed for the rescue of viruses lacking B1 and B12. In contrast, VRK2 functions as an antagonist of antiviral B12 signaling and thereby promotes the replication of ΔB1 but not ΔB1mutB12 virus.

FIG 8.

FIG 8

B12 shifts VRK1 kinase cascade signaling in the absence of B1/VRK2 regulation. (A) HAP1, VRK1KO, or VRK2KO cells were infected at an MOI of 3 with WT, ΔB1, or ΔB1mutB12 and harvested at 24 hpi for determination of viral yield. (B) Protein immunoblotting with anti-tubulin and anti-VRK1 in siVRK1-depleted HAP1 and VRK2KO cells. (C) Vaccinia DNA accumulation (left) and viral yield (right) measured for WT, ΔB1, and ΔB1mutB12 viruses at 24 hpi in transduced HAP1 and VRK2KO cell lines infected at an MOI of 3. The value of HAP1-siCTRL-WT was set to 1 for DNA accumulation assay. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Next, we sought to determine how vaccinia virus replication would be affected by the absence of both VRK1 and VRK2. HAP1 and VRK2KO cells were depleted of VRK1 via siVRK1 transfection. Following siVRK1 transfection of HAP1 and VRK2KO cell lines, VRK1 levels were depleted to approximately 15% of siCTRL-treated levels (Fig. 8B).

Based on the data presented in Fig. 8A, we predicted that VRK1 depletion in VRK2KO cells would result in no net change during ΔB1 virus infections as VRK1 only exhibits proviral activity in the absence of both B1 and B12. We also posited that during ΔB1mutB12 virus infection VRK1 depletion would result in decreased vaccinia virus replication if VRK1 also mediates rescue of ΔB1mutB12 virus in the absence of VRK2. Viral DNA accumulation and yield were determined in parental HAP1 and VRK2KO cells depleted of VRK1. Upon VRK1 depletion we observed no change in WT virus DNA accumulation or yield in either HAP1 or VRK2KO cells (Fig. 8C). However, we found that ΔB1mutB12 viral DNA accumulation was reduced almost 70% in VRK1-depleted VRK2KO cells compared to CTRL depleted VRK2KO cells; whereas VRK1 depletion did not significantly change ΔB1mutB12 viral DNA accumulation in HAP1 cells (Fig. 8C, left). Similarly, VRK1 depletion reduced ΔB1mutB12 viral yield almost 10-fold in VRK2KO cells but decreased viral yield to a lesser degree (2.7-fold) in parental HAP1 cells (Fig. 8C, right). Surprisingly, ΔB1 viral DNA accumulation was rescued 2.3-fold in VRK1-depleted compared to control VRK2KO cells (Fig. 8C, left). This rescue in viral DNA accumulation extended to viral yield with VRK1-depleted VRK2KO cells rescuing viral yield 79-fold compared to control VRK2KO cells (Fig. 8C, right). Together, these results confirm that VRK1 and VRK2 perform proviral functions during infections lacking vaccinia B1 and indicate that VRK2 can regulate B12-VRK1 signaling.

However, these studies also reveal that VRK1 signaling is not proviral in all contexts. Our data indicate that VRK1 may direct antiviral signaling in the absence of VRK2 and indicate that VRK2 likely impacts VRK1 signaling via pathways that we do know yet understand.

DISCUSSION

Poxviruses engage in complex manipulations of cellular signaling pathways to establish an environment conducive to viral replication. The vaccinia virus B1 protein kinase phosphorylates the cellular factor BAF, inhibiting the ability of BAF to otherwise interfere with cytoplasmic DNA replication. The antipoxviral activity of BAF is one of the many contributions made by BAF to maintain host genome integrity (4244). More recently, B1 was demonstrated to also suppress an antiviral activity of the vaccinia B12 pseudokinase. While B12 also decreases vaccinia virus DNA replication in the absence of B1, the mechanism by which B12 down regulates vaccinia virus DNA replication was unclear. To unravel the role of B12 during vaccinia infection, we began characterizing the B12 mechanism of action. Here, for the first time we describe the interactome of vaccinia B12 and reveal that B12 forms a complex with the cellular kinase VRK1. We then explore the impact of B12-VRK1 interaction on vaccinia replication, establishing B12 as a likely allosteric regulator of VRK1 that represses the ability of VRK1 to phosphorylate BAF. Finally, we also expand upon our previous characterization of VRK2 regulation of B12 (38), providing evidence that B1 and VRK2 are both modulators of the B12-VRK1 axis.

SInce B1 orthologs have not been compared functionally, we began our studies by positing that orthologs of B1 may differ in their ability to suppress BAF and/or B12 directed signaling and could therefore be of value in comparative studies dissecting the B12 mechanism of action. We compared B1 orthologs with varying percent identifies to vaccinia B1 from ectromelia (98% identity to B1), myxomavirus (47% identify), and goatpox virus (50% identity), and we assessed the ability of each ortholog to overcome BAF and B12 signaling. We predicted that orthologs of B1 from all of these viruses would all retain an ability to inhibit BAF as they replicate in cells expressing BAF; however, because myxomavirus and goatpox virus lack orthologs of B12 we postulated their B1 orthologs may be unable to suppress the B12 antiviral pathway. Interestingly, we found that B1 orthologs from vaccinia virus, ectromelia virus, myxomavirus, and goatpox virus all rescued ΔB1 virus yield in multiple cell lines, indicating that all can repress BAF and B12. This suggests that suppression of B12 signaling is a conserved function for B1, regardless of the presence of an equivalent to B12 within a virus’s genome. Two possible mechanisms may explain B1 governance of the B12 pathway. The first has been previously described and involves an upstream regulation of B12 by B1, consistent with evidence that B1 and B12 can be coimmunoprecipitated (38). The second mechanism is supported by our data here that B12 inhibits VRK1 activity, leading to the consideration that B1 homolog signaling may converge with the B12 axis downstream of B12 as an inherent consequence of their ability to inhibit BAF and perhaps other substrates. These two mechanisms are not mutually exclusive and are both presented as part of the working model shown in Fig. 9.

FIG 9.

FIG 9

Working model depicting how VACV B1 and B12 converge with cellular VRK kinase signaling in a manner critical for viral replication. This manuscript elucidates that B12 forms a complex with VRK1 and interferes with its downstream signaling. The presence of B12 inhibits viral DNA replication and is either mediated primarily by BAF in some cell types or likely involves additional VRK1 and VRK2 substrates as well in other cell types. The antiviral action of B12 can be observed in the absence of B1, indicating that B1 complements for inhibited VRK1 and/or that B1 may regulate B12 via a posited upstream pathway as suggested by the dotted line.

To elucidate how vaccinia B12 functions to impeded viral DNA replication, we first considered precedents from other pseudokinases. For example, pseudokinases can act as allosteric regulators of enzymatically active kinases, governing their interaction with downstream substrates (3437, 45). In search of candidate pathways that may be impacted by B12, we undertook an unbiased, mass spectrometry-based screen to determine the protein interactome of B12. Using an orthogonal approach involving three cell lines and two expression systems, we significantly refined the number of mass spectrometry-identified B12 interactors to one highly enriched candidate, the vaccinia virus-related kinase 1 (VRK1), which was further confirmed by immunoblotting of immunoprecipitated lysates. It is interesting that, prior to this report, we had found that B12 could interact with vaccinia B1, as well as with another VRK family member, VRK2 (38). To further investigate the interaction of B12 with VRK1 compared to VRK2, we performed immunoprecipitations in cell lines transduced to express similar amounts of VRK1 or VRK2 with identical epitope tags. When VRK expression was normalized, we calculated that B12 immunoprecipitated approximately 35 times more VRK1 than VRK2. Although we found these results somewhat surprising because of the considerable similarity between human VRK1 and VRK2, they indicate that B12 interacts with VRK1 in a manner unique from its interaction with VRK2 or B1. Notably, while the interactomes of the VRKs have been previously described (46), the formation of complexes containing multiple distinct VRK homologs has not been reported to date, including studies of the pseudokinase VRK3. Therefore, our ability to readily detect B12-VRK complexes containing either VRK1 or VRK2 suggests that B12 may be unique among VRK homologs in its mechanism of integrating with VRK signal transduction.

Having established that VRK1 from multiple species is a major interactor of B12, we next sought to determine whether this interaction was functionally connected to B12 repressive activity. We thus examined whether VRK1 overexpression or depletion affected vaccinia virus replication in CV1 cells. Comparisons of viral replication and spread revealed that ΔB1 virus but not ts24 or ts42 virus could be rescued by VRK1 overexpression and that VRK1 activity is necessary for ΔB1mutB12 virus rescue. These data suggest that B12 impairs the ability of VRK1 to perform proviral functions during infections lacking B1. Since VRK1 overexpression could rescue ΔB1 virus replication in CV1 cells, it may be that abundant VRK1 either serves as a molecular “sink” acting to dilute B12 activity or simply increases the amount of unbound active VRK1 in the cell. In either case, apparent saturation of B12 with excess VRK1 suggests that the stoichiometry between VRK1 and B12 is a critical determinant of the repression of VRK1 kinase activity. If true, it may be that cell type-specific differences in the expression of VRK1 explain previously observed differences in sensitivity to B1-B12 signaling (11, 1618, 23, 31, 47). Lastly, we confirmed that VRK1 was necessary for ΔB1mutB12 rescue in HAP1 lineage cells. Importantly, we observed that viral DNA accumulation and yield were reduced in VRK1KO cells infected with ΔB1mutB12 virus compared to parental HAP1 cells and could be rescued by VRK1 reconstitution. Together, these results confirm that VRK1 is necessary for the rescue of ΔB1mutB12 in multiple cell lines. Overall, these data support a model in which B12 serves as an allosteric regulator of VRK1 and preventsVRK1 from performing proviral functions during infections in which B12 signaling is not mitigated. When considering our previously reported model for VRK2 inhibition of B12 signaling during ΔB1 infection (38), these results now also suggest that B1 and VRK2 can mask the impact of B12 signaling on viral fitness by bypassing the contribution of VRK1.

Because VRK1 is a known BAF kinase in multiple organisms (25, 48, 49) and the mutation of B12 during ΔB1 virus infection results in increased BAF phosphorylation (31), we posited that B12 may specifically disrupt the ability of VRK1 to phosphorylate BAF. Therefore, we sought to determine whether VRK1 overexpression during ΔB1 infection could functionally mimic the increase in BAF phosphorylation we observed during ΔB1mutB12 infection and whether BAF phosphorylation could be decreased by VRK1 depletion or knockout during ΔB1mutB12 infection. Indeed, BAF phosphorylation was increased by VRK1 overexpression during ΔB1 infection and was decreased by VRK1 depletion in CV1 cells during ΔB1mutB12 infections. In addition, we examined BAF phosphorylation during ΔB1 and ΔB1mutB12 infections in parental HAP1 and VRK1KO cells. In these cell lines, BAF phosphorylation was also decreased by the loss of VRK1 during ΔB1mutB12 infection, indicating that B12 represses the ability of VRK1 to phosphorylate BAF in multiple cell lines. BAF phosphorylation was also reduced during ΔB1 infections in both HAP1 and VRK1KO cells. Cumulatively, these data are consistent with a model in which B12 represses the ability of VRK1 to phosphorylate BAF and that B12 impairment of VRK1 kinase activity can be offset by overexpression of VRK1.

Next, having established that VRK1 is necessary for the rescue of ΔB1mutB12 viral DNA accumulation and yield and that VRK1 is responsible for the increase in BAF phosphorylation observed during ΔB1mutB12 infection, we sought to address whether VRK1 was mediating the rescue of ΔB1mutB12 by increasing BAF phosphorylation. Indeed, BAF depletion in VRK1-depleted CV1 cells resulted in increased viral yields for both ΔB1 and ΔB1mutB12 infections compared to CV1 cells only depleted of VRK1, suggesting that B12 decreases viral replication by preventing VRK1 phosphorylation of BAF in CV1 cells. Given that the rescue of ΔB1 and ΔB1mutB12 viral yield in shBAF:siVRK1 cells was reduced compared to ΔB1mutB12 in shCTRL:siCTRL cells, this suggests that B12-VRK1 signaling also acts via a BAF-independent mechanism. The evidence supporting a BAF-independent mechanism for B12-VRK1 repression of vaccinia replication was more striking in HAP1 lineage cells wherein BAF depletion failed to rescue ΔB1 or ΔB1mutB12 viral DNA accumulation or yield in VRK1KO cells. These studies dissecting the B12-VRK1 signaling axis in multiple cell lines indicate that the role of B12 during vaccinia virus infections is cell type dependent, with B12-VRK1 inhibitory activity resulting predominantly from mitigation of BAF phosphorylation in CV1 cells, whereas B12 also functions to repress vaccinia replication in a VRK1-dependent manner in HAP1 lineage cells, but likely via dysregulation of other as-yet-unidentified VRK1 substrates in addition to BAF.

It is interesting to speculate that the BAF-independent pathway by which B12 downregulates vaccinia virus replication may be via one of our other B12 interactors. For example, glycogen phosphorylase (PYGL) was significantly enriched in all but the L929 WRHAB12 interactome and is a current avenue of study. In addition, ribosomal proteins were highly enriched during immunoprecipitations of WRHAB12-infected cells but not uninfected HAB12-expressing cells. Given the abundance of data regarding vaccinia regulation of translation (50) and the known association of B1 in phosphorylating components of the ribosomal complex (51), the ribosome may be an additional starting point for future studies examining the BAF-independent pathway by which B12 represses vaccinia virus replication. However, it may be that B12 activity fully derives from its impairment of VRK1 kinase activity. In such a case, investigation of VRK1 substrates other than BAF may be highly valuable. VRK1 governs numerous cellular processes (52, 53) and modifies multiple factors, including histones, XRCC5/6, and PARP (46, 5458). Since many of these proteins have all been studied in connection to vaccinia virus replication in other contexts (5963), they are intriguing future avenues of study.

Lastly, we compared mutant virus replication in cells lacking either VRK1 or VRK2 alone or when the levels of both are reduced. We uncovered evidence that VRK1 and VRK2 promote vaccinia DNA replication through distinct but intersecting pathways, as indicated in Fig. 9. For example, parallel depletion studies in HAP1 and VRK2KO cells revealed that VRK1 loss inhibited ΔB1mutB12 replication in VRK2KO cells to a greater magnitude than in parental HAP1 cells. Interestingly, we also found that VRK1 depletion correlated with a robust rescue of ΔB1 virus replication in VRK2KO cells. These data inform a model in which VRK1 is proviral in the absence of B12 (ΔB1mutB12 infection); however, it also indicates that when complexed with B12 during ΔB1 infection, the B12-VRK1 complex can mediate antiviral signaling in the absence of VRK2. Overall, these data support our hypothesis that VRK2 can regulate the B12-VRK1 signaling axis in HAP1 derived cell lines.

Although we have progressed substantially in our understanding of the signaling axis involving B12 through these studies, it remains unclear as to why orthopoxviruses harbor a protein capable of potent viral repression. Our ongoing working model to explain this apparent paradox synthesizes evidence from current and prior studies as follows. During WT vaccinia infections, B12 antiviral activity may be masked by B1 in two ways: first, via direct phosphorylation of B12 by B1 or VRK2 (38), and second, via B1-mediated phosphorylation of BAF (23, 31). However, data from this study indicate that regardless of the presence of B1, a B12-VRK1 complex forms during infection (Fig. 2 and data not shown). Since both B12 and VRK1 are predominantly found in the nucleus (22, 31), we posit that assembly of the B12-VRK1 complex may remodel nuclear signal transduction to favor viral replication. It is possible a B12-VRK1 complex contributes to host shutdown or immune signaling disruption and could thus be performing proviral functions in this compartment. While speculative at this point, other nuclear poxviral proteins have demonstrated abilities to disrupt signaling in this organelle (59, 60, 64, 65), providing precedents for this theory.

MATERIALS AND METHODS

Chemicals and reagents.

Chemicals were obtained from Sigma-Aldrich or Fisher Scientific. Primers were obtained from Integrated DNA Technologies. Codon-optimized constructs were obtained from GeneArt.

Cell culture.

Human near-haploid fibroblast HAP1 parental, vaccinia virus-related kinase 1 knockout (VRK1KO), and vaccinia virus-related kinase 2 knockout (VRK2KO) cell lines were obtained from Horizon Genomics, maintained in Iscove modified Dulbecco medium supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and penicillin-streptomycin, and incubated at 37°C in a 5% CO2 atmosphere. VRK1KO cells (catalog no. HZGHC000073c014) contain a 11-bp deletion in VRK1 exon 5. VRK2KO cells (catalog no. HZGHC000403c006) contain a 7-bp deletion in VRK2 exon 2. African green monkey kidney CV1 cells purchased from Invitrogen Life Technologies, human kidney 293T cells purchased from the American Type Culture Collection (ATCC), and mouse fibroblast L929 cells purchased from ATCC were all maintained in Dulbecco modified Eagle medium supplemented with 10% FBS and penicillin-streptomycin and incubated at 37°C in a 5% CO2 atmosphere.

Viruses and viral infections.

Wild-type vaccinia virus (WR strain), B1-deleted virus (ΔB1) (23), adapted B1-deleted virus (ΔB1mutB12) (31), WRHAB12 (31) virus, D5-mutant Cts24 (39), and E9-mutant Cts42 (40) were used for vaccinia virus infections. These viruses were expanded on BSC40, CV1, or CV1:3XFLAGB1 cells, purified by ultracentrifugation on a 36% sucrose cushion, and stored at −80°C in 1 mM Tris (pH 9). For vaccinia viral DNA accumulation and viral titer determination, confluent monolayers of cells were infected with WT, ΔB1, ΔB1mutB12, ts24, or ts42 virus at a multiplicity of infection (MOI) of 3 PFU per cell at 37°C for 24 h. Cells were harvested into 400 µl of phosphate-buffered saline (PBS). Cells were aliquoted (200 µl for viral DNA accumulation and 200 μl for viral titer determination) prior to downstream assays. For viral yield assays, following cell harvest, cells were pelleted and resuspended in 200 µl of 10 mM Tris (pH 9). Virus samples were freeze-thaw lysed three times and titrated on CV1 or CV1:3XFLAGB1 cells at 37°C with the exception of the 3XFLAGVRK1-overexpressing experiment that was titrated at 31.5°C.

For plaque assays, confluent monolayers of transduced (shBAF, 3XFLAGVRK1, 3XFLAGB1, 3XFLAGm142, 3XFLAGGTPV132, or 3XFLAGEMV152) or transfected (siVRK1) CV1 cells in 35-mm dishes were infected with WT, ΔB1, or ΔB1mutB12 virus at 300 PFU at 37°C for 48 to 72 h.

For protein immunoprecipitations, immunoblotting, and proteomic analysis of vaccinia virus-infected cells, confluent monolayers of cells were infected with vaccinia virus at the indicated MOIs and lengths of time prior to lysate collection.

Cloning of constitutive expression vectors.

pHAGE-HYG-3XFLAGB1 and pHAGE-HYG-3XFLAGVRK2A were described previously (38). pHAGE-HYG-3XFLAGm142, pHAGE-HYG-3XFLAGEMV152, and pHAGE-HYG-GTPV132 were produced by NheI and BamHI restriction digestion of a codon-optimized, 3×FLAG-tagged ORF for each construct (accession numbers: m142, NC_001132.2; EMV152, KJ563295.1; and GTPV132, AY077835.1) and cloning the product into NheI and BamHI digested pHAGE-HYG-MCS vector. pHAGE-HYG-3XFLAGVRK1 was produced through a two-step, overlap PCR. In the first step, 3×FLAG was amplified from a plasmid containing 3×FLAG with the primers F-FLAG (5′-GAGAGGATCCGCCACCATGGACTACAAAGACCATGACGG-3′) that contained a BamHI restriction site at the 5′ end of 3×FLAG and R-FLAG_VRK1 (5′-GCCAACTGACTCTGAAGAATTCATGTCATCGTCATCCTTGTAATCGATGTCA-3′) that contained the 3′ end of 3×FLAG and the 5′ end of VRK1. VRK1 was amplified from the VRK1 ORF with the primers F_FLAG_VRK1 (5′-TGACATCGATTACAAGGATGACGATGACATGAATTCTTCAGAGTCAGTTGGC-3′) that contained the 3′ end of 3×FLAG and the 5′ end of VRK1 and R_VRK1 (5′-GAGAGGATCCTTACTTCTGGACTCTCTTTCTGGTTCTTGAACGGGTCTGT-3′) that contained the 3′ end of VRK1 and a BamHI restriction digest site. In the second step, PCR products from these PCR reactions were combined and amplified into one PCR product (3XFLAGVRK1) with the F-FLAG and R_VRK1 primers, BamHI digested, and ligated into the multiple cloning site of BamHI digested pHAGE-HYG-MCS. The construct was verified by DNA sequencing.

Cloning of inducible B12 expression vector.

pCW57-PURO-HAB12 was produced by amplifying a codon-optimized, HA-tagged B12 ORF using the primers FHAB12 (5′-TAGATAGCTAGCGCCACCATGTATCCCTACGACG-3′) and RHAB12 (5′-ATAAGTGCTAGCTTAGTCCTGGATGAACAGCTTCCGC-3′) into the NheI site of the first multiple cloning site of pCW57-PURO-MCS1-2A-MCS2. The construct was verified by DNA sequencing.

Generation of stable cells expressing VRK1, VRK2, B1, m142, EMV152, and GTPV132.

Lentiviral expression vectors for respective genes were used to generate lentiviruses as described previously (23). For transduction, cells at 25% of confluence were seeded in 35-mm dishes. The next day, cell growth medium was replaced with 1 ml of lentivirus supernatant and incubated for 16 h. The medium was then replaced and incubated for an additional 24 h. Cells were then passaged in medium containing 500 µg/ml (HAP1, VRK1KO, and VRK2KO) or 200 µg/ml (L929 and CV1) of hygromycin to select for stable lentiviral integration. Protein expression was confirmed by immunoblotting using mouse anti-FLAG antibody.

Generation of stable cells expressing inducible B12.

The inducible lentiviral expression vector pCW57 (Addgene, catalog no. 71782) was used to generate lentivirus as described previously (23). For transduction, cells at 25% of confluence were seeded in 35-mm dishes. The next day, cell growth medium was replaced with 1 ml of lentivirus supernatant and incubated for 16 h. Medium was then replaced and incubated for an additional 24 h. Cells were then passaged in medium containing 0.5 µg/ml (HAP1) or 10 µg/ml (L929 and CV1) of puromycin to select for stable lentiviral integration. After selection, the cells were induced for 24 h with 5 µg/ml doxycycline to induce protein expression. Protein expression was confirmed by immunoblotting with mouse anti-HA antibody.

Generation of BAF-depleted cells.

Lentiviruses expressing BAF-specific short hairpin RNA (shRNA) or control shRNA have been described previously (19) and were used for stable depletion of BAF in HAP1, VRK1KO, and CV1 cells. For transduction, cells were seeded in 35-mm dishes. The next day, cell growth medium was replaced with 1 ml of lentivirus supernatant and incubated for 16 h. Medium was then replaced with fresh medium and incubated for an additional 24 h. Cells were then passaged in medium containing 0.5 µg/ml (HAP1 and VRK1KO) or 10 µg/ml (CV1) of puromycin to select for stable lentiviral integration. BAF depletion was confirmed by immunoblotting with a custom rabbit anti-BAF antibody.

DNA purification and qPCR.

Vaccinia viral DNA was extracted using a GeneJET whole-blood genomic DNA purification minikit (Thermo Scientific) according to the manufacturer’s protocol. Vaccinia viral DNA qPCR was performed using iTaq Universal SYBR Green Supermix (BioRad) by following a protocol described previously (66). Serial dilutions were included in each qPCR run to develop a standard curve and determine PCR efficiency of the primer sets in each experiment. qPCR analysis was performed using 10 ng of DNA and a 1 µM concentration of each primer. DNA quantification used the primers FHA (5′-CATCATCTGGAATTGTCACTACTAAA-3′) and RHA (5′-ACGGCCGACAATTATAATTAATGC-3′). For specified experiments using HAP1 lineage cells, GAPDH quantitation was performed using the primers FGAPDH (5′-AGGGCTGCTTTTAACTCTGGT-3′) and RGAPDH (5′-CCCCACTTGATTTTGGAGGGA-3′) for normalization.

siRNA depletion.

Small interfering RNAs (siRNAs) against VRK1 and nontargeting siRNAs were designed and ordered from Dharmacon. The siRNA sequences were as follows: VRK1#2 (5′-GCAAGGAACCUGGUGUUGAUU-3′), VRK1#3 (5′-AGGUGUACUUGGUAGAUUAUU-3′), and nontargeting siRNA (5′-CAGUCGCGUUUGCGACUGGUU-3′). Cells were transfected with 100 nM siRNA using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s protocol. Protein depletion was measured by immunoblot analysis at 3 days posttransfection, and the cells were used for the respective assays.

Immunoblotting.

To evaluate protein expression, cells were harvested, pelleted, and suspended in SDS sample buffer supplemented with protease and phosphatase inhibitors and nuclease (Pierce Universal Nuclease). Whole-cell lysates were resolved on 12% (VRK1, B1, m142, EMV152, GTPV132, HAB12, and tubulin) or 18% (phospho BAF and BAF) SDS-PAGE gels and transferred to polyvinylidene difluoride. The primary antibodies used are as follows: β-tubulin (1:150,000; Sigma, T7816), FLAG (1:5,000; Sigma, F1804), VRK1 (1:500; Santa Cruz, sc390809), VRK1 (1:500; Abcam, ab171933), BAF (1:3,000; custom), phospho BAF (1:1,000; custom), HA (1:1,000; BioLegend, catalog no. 901514), and HA (1:1,000; Cell Signaling, C29F4).

Proteomic analysis of immunoprecipitations.

To determine the protein interactome of B12, confluent monolayers of cells in 100-mm dishes were harvested in cold PBS and pelleted. Cells were lysed with a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1% Triton X-100 and then supplemented with protease and phosphatase inhibitors and nuclease (Pierce Universal Nuclease). After a brief centrifugation to remove insoluble material, B12 and its interacting proteins were pulled down from whole-cell lysates with Dynabead protein G anti-HA magnetic beads (Cell Signaling), eluted in SDS sample buffer supplemented with protease and phosphatase inhibitors by heating at 37°C overnight, separated (5 mm) via electrophoresis on a 12% SDS-PAGE gel, and then subjected to silver staining. Gel pieces corresponding to respective immunoprecipitations were washed with water and destained. Trypsin was added, and digestion was carried out overnight at 37°C. Peptides were extracted from the gel pieces and dried, and the digests were redissolved in 2.5% acetonitrile–0.1% formic acid. Each sample was run by nano-LC-MS/MS using a 2-h gradient on a C18 column (0.075 × 250 mm) feeding into a Q-Exactive HF mass spectrometer. All MS/MS samples were analyzed using Mascot (Matrix Science). Mascot was set up to search UniProt databases: Homo sapiens reference proteome (UP000005640), Chlorocebus sabaeus reference proteome (UP000029965), or Mus musculus reference proteome (UP000000589) and, when applicable, the vaccinia virus strain WR reference proteome (000000344) with added contaminants assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.060 Da and a parent ion tolerance of 10.0 ppm. Deamination of asparagine and glutamine, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine were specified in Mascot as variable modifications. Scaffold software was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at >80% probability by the Peptide Prophet algorithm with a Scaffold delta-mass correction. Protein identifications were accepted if they could be established at >99% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.

Definition of B12 interacting proteins.

For analysis of B12 interactomes, proteins with established identifications of >99% probability and which contained at least two identified peptides were assessed. Within Scaffold, samples containing HAB12 were compared to control samples using the Fisher exact test with the Benjamini-Hochberg multiple-test correction. Significantly enriched proteins from like data sets (infected or uninfected) were then compared. Proteins that were significantly enriched in each experiment within a data set type were considered B12 interactors, whereas proteins that were significantly enriched in only a subset of experiments were eliminated from analysis. Lastly, significantly enriched proteins from infected and uninfected experiments were compared to each other.

Protein immunoprecipitations.

To study the B12 interactome, cells (uninfected or 7 hpi) were harvested in cold PBS and pelleted. Cells were lysed with a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100 and supplemented with protease and phosphatase inhibitors and nuclease (Pierce Universal Nuclease). Following a brief centrifugation to remove insoluble material, B12 and its interacting proteins were pulled down from whole-cell lysates with Dynabead Protein G anti-HA magnetic beads (Cell Signaling), eluted in SDS sample buffer supplemented with protease and phosphatase inhibitors by heating at 95°C for 5 min, and then subjected to protein immunoblotting.

To study the interaction between B12 and 3XFLAGVRK1 and 3XFLAGVRK2A, stably expressing CV1-3XFLAGVRK1 and CV1-3XFLAGVRK2A cells (uninfected or 7 hpi) were harvested in cold PBS and pelleted. Cells were lysed in the above lysis buffer and insoluble material removed by centrifugation. For HA-specific immunoprecipitation, HA-B12 and its interacting proteins were pulled down from whole-cell lysates with anti-HA magnetic beads (Cell Signaling). For FLAG-specific immunoprecipitation, 3×FLAG-tagged proteins and their interacting proteins were pulled down with anti-FLAG magnetic beads (Sigma). Proteins were eluted from beads in SDS sample buffer by heat, denatured by heating at 95°C for 5 min, and subjected to protein immunoblotting.

Statistics.

All experiments were repeated at least in biological triplicate, and graphed data represent the mean of all experimental replicates. Error bars shown represent standard deviations from the mean for viral titer or standard errors of the mean for viral DNA accumulation unless otherwise stated. The P values indicated were calculated using two-tailed Student t tests as appropriate with Prism v8.00 for Windows (GraphPad). Representative immunoblots are shown.

ACKNOWLEDGMENTS

This research was supported through an NIH grant to M.S.W. (R01AI114653). A.T.O. and A.C.L. were supported partly through a training grant (T32AI125207).

The contents of the manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

We thank the University of Nebraska—Lincoln Center for Biotechnology for their help with proteomic methodologies.

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