Nuclear translocation of Sp100A suppresses multiple DNA viruses and limits HSV-1 lytic replication in vivo

Abstract

Sp100 (Speckled protein 100 kDa), a key component of promyelocytic leukemia (PML) nuclear bodies, plays a pivotal role in intrinsic and innate immunity. The predominant isoform, Sp100A, has been shown by our previous studies to shuttle between subcellular compartments to enhance innate immunity against RNA viruses and to circulate between cells via extracellular vesicles (EVs) to restrict herpes simplex virus 1 (HSV-1) spread. This study investigates the biological significance of the cyto-nuclear shuttling of Sp100A, a key component of PML nuclear bodies, in antiviral defense against DNA viruses, particularly herpes simplex virus 1 (HSV-1). We demonstrate that Sp100A effectively inhibits multiple DNA viruses in vitro, with its antiviral activity being critically regulated by phosphorylation at the S188 site (a nuclear import-mimicking mutant, S188D, is active, while the S188A mutant is not). Furthermore, DNA virus infection and type I IFN significantly induce Sp100A secretion via extracellular vesicles (EVs), which confers broad, non–IFN–mediated antiviral protection between cells. In a murine model, Sp100A expression significantly reduced HSV-1 lytic replication and clinical signs, but did not impair latency establishment or reactivation potential. These findings underscore the critical role of Sp100A's dynamic shuttling in antiviral defense, showing its activity is specifically restricted to the lytic phase of HSV-1. Sp100A's multifaceted antiviral properties highlight its potential as a novel therapeutic target for combating DNA virus infections.

Highlights

• •This study highlights Sp100A's multifaceted antiviral properties.
• •Its role in controlling HSV-1 through both nuclear translocation and EV-mediated immune defense.
• •Recombinant HSV-1 viruses expressing Sp100A or Sp100A-S188D showed reduced replication in mouse ganglia.
• •Sp100A hold promise as potential therapeutic agents for DNA virus infections.

Introduction

Despite significant progress in antiviral therapeutics, the rapid evolution or complex recombination among circulating DNA viruses such as herpes simplex virus (HSV), varicella-zoster virus (VZV), and adenovirus, presents ongoing challenges in virology and infectious disease treatment (Norberg et al., 2011, 2015; Krishnan and Stuart, 2021). The complexity of viral replication, latency, and immune evasion underscores the need for novel antiviral approaches (Beachboard and Horner, 2016; Zhu et al., 2023). In particular, the development of broad-spectrum antiviral strategies remains a promising area of research.

One such approach involves the use of extracellular vesicle-based delivery systems for drugs or biologically active components, offering a new avenue for targeting viral infections more effectively. Extracellular vesicles (EVs) are gaining increasing attention as potential delivery systems for antiviral therapies. These naturally occurring lipid bilayer structures, including exosomes and microvesicles, play a key role in cell-to-cell communication and immune modulation (EL Andaloussi et al., 2013; Zhang et al., 2019; Kalluri and Lebleu, 2020; Mukerjee et al., 2024). Recent research has explored their use to deliver biologically active materials, such as antiviral peptides, proteins, small molecules, and RNA, to combat viral infections (Nasiri Kenari et al., 2020; Sil et al., 2020; Van Eijndhoven et al., 2020; Wang et al., 2022). Previous studies have demonstrated that extracellular vesicles (EVs) derived from bone marrow can influence immune cell infiltration in the brain and exhibit antiviral effects against Japanese encephalitis virus (JEV) (Soni et al., 2022). Similarly, EVs released from HSV-1-infected cells can inhibit virus replication in recipient cells through a STING-dependent mechanism (Deschamps and Kalamvoki, 2018). In addition, EVs from cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) can stimulate immune responses via mitochondrial DNA (Jeon et al., 2019). EVs can also act as direct carriers of antiviral molecules. For example, exosomal microRNAs derived from umbilical mesenchymal stem cells (MSCs) have been shown to inhibit hepatitis C virus (HCV) infection (Qian et al., 2016), while miRNA-431-5p-enriched EVs derived from IFN-β-stimulated MSCs potently suppress Zika virus (ZIKV) replication by downregulating CD95 expression (Yuan et al., 2024). For example, EVs loaded with RNA interference molecules or CRISPR-based gene-editing tools have shown promise in silencing viral genes or modifying host factors critical for viral replication. Additionally, EVs can be engineered to carry antiviral drugs, enhancing their bioavailability and prolonging their effects at the infection site. EVs can also act as carriers for immunomodulatory agents, such as cytokines or small molecules that stimulate the host immune system to control viral infections. The ability of EVs to carry and deliver these agents offers a novel approach to managing both acute and latent viral infections. Though EV-based antiviral strategies are still in the early stages of development, but they offer a promising, versatile platform for targeted therapy.

Speckled protein 100 kDa (Sp100) is an integral component of the multifunctional promyelocytic leukemia protein (PML) nuclear bodies (NBs), also known as ND10, involved in regulating both intrinsic and innate immunity (Szostecki et al., 1990). There are at least two IFN-responsive elements in the promoter region of Sp100: the ISRE (-ACTTTCACTTCTCT-) and GAS (-TTCCAGGAA-) domains (Guldner et al., 1992; Grotzinger et al., 1996), Consequently, its expression is robustly upregulated in response to diverse viral infections. The Sp100 gene encodes several isoforms, including the major isoform Sp100A and three longer variants-Sp100B, Sp100C, and Sp100HMG-generated through RNA splicing of a single primary transcript (Negorev et al., 2009). These isoforms share a common N-terminal domain of 477 amino acids (aa), which includes a PML NBs targeting signal (29–152aa), a dimerization (DD) domain (1–182aa), a heterochromatin protein 1 (HP1) binding domain (287–334aa), a transcription activation domain (333–407aa), and a nuclear localization signal (NLS) sequence (444–450aa) (Szostecki et al., 1990; Xie et al., 1993; Bloch et al., 1996; Sternsdorf et al., 1999; Negorev et al., 2001). They differ in their C-terminal regions (Seeler et al., 1998; Seeler et al., 2001; Negorev et al., 2009). Sp100A is the shortest isoform lacking the C terminal domain and is believed to interact with DNA through association with other chromatin-binding proteins such as HP1 and Bright (Surdo et al., 2003; Negorev et al., 2009). Studies have demonstrated that Sp100A inhibits viral replication through multiple distinct mechanisms (Everett and Chelbi-Alix, 2007). During HSV-1 infection, Sp100A acts in concert with PML to suppress viral immediate-early gene expression and restrict the replication of ICP0-deficient viral strains (Everett et al., 2006, 2008). Conversely, HSV-1 counteracts this host defense via its ICP0 protein, which disrupts nuclear body structures and antagonizes Sp100A SUMOylation (Negorev et al., 2006). Additionally, Sp100A represses human cytomegalovirus (HCMV) promoter activity by modulating chromatin states (Newhart et al., 2013); however, this suppression is counteracted by the HCMV immediate-early protein IE1, which promotes viral gene expression by degrading Sp100A through the ubiquitin-proteasome pathway (Kim et al., 2011; Liu et al., 2017). In adenovirus and human papillomavirus (HPV) infections, Sp100A restricts viral replication foci formation and gene transcription (Berscheminski et al., 2014; Stepp et al., 2017; Ip et al., 2023). Importantly, the antiviral activity of Sp100A is not limited to the nucleus: cytosolic Sp100A responds to interferon (IFN) signaling and promotes activation of interferon-stimulated genes (ISGs), thereby enhancing broad-spectrum antiviral responses (Negorev et al., 2009). However, several viruses, including Epstein-Barr virus (EBV) and adenovirus, evade host immune defenses by interfering with Sp100A function, either by disrupting its SUMOylation or inducing its degradation (Seeler et al., 2001; Ryabchenko et al., 2023). For instance, adenovirus E1B–55K protein mediates the degradation of Sp100A and other host restriction factors via a SUMO-dependent pathway (Ip et al., 2023), whereas EBV utilizes Sp100A as a transcriptional coactivator to promote viral gene expression (Ling et al., 2005). In summary, Sp100A restricts viral invasion through multiple pathways, including chromatin regulation within the nucleus, transcriptional repression, and activation of cytosolic ISG signaling cascades (Stepp et al., 2013).

Previous studies have shown that an unSUMOylated cytosolic Sp100A, escaped targeting by virus encoded protein, was secreted from infected cells into the extracellular space through EVs. During this naturally occurred biological process, EVs carrying Sp100A appear to “arm” neighboring uninfected cells and enhance their resistance to HSV-1, a highly prevalent human pathogen and prototype member of Alphaherpesvirinae, Orthoherpesviridae (Gu and Roizman, 2003; Everett et al., 2009; Perusina Lanfranca et al., 2013; Ma et al., 2022). This discovery suggests that Sp100A may function not only as an intracellular antiviral factor but also as a mediator of extracellular immunity. Previous studies conducted by our team have demonstrated that Sp100A plays a crucial role in IFN-mediated responses against RNA viruses, with its nuclear-cytosolic shuttling being meticulously regulated by IFNβ signaling. Specifically, Sp100A undergoes phosphorylation at the Ser188 site in the cytoplasm in response to IFNβ, which facilitates its nuclear import and enhances the transcriptional activation of antiviral interferon-stimulated genes (ISGs) such as RIG-I, IFI16, and OAS2, thereby curbing RNA virus replication. In contrast, the Sp100A-S188A mutant, which is unable to be phosphorylated and translocate to the nucleus, exhibits no antiviral activity (Dong et al., 2022). This dynamic localization is thought to be essential for its antiviral function against RNA viruses, but its exact role in antagonizing DNA virus infections remains unclear.

While these findings provide valuable insights, several critical questions remain unresolved. These include the broader antiviral properties of Sp100A against other DNA viruses, the coordination of its cyto-nuclear dynamics in facilitating extracellular vesicle (EV)-mediated intercellular communication during HSV-1 lytic replication, and whether Sp100A plays a role in the HSV-1 latency cycle. To address these questions, this study investigates whether Sp100A exerts similar antiviral effects against other DNA viruses including HSV-1, HSV-2, VZV, and adenovirus, utilizing both in vitro studies in cultured cells and in vivo analyses in a murine model of HSV-1 infection and whether its EV-mediated communication relies on cyto-nuclear shuttling to mount an effective antiviral defense. We also explore the potential for EV-mediated transfer of Sp100A to uninfected cells and demonstrate that Sp100A co-fractionates with EVs can protect neighboring cells from viral infection, highlighting their potential as a novel antiviral strategy. These findings deepen our understanding of Sp100A's role in innate immunity and lay the groundwork for future therapeutic applications targeting DNA virus infections.

Results

Sp100A has a broad restrictive effect against multiple DNA viruses

To mutate the 188th amino acid (aa) of Sp100A, the codon sequence TCT was modified as shown in Fig. 1A to generate Sp100A-S188A and Sp100A-S188D, respectively. Sp100A, Sp100A-S188A, and Sp100A-S188D were expressed at comparable levels in transiently transfected HEp-2 cells (Fig. 1B). Previous work demonstrated that Sp100A significantly inhibits HSV-1(F), a laboratory strain, in both HEp-2 and Sp100 knockout (Sp100^−/−) cells (Ma et al., 2022). To determine whether this antiviral activity extends to circulating herpes simplex viruses, two clinical strains (HSV-2-c1 and HSV-2-c2) were isolated from patient genital swabs, plaque-purified, amplified in HEp-2 cells for limited passages, and serotyped by sequencing (Supplementary Fig. S1). These strains were then tested in Sp100A-overexpressing cells in both HEp-2 and Sp100^−/− backgrounds, with GFP-overexpressing cells as controls. Sp100A exhibited distinct antiviral activity in Sp100^−/− cells compared to GFP controls (Fig. 1C and D). Next, the role of Sp100A nuclear translocation during HSV-2 infection was assessed by measuring multi-cycle growth kinetics of HSV-2-c1 and HSV-2-c2 in Sp100^−/− cells transfected with plasmids encoding GFP, Sp100A, Sp100A-S188A, or Sp100A-S188D. Plateaued virus titers were compared to those from Sp100A-S188A-expressing cells at 48–72 h post-infection (hpi) for HSV-2-c1 and HSV-2-c2. While Sp100A-S188A was less potent than Sp100A and Sp100A-S188D against clinical HSV-2 isolates, it still showed greater inhibition on the virus than GFP (Fig. 1E).

Fig. 1.

Sp100A-mediated broad anti-DNA virus responses in vitro. A Schematics of functional domains of Sp100A and Ser 188 related mutagenesis strategies. B Expression of GFP, Flag-Sp100A, Flag-Sp100A-188A, and Flag-Sp100A-188D proteins in HEp-2 cells. HEp-2 cells were transfected with plasmids encoding GFP, Flag-Sp100A, Flag-Sp100A-188A, or Flag-Sp100A-188D for 24 h, followed by immunoblotting with anti-Flag antibody to confirm protein expression. C HEp-2 cells were transfected with GFP and Flag-tagged Sp100A plasmids for 24 h, then infected with HSV-2 at a multiplicity of infection (MOI) of 0.01. Viral titers were assessed by plaque assay at 48 h post-infection (hpi). Statistical analyses were performed using Student's t-test. D Sp100 knockout (Sp100^−/−) cells were transfected with GFP and Flag-tagged Sp100A for 24 h, then infected with HSV-2 at an MOI of 0.01. Cell-associated viral titers were quantified by plaque assays at 48 and 72 hpi. Statistical analyses were performed using Student's t-test. E Multicycle growth kinetics of two HSV-2 strains from distinct clinical isolates were assessed in Sp100^−/− cells infected at an MOI of 0.01, with cell-associated viral titers measured by plaque assay. Statistical significance was determined by comparison with the GFP group. Statistical analyses were performed using two-way ANOVA. F, G Sp100^−/− cells were transfected with GFP, Flag-Sp100A, Flag-Sp100A-188A, and Flag-Sp100A-188D for 24 h, then infected with VZV at an MOI of 0.01. Cell-associated viral titers (F) were determined by plaque assay at 48 hpi. The expression level of VZV ORF40 (G) in infected cells was quantified using qRT-PCR, normalized to GAPDH as described in material and methods. Statistical analyses were performed using one-way ANOVA. H 293A cells were transfected with pcDNA3.1, Flag-Sp100A, Flag-Sp100A-188A, or Flag-Sp100A-188D plasmids for 24 h, then infected with ADV-mCherry at an MOI of 0.01. mCherry-positive cells were analyzed by flow cytometry. Statistical analyses were performed using one-way ANOVA. I Quantitative qRT-PCR was performed to assess mCherry levels in these cells. The results were calculated as described in G. Statistical analyses were performed using one-way ANOVA. All experiments were performed in triplicate. Data presented as mean ± SD. ∗∗P < 0.01; ∗∗∗∗P < 0.0001; ns, not significant.

The antiviral activity of Sp100A and its mutants was further tested against the vaccine strain of Varicella-Zoster Virus (VZV). Both cell-associated plaque-forming units (CFUs) and viral genome concentrations in infected cells were measured. For VZV, disruption of Sp100A nuclear translocation (via S188A mutation) abolished its inhibitory effect, rendering Sp100A-S188A-expressing cells indistinguishable from GFP controls. In contrast, Sp100A and Sp100A-S188D suppressed viral amplification in Sp100^−/− cells (Fig. 1F, G).

To evaluate antiviral activity against a broader range of DNA viruses, a recombinant adenovirus carrying an mCherry reporter was tested in cells overexpressing pcDNA3.1, Sp100A, Sp100A-S188A, or Sp100A-S188D. Metrics included the percentage of infected cells, average viral protein expression per cell, and viral reporter transcript levels per cell (Fig. 1H and I). Similar to VZV, phosphorylation of Ser188—a post-translational modification (PTM) critical for nuclear translocation—was essential for Sp100A's anti-adenoviral activity.

In all, Sp100A exhibits broad antiviral activity against HSV-2, VZV, and adenovirus, primarily through Ser188 phosphorylation-dependent nuclear translocation.

Extracellular vesicles mediated Sp100A delivery restrict both DNA and RNA virus infection

Previous studies on Sp100A and HSV-1 have demonstrated that Sp100A, initially identified as a nuclear-localized protein, is actively secreted into the extracellular space by HSV-1-infected host cells. This secreted Sp100A appears to “arm” neighboring cells against incoming viral infections (Ma et al., 2022). A key question arising from these findings is whether the specific post-translational modification of the 188th serine residue (S188) of Sp100A influences its role in establishing an antiviral defense state in surrounding uninfected cells.

Our previous studies showed that upon type I interferon stimulation or viral infections, cytoplasmic Sp100A is actively translocated into the nucleus in a manner dependent on the phosphorylation of serine at position 188 (Dong et al., 2022). However, under unstimulated or merely transfected conditions, mutations at the 188th amino acid of Sp100A did not alter its overall distribution across subcellular and extracellular compartments (Fig. 2A). Second, the extracellular secretion of Sp100A demonstrated pronounced responsiveness to specific cellular stimuli, including infection with HSV-1, HSV-2, VZV, and adenovirus, as well as treatment with poly (dA:dT) or type I interferon. In contrast, no significant response was observed upon RNA virus infection (VSV), bacterial infection (E.coli.), or stimulation with poly (I:C) or LPS (Fig. 2B). Finally, extracellular vesicles (EVs) co-fractionated with Sp100A or its mutants were readily internalized by recipient cells (Fig. 2C). While EVs containing Sp100A or Sp100A-S188D exhibited mild, yet statistically significant effects against both a representative DNA virus (HSV-1) and a representative RNA virus (VSV), EVs derived from Sp100A-S188A-transfected HEp-2 cells failed to confer protection against VSV infection in recipient cells (Fig. 2D–F).

Fig. 2.

Extracellular vesicle-associated Sp100A mediates intracellular antiviral responses against DNA and RNA viruses. A HEp-2 cells were transfected with GFP, Flag-Sp100A, Flag-Sp100A-188A, and Flag-Sp100A-188D plasmids for 48 h. Cytosolic and nuclear fractions, along with culture medium, were collected at specified time points and extracellular vesicles ranging in sizes between 30 and 200 nm in diameter were isolated using a commercial exosome extraction kit according to the manufacturer's protocol. Aliquots (40 μL of 600 μL cytoplasm, 40 μL of 200 μL nuclei and 20 μL of 150 μL extracellular fractions) were immunoblotted with anti-Flag antibodies, using histone, GAPDH, calnexin, and TSG101 as markers for sample origin and purity. B HEp-2 cells were mock-infected or transfected with Poly (dA:dT) or Poly (I:C) for 48 h, infected with HSV-1, VSV, VZV, or Adenovirus at an MOI of 0.1 for 48 h, or infected with E. coli at an MOI of 300 for 48 h, or treated with LPS (2 μg/mL), or treated with IFN-β (1000 U/mL). Exosomes were isolated and immunoblotted with anti-Sp100 antibodies, with TSG101 as a loading control. C Sp100^−/− cells were incubated with exosome-enriched fractions for 2 h, followed by incubation with fluorophore-conjugated anti-Sp100 antibodies. D Sp100^−/− cells (4 × 10⁵) incubated with extracellular vesicles (EVs) were infected with 4000 PFU of HSV-1 at 37 °C for 2 h. E, F Sp100^−/− cells (4 × 10⁵) incubated with EVs were infected with 40,000 PFU of VSV-GFP at 37 °C for 2 h. Viral genome levels were assessed by qRT-PCR, normalized to GAPDH as described in material and methods. Viral titers were assessed by plaque assay in these VSV-GFP-infected Sp100^−/− cells. G–I Artificial vesicles were isolated from Sp100A and mutants-transfected HEp-2 cells according to the protocol in the Materials and Methods section. These vesicles were imaged with a JEM-1400 flash electron microscope (JEOL Ltd., Japan) and quantified by nanoparticle tracking analysis (NTA). J Sp100^−/− cells (4 × 10⁵) incubated with artificial vesicles were infected with 4000 PFU of HSV-1 at 37 °C for 2 h. K, L Sp100^−/− cells (4 × 10⁵) incubated with artificial vesicles were infected with 40,000 PFU of VSV-GFP at 37 °C for 2 h, and viral genome levels were assessed by qRT-PCR as described in F. Viral titers were assessed by plaque assay in these VSV-GFP-infected Sp100^−/− cells. Data presented as mean ± SD. Statistical analyses were performed using one-way ANOVA. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

To further explore the potential of Sp100A-associated EVs as an antiviral strategy, artificial EVs enriched with GFP, Sp100A, or its mutants (Sp100A-S188A or Sp100A-S188D) were generated from HEp-2 cells. These EVs were quantified, and their average sizes were examined using scanning electron microscopy (Fig. 2G, H, I). The detailed methodology for preparing artificial EVs from transiently transfected cells is described in the Materials and Methods section. Naïve HEp-2 cells were incubated with these prepared EVs and subsequently superinfected with HSV-1 or recombinant VSV-GFP at a low multiplicity of infection. Measurements of cell-associated HSV-1 titers (Fig. 2J), VSV genome copy numbers (Fig. 2K), and extracellular VSV-GFP virus titers (Fig. 2L) revealed that artificial EVs containing Sp100A and Sp100A-S188D mediated, though relatively moderate, repeatable and consistent antiviral effects compared to EVs containing GFP or Sp100A-S188A. It should be noted that at the current stage, the inhibitory effect mediated by EV-associated Sp100A was generally mild, future studies on how to harness and potentiate this innate system for therapeutic benefits are warranted.

Interestingly, the antiviral activity of Sp100A and Sp100A-S188D-enriched EVs did not correlate with transcriptional regulation of IFNβ or antiviral ISGs in recipient HEp-2 cells, as initially hypothesized. In fact, cells incubated with EVs containing GFP, Sp100A, or its mutants showed no significant differences in mRNA levels of IFNβ, IFI16, or ISG15, in contrast to the direct overexpression of these proteins in HEp-2 cells (Dong et al., 2022) (Supplementary Fig. S2A). This could at least be partially explained by the relative low level of Sp100A proteins that were received through the EV-delivery method in comparison with direct overexpression in cells.

These findings collectively suggest that Sp100A, particularly when modified at the S188 residue, plays a critical role in enhancing antiviral defenses through an extracellular vesicle-mediated mechanism that operates outside canonical IFN signaling pathways.

Sp100A nuclear translocation is required for its inhibition on HSV-1 in cells

To comprehensively investigate the restrictive effect of Sp100A and its mutants on HSV-1 during both in vitro and in vivo infections, recombinant HSV-1 viruses expressing Sp100A (rHSV-1-A), Sp100A-S188A (rHSV-1-A_S188A), or Sp100A-S188D (rHSV-1-A_S188AD) were constructed using a previously established HSV-1 BAC system and TK repaired in TK^−/− Vero cells. Expression of Flag-tagged wild-type Sp100A and its 188th amino acid mutants, as well as successful TK repair, were confirmed by immunoblot (Fig. 3A). The single- and multi-cycle growth kinetics of these recombinant viruses were assessed in both interferon-competent HEp-2 cells and IFN production-deficient Vero cells (Fig. 3B–E). At a low multiplicity of infection (MOI), cell-associated HSV-1 titers plateaued at 24–48 h post-infection (hpi) in both HEp-2 and Vero cells. Specifically, at 48 hpi in HEp-2 (Fig. 3B) and 24 hpi in Vero cells (Fig. 3D), rHSV-1-A_S188A (green line) reached a titer similar to that of rHSV-1 (blue line), both significantly higher than rHSV-1-A (red line). This suggests that the mutation of the 188th amino acid from serine (S) to alanine (A) abolished the protein's ability to restrict HSV-1. Consistent with this, changing the 188th amino acid from S to aspartic acid (D), mimicking a phosphorylation modification, enhanced the protein's inhibitory effect on HSV-1. When the initial infection ratio was increased to an MOI of 5, the differences between the recombinant viruses, though minimized, remained significant in HEp-2 and Vero cells (Fig. 3C and E).

Fig. 3.

Modulation of viral replication by Sp100A mutations on HSV-1. A HEp-2 cells were mock infected, or with the indicated viruses at an MOI of 5 for 6 h. Protein expression was evaluated by immunoblotting with anti-Flag, anti-β-actin, anti-TK, anti-ICP0, and anti-ICP8 antibodies. Note that the target proteins (Flag, TK, ICP0, and ICP8) were detected on separate blots. β-actin was used as a loading control and was run on a parallel blot using the same batch of protein lysates and identical loading amounts to confirm equal sample loading. B, C Growth kinetics of the indicated viruses in HEp-2 cells at MOIs of 0.01 (B) and 5 (C). D, E Growth kinetics of the indicated viruses in Vero cells at MOIs of 0.01 (D) and 5 (E). Data in Fig. 3B–E, statistical comparisons of viral titers were performed between group rHSV-1 and groups rHSV-1-A, rHSV-1-A_S188A and rHSV-1-A_S188D, as well as between group rHSV-1-A_S188A and groups rHSV-1-A and rHSV-1-A_S188D. Statistical analyses were performed using two-way ANOVA. F Plaque size comparison of the indicated viruses in HEp-2 and Vero cells. G, H Plaque size of each virus was quantified from 50 plaques using ImageJ and plotted. The data were normalized to the mean plaque size of the rHSV-1-A_S188A group. Statistical analyses were performed using one-way ANOVA. Data presented as mean ± SD. ∗∗∗∗P < 0.0001; ns, not significant.

The recombinant viruses were plated onto HEp-2 and Vero cells side by side with rHSV-1 to investigate their cell-to-cell spreading ability and the plaque sizes were imaged, measured and plotted. As shown in Fig. 3F and G, the average plaque size formed by rHSV-1-A and rHSV-1-A_S188D was significantly smaller than that of rHSV-1 and rHSV-1-A_S188A in both cell lines. Although previous studies, including earlier work from our group, demonstrated that Sp100A is actively secreted into the extracellular space via EVs to exert antiviral functions, these results suggest that the restrictive effect of exogenously expressed Sp100A and Sp100A-S188D from the HSV-1 genome may be primarily mediated by direct cell-to-cell spread.

High levels of Sp100A impeded HSV-1 replication and ameliorated disease severity in mice

To evaluate the antiviral role of Sp100A and its mutants in vivo, BALB/c mice were ocularly infected with recombinant HSV-1 viruses carrying Sp100A variants. Mice infected with rHSV-1 or rHSV-1-A_S188A exhibited high mortality within the first two weeks post-infection, and the survival curves revealed distinct outcomes among the groups (Fig. 4A). All infected mice experienced weight loss during the first 7–10 days post-infection (dpi), followed by gradual recovery (Fig. 4B). Notably, mice infected with rHSV-1-A (red line, Fig. 4B) showed minimal weight loss during early infection (1–7 dpi) and recovered significantly faster, reaching a body weight comparable to the mock group by 20 dpi. In contrast, mice infected with rHSV-1-A_S188A or rHSV-1 exhibited similar and more severe weight loss patterns, with delayed recovery to initial weight (IW) until 14 dpi. Mice infected with rHSV-1-A_S188D displayed intermediate weight recovery, performing slightly better than rHSV-1-infected mice at 10 and 14 dpi.

Fig. 4.

Impacts of Sp100A and its variations on pathogenesis of HSV-1 in mice. A BALB/c mice were infected with the indicated virus at 1 × 10⁵ PFU/eye. Survival curves were plotted as above. B Mice were weighed at the indicated days post infection (dpi). The weight of individual mouse was normalized to its initial weight at the time of infection (IW). P values in the chart were calculated using two-way ANOVA. C Periocular area of Mock or virus infected mice was recorded at 5, 10, and 14 dpi and representative images were shown. D, E Eye dryness was assessed by Phenol red thread test at 5, 10, and 14 dpi. Representative results were shown in (D) and total thread wetting results were shown in (E). Statistical analyses were performed using two-way ANOVA. F Viral DNA levels in the trigeminal ganglia of infected mice (n = 3) were quantified by qRT-PCR at the indicated time using primers targeting ICP27 gene. The results were normalized to Adipsin and calculated as described in material and methods. G HSV-1 DNA level in the trigeminal ganglia of infected mice (n = 8) at 7 dpi was quantified by qRT-PCR. HSV-1 genomes were normalized to Adipsin and the relative expression levels were calculated as described in E. Statistical analyses were performed using one-way ANOVA. Data in Fig. 4B are presented as mean ± SD, whereas data in Fig. 4F and G are presented as median with interquartile range (IQR). ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

As shown in Fig. 4C, mice infected with rHSV-1 or rHSV-1-A_S188A developed visible signs of infection by 5 dpi, including corneal cloudiness, swollen eyelids (leading to partial or complete eye closure), and periocular hair loss, which persisted until 14 dpi. In contrast, mice infected with rHSV-1-A or rHSV-1-A_S188D showed no distinguishable clinical symptoms compared to mock-infected mice. No neurological symptoms, such as seizures, were observed in any group. To assess tear production—a clinically relevant symptom of herpetic keratoconjunctivitis sicca—phenol red thread test was performed at 5, 10, and 14 dpi. Representative images of tear-wetted cotton strips and quantified wetting lengths are shown in Fig. 4D and E, respectively. While rHSV-1-A and rHSV-1-A_S188D-infected mice exhibited tear production levels comparable to mock-infected mice, rHSV-1 and rHSV-1-A_S188A-infected mice showed severe impairment in tear production as early as 5 dpi, persisting through 14 dpi (Fig. 4E).

To assess viral replication, mice were sacrificed at 1, 7, and 14 dpi, and HSV-1 genome levels in the ganglia were quantified by qPCR. Consistent with previous reports, HSV-1 replication in the ganglia peaked at 7 dpi and declined thereafter, stabilizing from 14 dpi onward (Fig. 4F). Initial experiments revealed variations in viral genome copies at 7 dpi, but statistical power was insufficient to confirm these differences due to the limited sample size. To enhance the robustness and statistical reliability of the findings, a larger cohort of mice (n = 8 per each group) was subsequently infected, and viral loads in the ganglia were re-evaluated at 7 dpi. As shown in Fig. 4G, rHSV-1 and rHSV-1-A_S188A replicated at significantly higher levels (approximately 10-fold) compared to rHSV-1-A and rHSV-1-A_S188D at 7 dpi.

In summary, Sp100A and its nuclear translocation-mimicking mutant, Sp100A-S188D, strongly inhibited HSV-1 replication in vivo, as evidenced by significantly reduced viral loads in the ganglia and milder clinical symptoms in the eye. In contrast, the nuclear translocation-incompetent mutant, Sp100A-S188A, failed to restrict viral replication or disease progression, mirroring the effects of wild-type rHSV-1. These findings highlight the critical role of Sp100A's cytosolic-to-nuclear shuttling capability in controlling HSV-1 infection in vivo, providing valuable insights for the development of antiviral strategies targeting this mechanism.

On-site expression of Sp100A has no impact on HSV-1 latency establishment nor reactivation

A series of experiments were conducted to investigate whether Sp100A plays a role in HSV-1 latency. HSV-1 in the ganglia enters latency around 14 days post-infection (dpi). Therefore, HSV-1 genome levels were assessed in ganglia post 14 dpi, the results are shown in Fig. 5A. Intriguingly, in contrast to its impact during the lytic replication phase, no discernible impact on viral genome levels were observed across the four experimental groups during latency establishment. To assess HSV-1 reactivation, mice were euthanized at 28 dpi, a time point when viral latency is considered fully established in mice, and ganglia were harvested for explant reactivation assays following established protocols (Du et al., 2011). In brief, paired ganglia from each mouse were processed as follows: one ganglion was cultured with anti-NGF for 24 h (referred to as anti-NGF in Fig. 5), while the contralateral ganglion was immediately frozen at −80 °C (referred to as 0H in Fig. 5). After 24 h, total DNA and RNA were extracted from both anti-NGF and 0H samples, and viral DNA levels (Fig. 5B and C) as well as transcription levels of the immediate-early gene ICP27 (Fig. 5D and E) were quantified by qPCR. In all groups, successful reactivation of latent virus was achieved, with viral genome copy numbers increasing 10- to 100-fold, accompanied by robust transcriptional activation of HSV-1 lytic genes. When the reactivated samples were normalized to their corresponding 0-h baseline values, no significant differences in the fold change of viral DNA levels or ICP27 transcription levels were observed across the experimental groups (Fig. 5C–E).

Fig. 5.

Reactivation dynamics of HSV-1 carrying Sp100A mutations. A HSV-1 DNA level in the trigeminal ganglia of infected mice (n = 8) at 14 dpi was quantified by qRT-PCR using primers targeting ICP27. The results were normalized to Adipsin and calculated as described in material and methods. B–E HSV-1 infected mice were sacrificed at 30 dpi and the trigeminal ganglia were samples immediately (0 h, 0hr samples) or cultured in 199V medium supplemented with anti-NGF at 2 μg/mL for 24 h (anti-NGF samples). Total DNA and total RNA were extracted from 0H and anti-NGF samples. Viral DNA levels, viral transcript levels were quantified using qRT-PCR primers targeting ICP27. In Fig. 5C and E, fold change was calculated as the ratio of gene expression levels in anti-NGF-treated samples (anti-NGF samples) to those in samples collected before anti-NGF treatment (0hr samples). DNA readings were normalized to Adipsin and RNA readings were normalized to Map2. The relative expression levels were calculated as in A. Data in Fig. 5B and D are presented as individual data points (scatter plots), whereas all other data are presented as median with IQR. Statistical analyses were performed using one-way ANOVA. ns, not significant.

These results demonstrate that Sp100A, despite its critical role in suppressing lytic replication, does not influence the establishment, maintenance, or reactivation of HSV-1 latency.

Discussion

In this study, we explored the antiviral activity of Sp100A and its phosphorylation-dependent variants against multiple DNA viruses including HSV-1, HSV-2, VZV, and adenovirus. First, we confirmed that Sp100A overexpression in HEp-2 cells robustly restricts HSV-1 replication, consistent with previous reports (Ma et al., 2022). This inhibitory effect was further confirmed against two clinical HSV-2 strains, which showed a marked reduction in viral titers in Sp100^−/− cells overexpressing Sp100A, but not in control GFP-expressing cells (Fig. 1C and D). Additionally, the point mutations in Sp100A (S188A and S188D) indicated that Ser188 phosphorylation enhances antiviral activity, as Sp100A-S188D exhibited stronger inhibitory effects against HSV-2 and VZV compared to the non-phosphorylatable S188A variant (Fig. 1E, F, G). This suggests that the phosphorylation of Ser188 is a critical determinant of Sp100A's antiviral potency. Our findings indicate that Sp100A exhibits broad inhibitory effects against these viruses. Importantly, the mutation of Sp100A at Ser188 (S188A) significantly reduced its antiviral efficacy, suggesting that the phosphorylation of this residue is critical for its function.

The study also expands our understanding of Sp100A's antiviral mechanisms by revealing its potential role in extracellular vesicle (EV)-mediated immune responses, which can extend the antiviral defense to neighboring cells. Sp100A and its phosphorylation-mimicking variant, Sp100A-S188D, were found efficiently co-fractionates with extracellular vesicles and could protect recipient cells from both DNA and RNA virus infections, including HSV-1 and VSV (Fig. 2D, E, F). Interestingly, Sp100A-S188A-containing EVs did not confer protection against VSV, further emphasizing the phosphorylation-dependent mechanism of action for Sp100A's antiviral activity. This observation aligns with previous findings suggesting that Sp100A is actively secreted during HSV-1 infection to confer protection to neighboring cells (Ma et al., 2022). This highlights the potential of Sp100A-loaded EVs as a novel antiviral strategy, which may circumvent viral evasion mechanisms that target interferon responses and offer a broader spectrum of protection against viral infections. It is important to note that the antiviral effect mediated by the natural exosomes we isolated was observed to be modest. This is likely a reflection of their physiological state in vivo. Therefore, the significant, albeit low-level, activity we detected underscores the existence of a previously underappreciated arm of the host antiviral response. This finding provides a compelling rationale for the future development of engineered exosomes enriched with antiviral cargo to achieve a potent therapeutic effect. Further studies are warranted to fully elucidate the precise molecular pathways through which Sp100A-enriched EVs mediate antiviral defense.

The antiviral effects of Sp100A and its variants, previously observed in cell culture, were further validated in a murine model of ocular HSV-1 infection. Overexpression of Sp100A or its phosphomimetic mutant, Sp100A-S188D, reduced viral loads in infected mice by approximately 10-fold or more (Fig. 4G) and significantly alleviated disease severity, as evidenced by better survival rates, reduced weight loss and improved overall clinical manifestations (Fig. 4A, B, C). In contrast, the nuclear translocation-incompetent mutant, Sp100A-S188A, failed to confer similar protection. Remarkably, Sp100A overexpression nearly eliminated hallmark symptoms of HSV-1 infection, including eye dryness, periocular inflammation, and hair loss, in ocularly infected mice (Fig. 4C). These findings highlight the substantial therapeutic potential of Sp100A in managing acute and chronic/recurrent HSV-1 ocular infections and their associated clinical manifestations.

While Sp100A robustly inhibits HSV-1 replication during the acute phase of infection, it has no discernible impact on the establishment, maintenance, or reactivation potential of latent virus in ganglia (Fig. 5A–D). This demonstrates that Sp100A's antiviral activity is confined to the lytic phase of infection. Such a targeted mechanism provides a promising strategy to control active viral replication without disrupting latent viral reservoirs. More notably, an intriguing implication of our findings is that transient HSV-1 replication in mouse ganglia—which drives an approximately 1000-fold increase in viral genome burden during the first week post-infection (Fig. 4F)—maybe a distinct process from the establishment of latency within the same tissue. These results imply that, at least in the murine models, the decision of individual viral genomes—or the proportion of total genomes in the ganglion—to enter latency is independent of the extent of transient viral replication in the same ganglion. This uncoupling of lytic replication and latency establishment suggests a critical regulatory checkpoint in HSV-1 infection dynamics, offering new avenues for understanding viral persistence and developing targeted antiviral therapies.

Material and methods

Cell lines and viruses

HEp-2, HEK293T, 293A, and Vero cells were originally obtained from Prof. Bernard Roizman (the University of Chicago), ARPE-19 cells were obtained from Shanghai Anwei Biotechnology Co., Ltd, which were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Catalog No. 10-013-CVRC, Corning, USA) supplemented with 10% fetal bovine serum (FBS) (Catalog No. 10270-106, Gibco, USA). The Sp100^−/− cell line was used in our laboratory (Xu and Roizman, 2017). HSV-1(F), referred to as HSV-1 in this study, served as the prototype laboratory strain. Recombinant viruses rHSV-1, rHSV-1-A, rHSV-1-A_S188A, and rHSV-1-A_S188D were generated using a bacterial artificial chromosome (BAC)-based system, subsequently rescued, and purified in Vero cells as described previously (Stavropoulos and Strathdee, 1998).

Two clinical HSV-2 strains, provided by the Seventh Affiliated Hospital of Sun Yat-sen University, were characterized by plaque assays and sequencing. All HSV-1 and HSV-2 viruses used in this study were amplified in HEp-2 cells and titrated in Vero cells using plaque assays. The CFU of the vaccine strain VZV was quantified in ARPE-19 cells. Clinical HSV-2 samples were collected at the Seventh Affiliated Hospital of Sun Yat-sen University with a sterile swab from mucocutaneous genital lesions and from previously involved mucocutaneous sites in male patients. After sampling, specimens were transferred to the laboratory on ice for isolation, plaque purification and amplification.

Patient Information: HSV-2-c1 was collected from a 47 years old male, newly diagnosed with genital herpes and HSV-2-c2 was collected from 49 years old male, newly diagnosed with genital herpes. Both the patients were previously healthy.

HSV plaque assay

Infected cells were harvested, subjected to three freeze-thaw cycles, and briefly sonicated to release cell-associated viruses. Titration was performed in triplicate on Vero cells using 10-fold serial dilutions of virus stocks.

VZV plaque assay

Cells were harvested with trypsin, and titers were measured using ARPE-19 cells.

VSV-GFP genomes quantification

Total RNA was extracted from infected cells, and reverse transcription was performed using positive sensed VSV-GFP genome specific primer (5′-ATGGTGAGCAAGGGC-3′). Quantification of VSV-GFP genomes was carried out by qPCR using primers targeting the GFP (F: 5′-AGTCCGCCCTGAGCAAAGA-3′, R: 5′-TCCAGCAGGACCATGTGATC-3′).

Antibodies and drugs

Antibodies and drugs used in this study included the following: rabbit polyclonal anti-Flag antibody (Catalog No. 80010, Proteintech, USA), rabbit polyclonal anti-Sp100 antibody (Catalog No. GTX131569, GeneTex, USA), mouse monoclonal anti-Flag antibody (Catalog No. AB0008; Abways, Beijing, China), mouse monoclonal anti-β-actin antibody (Catalog No. 1000166; Sino Biological, Beijing, China), rabbit monoclonal anti-GAPDH antibody (Catalog No. AB0037, Abways Technology, Beijing, China), mouse monoclonal anti-Histone H3 antibody (Catalog No. 100005-MM01, Sino Biological, Beijing, China), rabbit polyclonal anti-TSG101 antibody (Catalog No. 28283-1-AP; Proteintech; USA), mouse monoclonal anti-calnexin antibody (Catalog No. sc23954, Santa Cruz, USA), mouse monoclonal anti-ICP0 antibody (Catalog No. sc53070, Santa Cruz, USA), mouse monoclonal anti-ICP8 antibody (Catalog No. ab20194, Abcam, UK), rabbit polyclonal anti-TK antibody (custom-made, GenicBio Limited, Shanghai, China), Alexa Fluor 594-labeled goat anti-rabbit IgG (H + L) (Catalog No. 2165334, Invitrogen, USA), goat anti-mouse IgG-HRP (Catalog No. 31430, Invitrogen, USA), goat anti-rabbit IgG (H + L)-HRP (Catalog No. 32460, Invitrogen, USA), and a protease inhibitor cocktail (Catalog No. EO0492, Thermo Scientific, USA).

Plasmids and transient transfection

Flag-tagged human Sp100A was PCR-amplified, cloned into pcDNA3.1 vectors and verified by sequencing. The PCR primers for the Sp100 and mutants were Flag-Sp100-F (5′-AACTTAAGCTTGCCACCATGGACTACAAAGACGATGACGACAAGGCAGGTGGGGGCGGCGACCTGAGCAC-3′) and Sp100A-R (5′-TGTGCTGGATATCCGCCTAATCTTCTTTACCTGACCCTCTTC-3′). JetPRIME (Catalog No. 114-15, Polyplus, France) was used for transfections in HEp-2 and other mammalian cell lines, following the manufacturer's instructions.

Western blotting

Cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS) supplemented with 10 μL/mL protease inhibitor. Protein concentrations were determined using the BCA Protein Assay Kit (Catalog No.GK10009, GLPBIO, USA). Equal amounts of protein were resolved by SDS-PAGE on 10% polyacrylamide gels and transferred onto PVDF membranes.

Membranes were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature, and then incubated with primary antibodies at 4 °C overnight. After washing three times with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Catalog No.34580, Thermo Fisher Scientific, USA) and imaged with a chemiluminescence detection system.

Collection of EVs and preparation of artificial vesicles

Cells (2 × 10⁷) at 50% confluence were transfected with GFP, Flag-Sp100A, Flag-Sp100A-188A, or Flag-Sp100A-188D constructs. At 50 h post-infection (hpi), culture medium was collected and subjected to sequential centrifugation at 300 ×g for 5 min, 2000×g for 20 min, and 10,000 ×g for 30 min. Exosomes were isolated using an exosome precipitation kit (Catalog No. EXOTC10A-1; System Biosciences; USA) and centrifuged for 30 min at 1500 ×g at 4 °C, then resuspended in PBS.

Cells were collected with trypsin, resuspended in PBS with protease inhibitors (PI). The cell suspension was homogenized on ice for 200 strokes. The homogenate was centrifuged at 1000×g for 5 min, and the supernatant was collected. The supernatant was subsequently centrifuged at 3000×g for 5 min, followed by centrifugation at 15,000 ×g for 30 min. The precipitate was resuspended in 1 mL PBS containing PI. Finally, the resuspended solution was passed through 0.8 μm filters twice and 0.45 μm filters seven times to generate artificial vesicles.

Nanoparticle tracking analysis

Extracellular vesicles (EVs) were quantified by nanoparticle tracking analysis (NTA) using a NanoSight NS300 instrument (Malvern, United Kingdom) equipped with a 488-nm laser. NTA 3.3 software was used to analyze vesicle size and concentration.

Transmission electron microscopy

Purified extracellular or artificial vesicles were centrifuged at 15,000 ×g, resuspended in PBS, and adsorbed onto a carbon-coated grid. The samples were negatively stained with 1% uranyl acetate for 1 min, and images were acquired using a JEM-1400 transmission electron microscope (JEOL LTD, Japan).

Subcellular fractionation

Subcellular fractionation was performed as previously described. Briefly, cells (1 × 10⁶) were collected, pelleted at 1000 ×g for 5 min, resuspended in ice-cold Buffer 1 (150 mM NaCl, 50 mM HEPES [pH 7.4], 25 mg/mL digitonin, 10 μL/mL protease inhibitor), incubated at 4 °C for 30 min, and centrifuged at 4600 rpm for 5 min to obtain the cytosolic fraction. Pellets were subsequently washed, resuspended in Buffer 2 (150 mM NaCl, 50 mM HEPES [pH 7.4], 1% [vol/vol] NP-40, 10 μL/mL protease inhibitor) and incubated for 30 min on ice, and centrifuged at 8700 rpm for 5 min to isolate membrane-associated components. Final pellets were resuspended in Buffer 3 (150 mM NaCl, 50 mM HEPES [pH 7.4], 0.5% [wt/vol] sodium deoxycholate, 0.5% [wt/vol] SDS, 1 mM dithiothreitol [DTT], 10 μL/mL protease inhibitor) on ice for 30 min, sonicated briefly, and the resulting solution contained nuclear proteins and membranes.

Immunofluorescence staining and immunoblot analyses

For immunofluorescence, cells grown on slides were washed, fixed, permeabilized in methanol at −80 °C overnight, and blocked in PBS-TBH (10% FBS, 1% bovine serum albumin [BSA], 1 PBS, 0.1% Triton X-100) at room temperature for 30 min and incubated with primary antibodies at appropriate dilutions in PBS-TBH overnight at 4 °C or for 1 h at 37 °C, followed by fluorophore-conjugated secondary antibodies. Slides were mounted with DAPI (Catalog No. 104139, Abcam, UK). Immunoblotting was performed by separating extracts on SDS-PAGE, transferring onto PVDF membranes, blocked with 5% BSA in PBS with 1% Tween 20, incubated with primary and secondary antibodies, followed by chemiluminescent (catalog number 34580, Thermo Fisher, USA) detection.

Flow cytometry

AdV-mCherry-infected 293A cells were collected, washed in PBS, and analyzed using flow cytometry (BECKMAN COULTER, USA).

DNA/RNA extraction, qRT-PCR, and HSV-1 genome quantification

Total DNA/RNA was extracted using an Omega DNA/RNA extraction kit (catalog number R6731-01, USA) according to the manufacturer's protocol. The gene expression level was quantified by quantitative real-time PCR (qRT-PCR) (StepOnePlus, Thermo Fisher, USA) using a SYBR green detection system (catalog number AG11701, Accurate Biology, Hunan, China). Relative DNA expression was quantified by quantitative real-time PCR using a SYBR green detection system. Viral DNA was measured using primers targeting ICP27, and host DNA was quantified using adipsin primers. The following primers were used: ICP27–F (5′-CGGGCCTGATCGAAATCCTA-3′), ICP27-R (5′-GACACGACTCGAACACTCCT-3′), human GAPDH-F (5′-GAAGGTGAAGGTCGGAGTC-3′), and human GAPDH-R (5′-GAAGATGGTGATGGGATTTC), murine Adipsin-F (5′-AGTGTGCGGGGATGCAGT-3′), murine Adipsin-R (5′-ACGCGAGAGCCCCACGTA-3′), murine Map2-F (5′-GCAAGAGCCCAGAGAAACGT-3′), murine Map2-R (5′-GATACACCCCTGCGAGGAG-3′).

Target gene expression was quantified using the 2^−△CT method. The △C_T was calculated by subtracting the C_T of the reference gene (GAPDH, Map2 or Adipsin as indicated in the figure legends) from the C_T of the target gene (△C_T = C_T, _Target - C_T, _Reference). Relative expression levels were expressed as 2^−△CT × 10⁶, where the scaling factor (10⁶) was used to facilitate data presentation and statistical analysis.

Virus infections

Cells at 80%–90% confluence were infected with HSV, VZV or adenovirus at the indicated multiplicity of infection (MOI) in DMEM at 37 °C. Cells were incubated with HSV for 2 h, VZV for 12 h, and adenovirus for 4 h, washed with PBS, and maintained in DMEM containing 1% FBS.

Animal experiments

Six-week-old BALB/c female mice (Bestest Biotechnology Co., Ltd., Zhuhai, People's Republic of China) were infected with recombinant HSV-1 (1 × 10⁵ PFU/eye). On designated days, mice were monitored for weight and tear secretion using phenol cotton thread (Catalog No. PC3017, Jingmin, Tianjin, China). Humane endpoints were observed if weight loss exceeded 25%. Mice were euthanized at specified times, and DNA/RNA from trigeminal ganglia samples was extracted as described. All procedures were approved by the Ethics Committee, School of Medicine, Sun Yat-sen University, and adhered to applicable regulations.

Phenol red thread test

The phenol red thread test was performed to quantify tear production in mice as previously described (Yin et al., 2021). Briefly, mice were gently restrained or anesthetized using inhaled isoflurane to minimize stress and movement during the procedure. A 25-mm-long phenol red impregnated thread with 3-mm bent end thread was carefully inserted into the lower conjunctival sac of the mouse's eye, avoiding contact with the cornea to prevent irritation. The thread was left in place for a standardized period of 20 s, during which tears wetted the strip by capillary action. After removal, the length of the wetted area was immediately measured in millimeters (mm) using calipers or a ruler. All tests were conducted under controlled environmental conditions to minimize variability, and baseline tear production values were established for the specific mouse strain used in the study. The procedure was performed bilaterally, and measurements were repeated to ensure consistency.

Statistical analysis

Data are presented as mean ± standard deviation (SD), analyzed using GraphPad Prism 6.0. Two-tailed unpaired Student's t-test, one-way ANOVA and two-way ANOVA were used to calculate P values. In the figures, P values of 0.05 are marked as “∗”, P values of 0.01 are marked as “∗∗”, P values of 0.001 are marked as “∗∗∗”, and P values of 0.0001 are marked as “∗∗∗∗”.

Data availability

All relevant data are within the paper and its supplementary materials. Additional information or materials are available upon request by contacting the corresponding author.

Ethics statement

The procedures for the care and use of animals were approved by the Ethics Committee of the School of Medicine of Sun Yat-sen University. All applicable institutional and governmental regulations concerning the ethical use of animals were followed.

Author contributions

Yilei Ma: investigation, data curation, writing-review & editing. Weidong Li: investigation, data curation, writing-review & editing. Jialing Li: investigation, data curation. Xiao Zhang: investigation, data curation. Xiuyan Guo: investigation, data curation. Zhu Li: resources. Yunsheng Xu: resources. Pei Xu: conceptualization, data curation, writing-original draft, writing-review & editing.

Conflict of interest

The authors declare that they have no conflict of interest.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Figure S1.

A Representative images of plaque purification of clinical strains-1 of HSV-2 (HSV-2-c1) in Vero cells. B Representative images of plaque purification of clinical strains-2 of HSV-2 (HSV-2-c2) in Vero cells. C, D The gG and gB genes of the two clinical isolates were sequenced and compared to representative HSV-1 and HSV-2 strains.

Supplementary Figure S2.

A Sp100^−/− cells were incubated with purified exosomes for 8 h, and IFN-β, IFI16, ISG15 mRNA levels were quantified by qRT-PCR. B Sp100^−/− cells (4 × 10⁵) incubated with artificial vesicles isolated from HEK293T cells were infected with 40,000 VSV-GFP at 37 °C for 2 h. Viral genome levels were assessed by qRT-PCR, normalized to GAPDH and calculated as described in material and methods. Data presented as mean ± SD. Statistical analyses were performed using one-way ANOVA. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.