All VZV-infected PBMCs  and V-CD8+ showed significant elevations in PD-L1 expression compared to uninfected cells. VZV induced PD-L2 expression in B cells and V-CD8+. Only VZV-infected CD8+ T cells, NKT cells and V-CD8+ upregulated PD-1 expression, the immunoinhibitory receptor for PD-L1/PD-L2. VZV induced CTLA-4 expression only in V-CD8+ and no significant changes in LAG-3 or TIM-3 expression were observed in V-CD8+ or PBMC T cells. To test whether PD-L1, PD-L2 or CTLA-4 regulates V-CD8+ effector function, autologous PBMCs were VZV-infected and co-cultured with V-CD8+ cells in the presence of blocking antibodies against PD-L1, PD-L2 or CTLA-4; ELISAs revealed significant elevations in IFNγ only upon blocking of PD-L1. Together, these results identified additional immune cells that are permissive to VZV infection ; along with a novel mechanism for inhibiting CD8+ T cell effector function through induction of PD-L1 expression.Varicella zoster virus (VZV) is a lymphotropic alpha-herpesvirinae subfamily member that produces varicella on primary infection and causes zoster, vascular disease and vision loss upon reactivation from latency. VZV-infected peripheral blood mononuclear cells (PBMCs) disseminate virus to distal organs to produce clinical disease. To assess immune evasion strategies elicited by VZV that may contribute to dissemination of infection, human PBMCs and VZV-specific CD8 + T cells, NKT cells and VZV-specific CD8+ T cells. Blocking PD-L1 during co-culturing of VZV-specific CD8+ T cells with autologous VZV-infected PBMCs enhanced IFNγ levels almost 2-fold compared to isotype controls. These results indicate that blocking PD-L1 expression during varicella or zoster may restore CD8+ T cell effector function, enabling effective clearance of virus-infected cells to reduce viral spread and multisystem disease.The burden of disease produced by VZV is significant, since 90% of the world population harbors latent virus. At least 50% of infected individuals will reactivate by 85 years of age to develop zoster, which is an established risk factor for stroke and myocardial infarction, as well as multisystem diseases with or without rash. VZV-infected PBMCs disseminate virus to distal organs to produce clinical disease. PD-L1 is an immunoinhibitory protein that interacts with PD-1, its receptor, expressed mainly on T cells to prevent their activation and subsequent clearance of virus- or malignantly transformed cells. We show here that all peripheral blood mononuclear cells have a dramatic induction of PD-L1 expression upon infection with VZV, which is combined with the induction of PD-1 expression on CD8 Varicella zoster virus (VZV) is a lymphotropic, human alpha-herpesvirinae subfamily member that produces varicella (chickenpox) upon primary infection, after which virus establishes latency in ganglionic neurons along the entire neuraxis . With ag+ T cell function during chronic viral infection [The mechanism(s) that facilitates spread of VZV-infected PBMCs without effective immune clearance is unknown; however, upregulation of immunoinhibitory proteins may be important. Therefore, we hypothesized that VZV infection of PBMCs induces immunoinhibitory proteins to evade immune clearance. Such cell surface proteins normally act to halt an immune response after clearance of damaged or infected cells so that healthy cells are not harmed by inflammatory cytokines or activated immune cells –10. For nfection . Finallynfection .+ T cell immune responses [+ T cells [+ and CD8+ T cells, such as PD-1, CTLA-4, TIM-3 and LAG-3 [Cancer- and virus-mediated upregulation of these immunoinhibitory proteins to evade clearance is well-established. Cancer cells induce the expression of immunoinhibitory ligands, such as PD-L1 and PD-L2, in the tumor microenvironment to prevent immune cell activation, enabling progression or metastasis. In that context, therapeutic targeting of immunoinhibitory proteins, mainly PD-1/PD-L1 and CTLA-4, has revolutionized cancer therapy . Herpes esponses . PD-L1 e T cells . In addi T cells . While E T cells . Finallynd LAG-3 –23.While earlier studies showed that VZV infection of human tonsillar T cells induces PD-1 expression  and that+ T cells  and CD8+ T cells were the least permissive to infection   or vaccine strain (vOka) and analyzed 48 h later for expression of VZV surface glycoprotein E (gE) using flow cytometry . The cel: 4–17%) . Monocyt: 4–17%) . Next, N T cells . Results T cells .To confirm cell surface staining of VZV gE, we infected human PBMCs using cell-associated GFP-expressing strain of VZV (VZV-GFP). Aside from B cells being slightly more permissive to VZV-GFP infection than NKT, the preferential infection rates of VZV-GFP were similar to surface staining of VZV gE shown using both Ellen and vOka strains , further2 petri dishes for each time point with VZV-infected HFLs that were infected with identical titers to allow the entire PBMC populations to be harvested and analyzed. Monocytes were 87%, 88% and 71% VZV-gE+ at 24, 48 and 72 hpi with a significant decrease in surface VZV-gE expression from 48 and 72 hpi (P = 0.006)  and harvested PBMCs at 24, 48 and 72 h post infection (hpi) and analyzed for surface VZV-gE expression. PBMCs were co-cultured on separate 10cm= 0.006) . B cells= 0.006) . Interes= 0.006) ; yet no + T cells and CD4+ T cells  which was significantly higher than all other PBMC subsets analyzed aside from B cells . B cells T cells . NK cellhi cells .+ and CD8+ T cells being the least permissive to infection.Taken together, these results indicate that both the VZV Ellen and vaccine strain can infect all immune cell populations in the PBMC pool with preferential infection of monocytes, followed by NK cells, NKT cells and B cells; with CD4+ T cells, CD8+ T cells and HFLs showed nuclear VZV ORF63 expression indicative of productive infection , anti-PD-L2 (αPD-L2) or anti-CTLA-4 (αCTLA-4) blocking antibodies or isotype controls. Since no induction of LAG-3 or TIM-3 expression was observed these immunoinhibitory proteins were not blocked. After 24 h of co-culturing autologous PBMCs with VZV ORF34- or ORF18-specific CD8+ T cells, cell culture supernatants were harvested and analyzed for IFNγ levels using ELISA (+ T cells had significant elevations in IFNγ levels as compared to their uninfected counterparts (P<0.0001). Importantly, only blocking of PD-L1 induced significant elevations in IFNγ levels when compared to isotype controls (P<0.0001 for both). Virtually no changes in IFNγ levels were observed when PD-L2 or CTLA-4 was blocked compared to isotype controls for either VZV ORF34- or ORF18-specific CD8+ T cells co-cultured with VZV-infected PBMCs. Also, cell culture supernatants from autologous PBMCs, from the same donor from whom the VZV-specific CD8+ T cells were derived, co-cultured with uninfected- and VZV-infected HFLs were analyzed for IFNγ levels and virtually no IFNγ secretions were detected during co-culturing with uninfected- or VZV-infected HFLs  or isotype controls for 24 h and cell culture supernatants were harvested and analyzed for IFNγ levels using ELISA. Neither VZV ORF34- nor ORF18-specific CD8+ T cells showed a change in IFNγ levels between isotype and αPD-L1 treatments when co-cultured with uninfected HBVAFs, whereas PD-L1 blockade in both ORF34- and ORF18-specific CD8+ T cells showed significant elevations in IFNγ levels as compared to isotype controls during co-culture with VZV-infected HBVAFs   .+ effector T cell functioning in response to VZV.In summary, blocking only PD-L1 enhanced CD8Herein, we examined the ability of VZV to infect human PBMCs and modulate expression of multiple immunoinhibitory proteins in a cell-type specific manner. Strikingly, VZV productively infected all PBMC subsets and we report here for the first time the productive infection of monocytes, B cells and NKT cells. While VZV has been widely regarded as a T cell tropic virus , a recenDuring primary VZV infection, viral particles are inhaled and the first site of replication is tonsils where it is presumed that tonsillar T cells become infected and subsequently traffic virus to skin promoting the vesicular rash associated with varicella/chickenpox . However+ T cell activation. VZV infection of monocytes would presumably prevent monocyte maturation and subsequent CD4+ T cell activation that is absolutely required for controlling VZV infection along with simian varicella virus (SVV) [The preferential targeting of VZV infection towards innate immune cells (monocytes and NK cells) shown here would have several detrimental implications on the host immune response. First, an adaptive T cell or B cell immune response directed against VZV during primary infection takes approximately 2 weeks . Therefous (SVV) , 38. Intus (SVV) , while aus (SVV) , all of A caveat to the primary infection model of SVV in African green macaques was that the SVV-GFP virus used in these studies was severely attenuated compared to wild-type virus with regards to viral DNA found in PBMCs (approximate 1000-fold decrease) . We also+ T cells. The significant induction in IFNγ secretions during PD-L1 blockade in both VZV-specific CD8+ T cell lines shown here are similar to results in lymphocytic choriomeningitis virus (LCMV)-infected mice, where only anti-PD-L1 blockade and not anti-CTLA-4 rescued CD8+ T cell effector function [+ T cells responsible for killing VZV-infected cells as the infected VZV-specific CD8+ T cells probably do not elicit the same anti-viral immune response as their uninfected counterparts. Further supporting this notion, a bystander effect for PD-1 and CTLA-4 induction was only observed in VZV-specific CD8+ T cells and not in CD8+ T cells from healthy donor PBMCs which would contain <0.01% of VZV-specific CD8+ T cells as previously reported [+ T cell effector function. For example, both VZV-specific CD8+ T cell lines had modest 30–50% inductions in CTLA-4 and PD-1 expression levels in bystander cells compared to uninfected cells. However, VZV-infected PBMCs had a 2- to 14-fold induction in PD-L1 expression levels compared to uninfected PBMCs, which can explain the enhanced IFNγ levels observed when blocking PD-L1 and not CTLA-4. Induction of PD-L2 expression by VZV was minimal in PBMCs compared to PD-L1 levels, which can explain the lack of enhanced IFNγ levels observed when blocking PD-L2.Another striking finding was the induction of PD-L1 expression by VZV in all subsets of human PBMCs examined, along with the enhancement of T cell effector function after blocking PD-L1 in VZV-specific CD8function . We usedreported . Therefo+ T cells from zoster patients when compared to healthy controls [+ T cells that could clearly aid in immune suppression during zoster. VZV-infected PBMCs are present during both varicella and zoster but at a very low rate  [+ T cells, which may potentially amplify immune suppression during varicella or zoster especially considering that monocytes and CD4+ T cells represent the vast majority of immune cells in the PBMC pool (approximately 70–80%).The induction of the PD-1:PD-L1 pathway during viral infection has been well documented and PD-1 expression has been shown to be increased in CD8controls . We repo0 cells)  which wo+ T cell responses [Aside from zoster, VZV reactivation can also produce VZV vasculopathy presenting as stroke, giant cell arteritis and granulomatosis aortitis . Intriguesponses . Results+ T cells and NKT cells could greatly diminish the effectiveness of the immune system in clearing virus. The enhanced CD8+ T cell effector function after PD-L1 blockade during VZV infection shows that VZV-mediated immune suppression can be reversed to attenuate virus dissemination and multisystem disease associated with VZV.Overall, our comprehensive examination of VZV infection in human PBMCs shows that all immune cell populations are permissive for VZV infection. The ability of these immune cells to transmit virus combined with the induction of PD-L1 expression in all PBMCs provides novel immune evasion strategies elicited by VZV infection. Induction of PD-L1 in PBMCs combined with the induction of PD-1 expression on CD82 cell culture dishes. There are 8.0 x 10^6 HFLs/HBVAFs on a confluent monolayer in 10-cm2 cell culture dishes, so approximately 1.25 PBMCs:1 HFL were co-cultured together. To confirm immunoinhibitory protein staining in flow cytometry, PBMCs were cultured alone with- or without treatment with PMA/Ionomycin  for 48 h before harvesting. Polyclonal VZV-specific CD8+ T cell lines that recognize specific epitopes in either VZV ORF34 or ORF18 proteins in the context of HLA A*0201 [+ T cell lines were obtained from the same donor, such that autologous PBMCs were used as antigen presenting cells in some experiments. Polyclonal HSV-2-specific CD8+ T cell lines that recognize specific epitopes in either HSV-2 UL47 or UL49 proteins in the context of HLA-A*0201 (UL47) [Human fetal lung fibroblasts (HFLs)  and human brain vascular adventitial fibroblasts (HBVAFs)  were cultured in basal fibroblast medium supplemented with 2% fetal bovine serum (FBS), 1% fibroblast growth serum and 1% 100X penicillin-streptomycin (PS) (Sciencell). Cells were grown to approximately 80% confluency and then infected with cell-associated viruses (40 pfu/ml) for 48–72 h. The VZV Ellen strain and vOka strains were used along with VZV-GFP that has been previously described . At the A A*0201  were tet1 (UL47)  and HLA-1 (UL47)  were tetFrozen PBMCs from six healthy donors of age 29–50 years were obtained commercially from Astarte Biologics . Whole blood from twelve healthy donors of age 22–40 years were obtained commercially from Bonfils . Informed consent was obtained by the source companies identified above from all adult subjects providing PBMCs and whole blood for this research project; samples were de-identified by the company prior to our receipt.For flow cytometry and FACS sorting experiments, fluorochrome-conjugated antibodies against the following antigens were used: CD45 , CD3 , CD56 , CD8 , CD4 , CD19 , CD14 , HLA-DR , PD-L1 , LAG-3 , TIM-3 , HLA-ABC , Isotype   ; CD4  and PD-L2  ; CTLA-4 , PD-L1 , PD-1  ; VZV gE  conjugated in house to R-PE as previously described . For imm+ T cells were harvested, washed with cold PBS and live/dead aqua-stained on ice as per the manufacturer’s protocol (ThermoFisher). Cells were then resuspended in FACS buffer  with addition of antibodies on ice for at least 30 min. Then, cells were fixed in 1% paraformaldehyde (ThermoFisher) or fixed and permeabilized to stain for intracellular CTLA-4, using fixation/permeabilization kit (ThermoFisher). For FACS staining of PBMCs, after live/dead staining and washing, cells were cultured with human TruStain FcX (Biolegend) and True-Stain Monocyte Blocker (Biolegend) for 5 min on ice per manufacturer’s instructions before the addition of fluorescence-conjugated antibodies. Fluorescence-minus-one (FMO) controls were used for all immunoinhibitory proteins as were isotype controls for VZV gE and CTLA-4 staining. For flow cytometry staining of HBVAFs, cells were harvested with a citrate buffer as previously described [PBMCs or VZV-specific CD8escribed  and thenHuman PBMCs were harvested on ice, washed with FACS buffer and then resuspended in FACS buffer with the addition of antibodies for 30 min on ice. No viability stain was conducted on the sorted cells, however, PBMCs were stained for live/dead aqua as described above before the sorting process to confirm viable cells (All PBMCs sorted were >90% viable). PBMCs were sorted using a FACS-Aria cytometer (BD Immunocytometry Systems). All sorted immune cell subsets were >93% pure based upon flow cytometry analyses. VZV-infected PBMCs were sorted based upon VZV-gE+ along with their lineage markers described in After FACS sorting of PBMCs, individual VZV-infected immune cell subsets  were resuspended in complete RPMI media and added to semi-confluent monolayers of HFLs in 96-well plates. After 3 days of co-culturing, HFLs were washed with PBS and harvested using trypsin (Sciencell) then plated on ibidi 24-well μ-Plate  for an additional 48 h to allow viral infection to spread. In some experiments, sorted PBMCs were washed in citrate buffer  for 3 min, then washed with FACS buffer before co-culturing with HFLs to confirm no potential virus was “stuck” to PBMCs, ensuring that productive viral transmission occurred from VZV-infected PBMCs as previously described , 32. ForHFLs were propagated as described above in an ibidi 24-well μ-Plate (ibidi) and analyzed for immunofluorescence as previously described . BrieflyTotal RNA was extracted from sorted uninfected- and VZV-infected human PBMCs along with from uninfected- and VZV-infected HFLs using the Direct-zol RNA miniprep kit . Enzyme degradation of residual DNA was completed using the Turbo-DNA free kit (ThermoFisher). First strand cDNA synthesis was completed using the Transcriptor first strand cDNA synthesis kit . RNA extraction, DNase treatment and cDNA synthesis were all performed according to the respective manufacturer’s instructions. For q-RT-PCR amplification, primer and probe sets for VZV ORF63 and ORF68 were used as previously described ; along w+ T cells (2.0 x 10^6 cells/well) for 24 h before harvest of cell culture supernatants for IFNγ ELISA analyses. For PD-L1, PD-L2 and CTLA-4 blockade experiments using PBMCs from the same donor from whom the VZV-specific CD8+ T cells were derived, frozen PBMCs were co-cultured with uninfected- or VZV-infected HFLs for 48 h as described above and only non-adherent cells were harvested, washed in FACS buffer, seeded in 96-well plates (1.0 x 10^5 cells/well) and treated with either anti-PD-L1 , anti-PD-L2 , anti-CTLA-4  or isotype control  for 1 h before co-culture with VZV-specific CD8+ T cells (1.0x10^5 cells/well) for 24 h. Cell culture supernatants were then harvested for IFNγ ELISA analyses.For PD-L1 blockade experiments in HBVAFs, HLA A*0201 (+) HBVAFs were grown in 12-well plates at an approximate density of 5.0 x 10^5 cells/well, uninfected- or VZV-infected, treated with anti-PD-L1  or isotype control  for 30 min, and co-cultured with VZV-specific CD8Levels of IFNγ in cell culture supernatants were measured using the Meso Scale Discovery human IFNγ kit  according to the manufacturer’s instructions. IFNγ levels were calculated by reference to a standard curve and all samples were analyzed in duplicate.t-test, repeated measures (RM) one-way ANOVA with the Greenhouse-Geisse correction and Tukey posttest, and ordinary one-way ANOVA with the Tukey posttest.Statistical analysis was performed using GraphPad Prism . Statistical significance was determined using the Student’s unpaired S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S1 Fig-CD56+, NKT = CD3+CD56+, B cells = CD3-CD56-CD19+HLA-DR+, CD4+ T cell = CD56-CD3+CD4+CD8-, CD8+ T cell = CD56-CD3+CD8+CD4-. Live myeloid cells monocytes = CD3-CD56-CD19-CD14hi,HLA-DR+. FSC = forward scatter and SSC = side scatter.After 48-h co-culture of PBMCs with uninfected- or VZV-infected HFLs, cells were harvested on ice, washed with PBS and stained using live/dead aqua followed by cell surface staining before flow cytometry analyses. Flow cytometry gating scheme, were sequentially gated by singlets, FSC/SSC for size, and gated for live/dead aqua-negative (live lymphocytes), followed by cell surface staining for CD3, CD56, CD19, CD14, CD4, CD8 and HLA-DR. NK = CD3(TIF)Click here for additional data file.S2 Fig(A) Representative flow cytometry plots of live monocytes, NK cells, NKT cells, B cells, CD4+ T cells and CD8+ T cells, examining GFP expression. (B) Frequency of live GFP+ monocytes, NK cells, NKT cells, B cells, CD4+ T cells and CD8+ T cells from 5 healthy donors with bar graphs representing average % VZV-GFP+ cells ± SD. *P<0.05, **P<0.01; # above monocytes represents P<0.01 for significant increases in % VZV-GFP+ monocytes compared to all other immune cell populations analyzed. Statistical significance was determined using RM one-way ANOVA with the Greenhouse-Geisser correction and Tukey posttest.Human PBMCs were co-cultured with uninfected- (UI) or VZV-GFP-infected HFLs for 48 h then harvested and analyzed using flow cytometry. (TIF)Click here for additional data file.S3 FigHuman PBMCs were co-cultured with uninfected- (UI) or VZV-infected HFLs (Ellen strain) for 24, 48 and 72 h then harvested and analyzed using flow cytometry. Bar graphs represent average % VZV-gE+ immune cells ± SD. *P<0.05 and **P<0.01 for significant decreases in % VZV-gE+ immune cells compared to various time points analyzed. Results representative of 4 independent experiments using PBMCs from 4 different healthy controls. Statistical significance was determined using RM one-way ANOVA with the Greenhouse-Geisser correction and Tukey posttest.(TIF)Click here for additional data file.S4 Fig+ T cells were co-cultured with uninfected (UI) or VZV-infected HFLs for 48 h then harvested and analyzed using flow cytometry. (A) Representative flow cytometry gating scheme for VZV gE low expressing cells  and VZV gE high expressing cells . (B) Summary of % VZV gE+hi cells in monocytes, B cells, NK cells, NKT cells, CD8+ T cells and CD4+ T cells. (C) Summary of % VZV gE+hi cells in VZV ORF34- or ORF18-specific CD8+ T cells compared to CD8+ T cells from human PBMCs. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; # above monocytes represents P<0.01 for significant increases in % VZV gE+hi cells compared to all other immune cell populations analyzed except for B cells which was not significant. Statistical significance was determined using RM one-way ANOVA with the Greenhouse-Geisser correction and Tukey posttest.Human PBMCs, VZV ORF34- or ORF18-specific CD8(TIF)Click here for additional data file.S5 Fig+ T and CD8+ T cells were sorted using flow cytometry. Individual sorted immune cells were then co-cultured with uninfected HFLs. After 5 days of co-culture, flow cytometry analyses of VZV gE expression in HFLs revealed productive infection of HFLs by all individual immune cell populations analyzed. Negative controls were provided by isotype staining of VZV-infected HFLs. Results representative of PBMCs from 2 healthy donors.Human PBMCs were co-cultured with VZV-infected HFLs for 48 h, then VZV-infected monocytes, NK, NKT, B cells, CD4(TIF)Click here for additional data file.S6 Fig+CD8+ T cells. Black histograms represent untreated PBMCs and red histograms represent PMA/Ionomycin treated PBMCs. Results representative of duplicate experiments from 2 healthy individual PBMCs.Human PBMCs were cultured alone or in the presence of PMA/Ionomycin for 48 h then harvested and analyzed for PD-1, PD-L1, PD-L2, CTLA-4, LAG-3 and TIM-3 expression in live CD3(TIF)Click here for additional data file.S7 Fig+ HBVAFs were uninfected- (UI) or VZV-infected (VZV) for 72 h and then cultured alone or co-cultured with either HLA-A*0201-restricted VZV ORF18 specific CD8+ T cells (ORF18), or HLA-A*0201-restricted HSV-2 UL47-specific CD8+ T cells (UL47) or HLA-B*0702-restricted HSV-2 UL49-specific CD8+ T cells (UL49) for 24 h. In addition, autologous PBMCs from the same donor from whom the ORF18 CD8+ T cells (PBMC) were derived were co-cultured for 24 h with UI- or VZV-infected HBVAFs. Then cell culture supernatants were harvested and analyzed for IFNγ levels using ELISA. Results are representative of 3 independent experiments with bar graphs representing average IFNγ levels (pg/ml) ± SD. # represents P<0.0001 for significant inductions of IFNγ compared to all other experimental conditions. Statistical significance was determined using ordinary one-way ANOVA with the Tukey posttest.HLA-A*0201(TIF)Click here for additional data file.S8 Fig(A) Representative flow cytometry plots of VZV gE and MHC-I expression in VZV-infected HBVAFs stained with isotype for MHC-I (Iso), uninfected HBVAFs (UI) or VZV-infected HBVAFs. VZV gE negative bystander cells (Bys) or VZV gE positive cells (V+). (B) Representative flow cytometry plots for MHC-I mean fluorescence intensity (MFI) expression levels in HBVAFs. Iso = black histograms, UI = grey histograms, Bys = blue histograms and V+ = Red histograms. (C) Summary of MHC-I MFI expression levels in UI, Bys, V+ and Iso stained HBVAFs. Results are representative of three independent experiments. Bar graphs represent average MHC-I MFI levels ± SD. **P<0.01 and ****P<0.0001. # represents P<0.0001 for significant increases in MHC-I MFI levels in UI, Bys and V+ HBVAFs compared to Iso stained HBVAFs. Statistical significance was determined using ordinary one-way ANOVA with Tukey posttest.HBVAFs were uninfected or VZV-infected (Ellen strain) for 72 h then harvested and analyzed for VZV gE and MHC-I expression using flow cytometry. (TIF)Click here for additional data file.S9 Fig+ T cells were co-cultured with uninfected- or VZV-infected HLA-A*0201+ HBVAFs for 24 h in the presence of PD-L1 blocking antibody (αPD-L1) or isotype controls. Then cell culture supernatants were harvested and analyzed for IFNγ levels using ELISA. Results are representative of 3 independent experiments with bar graphs representing average IFNγ levels (pg/ml) ± SD. **P<0.01, ***P<0.001. NS = not significant. Statistical significance was determined using RM one-way ANOVA with the Greenhouse-Geisser correction and Tukey posttest.VZV ORF34- or ORF18-specific CD8(TIF)Click here for additional data file.


Scientific Reports 10.1038/s41598-017-18549-7, published online 09 January 2018Correction to: This Article contains an error in Figure 6 where the white background has been erroneously changed to black, which obscures multiple labels. The correct Figure 6 appears below as Figure


Scientific Reports 10.1038/s41598-018-22131-0, published online 06 March 2018Correction to: In Figure 3A, the image taken at 5 days of germination under salt stress conditions is incorrect. The correct Figure 3 appears below as Figure


SMN1. It is widely posited that defective gene expression underlies SMA. However, the identities of these affected genes remain to be elucidated. By analyzing the transcriptome of a Caenorhabditis elegans SMA model at the pre-symptomatic stage, we found that the expression of numerous nuclear encoded mitochondrial genes and vacuolar H+-ATPase genes was significantly down-regulated, while that of histone genes was significantly up-regulated. We previously showed that the uaf-1 gene, encoding key splicing factor U2AF large subunit, could affect the behavior and lifespan of smn-1 mutants. Here, we found that smn-1 and uaf-1 interact to affect the recognition of 3′ and 5′ splice sites in a gene-specific manner. Altogether, our results suggest a functional interaction between smn-1 and uaf-1 in affecting RNA splicing and a potential effect of smn-1 on the expression of mitochondrial and histone genes.Spinal muscular atrophy (SMA) is a severe motor neuron degenerative disease caused by loss-of-function mutations in the survival motor neuron gene  SMN1)  is a severe congenital motor neuron degenerative disease caused by loss-of-function mutations in the survival motor neuron gene 1 (SMN1) . In addipression . SMN is  elegans .SMN deficiency and subsequent aberrant gene splicing underlie the pathogenesis of SMA  . snRNPs s of SMA . HoweverSMN may interact with numerous genes in Drosophila and C. elegans  and small subunit (UAF-2) interact with each other to bind the pyrimidine tract preceding the 3′ splice site and the conserved 3′ splice site  begins with the recognition of 5′ splice sites by the U1 snRNP and of 3′ splice sites by the U2AF large and small subunits . Specifiice site .C. elegans  that causes conditional lethality at higher temperatures and affects alternative splicing (uaf-1 interacts with smn-1 found that this uaf-1 mutation (n4588) and a derived uaf-1 mutation  (smn-1(ok355) mutants at late developmental stages when smn-1 mutants exhibit severe defects in behavior and lifespan (uaf-1(n4588) can decrease or increase the recognition of 3′ splice sites in a gene-specific manner (smn-1(ok355) mutants, which might compensate the defects in snRNPs and lead to the suppression   can partlifespan . Since uc manner , we postpression . In thissmn-1 affects gene expression in C. elegans. ok355 is a null mutation in smn-1 that deletes the majority of the coding regions of the gene (smn-1(ok355) mutants have similar behavior, body size and survival rate at early larval stages compared to wild type  for RNA-Seq analyses, postulating that we might observe early-stage changes of gene expression caused by smn-1 deficiency.It was unclear how the gene . smn-1 and | log2_ratio|≥ 1 from pairwise comparisons. The heat map indicates that smn-1(ok355) mutants exhibited apparently different gene expression profiles compared to wild type (smn-1(ok355) mutants, including 1564 up-regulated and 1891 down-regulated ones  of smn-1 transcripts were barely detectable in all smn-1(ok355) samples compared to wild type or uaf-1(n4588) samples  with ild type . Three t samples .smn-1, we performed KEGG (Kyoto Encyclopedia of Genes and Genomes1) pathway analyses on the DEGs of smn-1(ok355) mutants. Twenty-five significantly affected pathways were identified  and two pathways enriched with genes encoding nucleosome proteins   .smn-1(ok355) mutants include phenylalanine metabolism, retinol metabolism, ATP-binding cassette (ABC) transporters and the pentose phosphate pathway  (tdc-1) and octopamine (tdc-1) (ugt genes encoding UDP glycosyltransferases (UGT), followed by seven cyp genes encoding cytochrome P450 proteins (2), while the others were similar to homologs localized on the plasma membrane or other organelles (The other four KEGG pathways significantly affected in  pathway . Two DEG (bas-1) , tyramin (tdc-1)  (Supplem (tdc-1) . Among tproteins . UGT andproteins . Among tganelles . The penganelles , a functConsidering that the same gene can be assigned to different KEGG pathways, we further sorted these DEGs to a non-redundant list . In the 3) analyses of the DEGs showed that mitochondrial components were the most significantly affected group  mutants based on the hypergeometric test , a closer examination of the RNA-Seq results identified a non-redundant list  of DEGs annotated to GO terms related to lifespan, motor functions and/or neuron activities  was found to be significantly up-regulated in smn-1(ok355) mutants based on RNA-Seq results  tails [poly(A)-], and the replication-independent type, which produces transcripts with poly(A) tails [poly(A)+] (SMN-deficient cells showed that defective cleavage between the 3′ stem-loop end structure and the poly(A) signal (poly(A)+ transcripts +]  +] . For rep) signal  by the Unscripts .his-72, which encodes an H3 histone and is replication-independent, was not identified as a DEG based on RNA-Seq  mutants . RT-qPCR experiments confirmed that the expression levels of the four selected genes  were sig mutants .poly(A)+ transcripts of replication-dependent histone genes in smn-1(ok355) mutants (poly(A)+ transcripts or total transcripts  of four such genes. RT-qPCR experiments confirmed that the poly(A)+ transcripts were significantly up-regulated [his-3 (poly(A)+)].The RNA-Seq results suggested up-regulation of  mutants . To valismn-1(ok355) mutants  (smn-1(ok355) homozygotes compared to smn-1(ok355)/+ heterozygotes . Therefore, a transcript-independent mechanism(s) might be employed to regulate histone protein levels in vivo.To investigate whether more histone transcripts would lead to increased protein expression, we generated transgenic animals expressing a ransgene . Consist1(ok355) , the expozygotes . Howeverozygotes . Similaruaf-1(n4588) and uaf-1(n4588 n5127) mutations can partially suppress the defective behavior and lifespan of smn-1(ok355) mutants at late developmental stages when the defects are obvious (uaf-1(n4588) affects gene expression, we performed RNA-Seq on uaf-1(n4588) single and smn-1(ok355); uaf-1(n4588) double mutants at the pre-symptomatic stage (We previously found that the  obvious . To undeic stage .smn-1(ok355); uaf-1(n4588) double mutants, the major KEGG pathways identified in smn-1(ok355) single mutants were no longer significant (smn-1(ok355) single mutants but not DEGs in the double mutants . One gene appeared to be a real negative in the double mutants (T22B7.7).In nificant , S9, thonificant , S6. We  mutants . Five of mutants , while t mutants , T22B7.7uaf-1(n4588) single mutants were similar to that of wild type (uaf-1(n4588) mutants, including 80 up-regulated and 153 down-regulated ones (uaf-1(n4588) single and smn-1(ok355); uaf-1(n4588) double mutants identified 1,743 DEGs, among which 1,010 were up-regulated and 733 down-regulated (The gene expression profiles of ild type . 233 DEGted ones . No KEGGted ones . A compaegulated .smn-1(ok355); uaf-1(n4588) double mutants with smn-1(ok355) single mutants and identified 349 DEGs ; uaf-1(n4588) vs. smn-1(ok355) comparison exhibited expression changes opposite to that in smn-1(ok355) vs. N2 comparison (smn-1(ok355) was increased in the double mutants, while that of 121 up-regulated genes in smn-1(ok355) was decreased in the double mutants. Though it is interesting to speculate that some of these expression changes might underlie the suppression of smn-1(ok355) defects by uaf-1(n4588), caution has to be exercised in interpreting these results before the expression is validated and extensive analyses of gene functions are performed.One hundred and fifty-two of the 349 DEGs in mparison , among wsmn-1(ok355) mutants, among which 18 genes (cpt-2 gene (smn-1(ok355) mutants  affected alternative splicing, we examined nine genes (uaf-1(n4588) mutant embryos. These genes were recently reported in a study about the interaction of uaf-1 with the RNA-binding motif gene rbm-5 (To further investigate how ne genes  that exhne rbm-5 .smn-1(ok355) could apparently increase the intron retention of the abt-5 gene (flp-1 gene (smn-1(ok355) also appeared to act together with uaf-1(n4588) to increase the intron retention of the gcy-11 gene (smn-1(ok355) completely suppressed the recognition of a cryptic 5′ splice site within the zipt-2.4 gene in uaf-1(n4588) mutants, though smn-1 had no effect by itself (smn-1(ok355) did not alter their splicing in either wild-type or uaf-1(n4588) background  caused reduced expression of multiple mitochondrial genes in C. elegans.Mitochondrial dysfunctions have been described in human iPSC and zebrafish models of SMA . DefectiSMN on mitochondrial functions and gene expression remain to be understood. A recent study in C. elegans found that SMN-1 promotes the localization of mitochondria to the I bands of body wall muscles, which requires the Arp2/3 actin-nucleation complex  mutants might compromise the luminal acidity of these vesicles, causing defects in endosome, lysosome and/or synaptic vesicles. Hence, it is plausible that reduced V-ATPase expression could contribute to the vesicle defects in SMN mutants.Defects in the morphology, number and endocytosis of synaptic vesicles were described in human , mouse  and C. e elegans  models o elegans . The redsmn-1(ok355) mutants (smn-1(ok355) mutants.Multiple genes related to lifespan, motor functions and neuronal activities were also affected in  mutants . The rel mutants . Though  mutants . Furtherpoly(A)- transcripts (SMN deficiency leads to defective U7 snRNP formation and increased levels of poly(A)+ transcripts of replication-dependent histone genes (poly(A)+ transcripts of replication-dependent histone genes were similarly up-regulated in smn-1(ok355) mutants. However, this up-regulation might not involve defective U7 snRNP formation like that in mouse, as conserved U7 snRNA sequence was not identified in the C. elegans genome by bioinformatics analyses + transcripts, the total transcripts of three out of four replication-dependent histone genes, as well as four replication-independent histone genes, were also up-regulated in smn-1(ok355) mutants  caused reduced recognition of weak 3′ splice sites in two genes (tos-1 reporter transgenes (smn-1(ok355) mutants  mutants remains to be elucidated. It is plausible that smn-1 deficiency causes defective splicing of these transcripts and as a result generates pre-mature stop codons. Such transcripts are often degraded by non-sense-mediated mRNA decay (NMD) (smn-1 deficiency might cause reduced transcription or transcript stability of these genes. Future studies in NMD mutant backgrounds (The molecular mechanism underlying the down-regulated expression of genes in ay (NMD) , causingay (NMD) . Intereskgrounds  should pC. elegans, knocking down smn-1 specifically in motor neurons can cause degeneration  mutants was reduced to ∼50% of the wild-type levels. Though not dramatic, this level of change appears reasonable, as it was detected at the pre-symptomatic stage of smn-1(ok355) mutants when a residual amount of maternal smn-1 product was probably still expressed. The defects in gene expression might become more severe when the mutants enter later developmental stages, in which the maternal smn-1 will be completely depleted. The expression of the plst-1 gene provides such an example (see below). In addition, if any of these DEGs contribute to the defects of smn-1(ok355) mutants, it is more likely that they do so together with other DEGs, while the effects of an individual or a few genes could be weak. Hence, it is important to identify a comprehensive list of DEGs before we can have a more impartial understanding of the molecular changes related to the defects of smn-1(ok355) mutants.Our validation of the down-regulated DEGs –D indicaPLS3 gene was identified as a protective modifier of SMA  mutants at later developmental stages (3 days or 5 days after the L1 stage) , suggesting that plst-1 expression might not be severely affected at the pre-symptomatic stage of smn-1(ok355) mutants.The human r of SMA . We prev1 stage) . In the poly(A)+ mRNAs were examined by the RNA-Seq experiments . We therefore used RT-qPCR to quantify their expression. The results showed that U2 and U4 were significantly up-regulated in smn-1(ok355) mutants (smn-1(ok355) mutants at later developmental stages  mutants at the pre-symptomatic stage. Nevertheless, these results suggest that smn-1 can exert differential effects on the expression of U snRNAs and uncovering the underlying mechanism might facilitate our understanding of SMN functions.The expression levels of U snRNAs could not be extracted from the RNA-Seq results because only  mutants . In prevl stages . Though smn-1 was required for the recognition of a cryptic 5′ splice site in uaf-1(n4588) background (zipt-2.4)  . Such anm-5 gene , which em-5 gene . We postsmn-1(ok355) by uaf-1(n4588) (smn-1(ok355) might be ameliorated by uaf-1(n4588), which would lead to the suppression. Under an alternative circumstance, the altered splicing or expression of a new set of genes might impact the phenotype of smn-1(ok355); uaf-1(n4588) double mutants, leading to the suppression.We hypothesize two potential mechanisms that underlie the suppression of 1(n4588) . Under oabt-5 gene (uaf-1(n4588) suppressed the defective splicing at the 3′ splice site in smn-1(ok355) mutants. We found that uaf-1(n4588) also suppressed the up-regulated expression of two genes in smn-1(ok355) background (uaf-1(n4588) can increase or decrease the recognition of 3′ splice sites in a gene-specific manner (uaf-1(n4588), which might generate splicing isoforms that are degraded by NMD. Alternatively, uaf-1(n4588) might affect a gene that is required for the normal expression of these genes. Similarly, expression studies in an NMD mutant background should help dissect these potential mechanisms.It appears that the splicing of the t-5 gene  is consickground . Considec manner , such a smn-1 deficiency in C. elegans. The splicing of a subset of genes was also partially affected at this stage. Though the molecular mechanisms underlying this phenomenon remain to be investigated, our results, together with findings in other SMA models, suggest a cross-species effect of SMN on mitochondrial and histone gene expression. Future examination of the expression of more genes and analyses of their functions should further the understanding of SMN functions.In short, we identified the defective expression of a broad spectrum of genes that encode mitochondrial, V-ATPase, histone, UGT and cytochrome P450 proteins as a probable pre-symptomatic effect of Caenorhabditis elegans strains were grown at 20°C, unless otherwise indicated. N2 (Bristol) was the reference wild-type strain. Strains used in this study include:smn-1(ok355) I/hT2[bli-4(e937) let-?(q782) qIs48]  uaf-1(n4588) III (MT16492 588) III smn-1(ok355) I/hT2[bli-4(e937) let-?(q782) qIs48] ; uaf-1(n4588) IIICSM1111 smn-1(ok355) I/hT2[bli-4(e937) let-?(q782) qIs48] ; macEx588CSM1112 smn-1(ok355) I/hT2[bli-4(e937) let-?(q782) qIs48] ; macEx589CSM1113 smn-1(ok355) I/hT2[bli-4(e937) let-?(q782) qIs48] ; macEx590CSM1114 CSM1111 was grown at 15°C before being analyzed at 20°C.smn-1(ok355) homozygous mutants arrest at late larval stages and were maintained as heterozygotes using the balancer hT2 . Heterozygous mutants or wild-type animals were synchronized by bleaching and allowed to grow for 2 days post the L1 larval stage. For smn-1(ok355) single mutants or smn-1(ok355); uaf-1(n4588) double mutants, ∼2,500 individuals were picked for each biological replicate under a fluorescence dissecting microscope (Olympus SZX16). Animals were washed with M9 three times and kept in M9 for 3 h to remove intestinal bacteria. Total RNAs were extracted using TRI Reagent Solution according to manufacturer’s instructions (Invitrogen).RNA-Seq was performed by Annoroad Gene Technology (Beijing). Specifically, mRNAs were enriched with oligo(dT) beads. The constructed libraries were sequenced as 150 bp paired-ends on an Illumina platform. Raw data was filtered by Perl scripts using the following criteria: (1) reads containing more than five adapter-polluted bases were filtered out; (2) reads with the number of low quality bases  in more than 15% of total bases were removed; (3) reads with the number of N bases accounting for more than 5% were removed; (4) both reads would be filtered out if either one of the paired-end reads was adaptor-polluted.C. elegans genome index, and clean data was mapped to the reference genome (WS235) using TopHat v2.0.12. Differential gene expression analysis was performed by DESeq v1.14.0. The p-value was adjusted by Benjamini and Hochberg’s method as q-value for controlling false discovery rate (FDR). Genes with q ≤ 0.05 and | log2_ratio|≥ 1 were identified as DEGs.More than 39 million mapped reads and more than 6 GB of clean bases were obtained for each sample . Bowtie/4, a package of software programs for analyzing alternative splicing events from RNA-Seq data. The ratio of a given alternative splicing event was determined by the following formula, e.g., the percentage of an intron retention event = FPKMwithintron/(FPKMwithintron + FPKMwithoutintron). The ratios of the same splicing event were compared between genotypes to identify potential splicing differences.Alternative splicing events were predicted by ASprofile v1.0.4C. elegans genome (WS235) with annotated functions in the two databases as the background gene set. P-value for the enrichment level was calculated and adjusted as q-value. KEGG or GO terms with q ≤ 0.05 were considered significantly affected by the DEGs.KEGG and GO enrichment analyses on the DEGs were implemented by hypergeometric test using all genes in the smn-1(ok355); uaf-1(n4588) double mutants and three biological replicates for uaf-1(n4588) or smn-1(ok355) single mutants were analyzed by RNA-Seq. We examined the correlation coefficients between the biological replicates for each genotype (smn-1(ok355); uaf-1(n4588), replicates of all other genotypes were highly correlated (r > 0.9). The coefficient for the two replicates of smn-1(ok355); uaf-1(n4588) mutants was 0.8260  and smn-1(ok355); uaf-1(n4588) mutants, ∼250 animals were picked for each biological replicate. Total RNAs were prepared using TRI Reagent Solution (Invitrogen) with 15 μg yeast tRNA (Invitrogen) added as co-precipitant, followed by RNase-Free DNase I (TAKARA) digestion. cDNAs were synthesized with random hexamers using Maxima First Strand cDNA Synthesis Kit (Thermo Scientific) or with oligo(dT)20 primers (only for replication-dependent histone genes) using SuperScript IV First-Strand Synthesis System (Invitrogen). 2 μg total RNA (including yeast tRNA as co-precipitant) from each sample was used for RT experiments. qPCR was performed using Maxima SYBR Green qPCR Master Mix (Thermo Scientific) on a Bio-Rad CFX96 real-time cycler. The reaction mix contains 2 μL of diluted template  and 0.3 μM of each primer in a final volume of 15 μL. Thermal cycling was performed using a two-step cycling protocol: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Bio-Rad CFX Manager v3.0 was used to calculate the relative expression level of each gene with tba-1 as the reference gene. Melting curve for each sample contained one single peak. Alternative splicing was detected using the following PCR protocol: 90 s at 94°C, followed by 40 cycles of 15 s at 94°C, 15 s at 60°C and 30 s at 72°C, with a final extension of 3 min at 72°C. The proportion of each splice isoform was quantified using ImageJ 1.52a. Three biological replicates were analyzed for each genotype. PCR primers are listed in Synchronized animals were grown on NGM plates for 2 days post L1. For his-41 expression plasmid was constructed using the In-Fusion HD Cloning Kit (Clontech) following the manufacturer’s instruction. First, the genomic fragment containing the full-length his-41 CDS and a 1.1 kb endogenous promoter was amplified and subcloned into pMD18T vector (Takara). Second, the mCherry gDNA from the pCFJ90 (myo-2p::mCherry) plasmid (his-41 stop codon) were amplified and subcloned into pMD18T-his-41p::his-41 gDNA backbone using the KpnI site. plasmid  and a 3′his-35 and hil-3 expression plasmids were constructed in a similar manner. For his-35, a 1.3 kb promoter and a 1.2 kb 3′ UTR were used. For hil-3, a 1.8 kb promoter and a 0.6 kb 3′ UTR were used. All plasmids were verified by sequencing. Primers for plasmid construction are listed in The myo-3p::GFP as co-injection marker.Germline transgene experiments were performed as described . The tra3 in M9 buffer. Images were captured with Zeiss 880 confocal microscope (10× objective). Total fluorescence intensity was quantified as un-calibrated OD, and fluorescence density was calculated using total intensity divided by area using ImageJ 1.52a.Synchronized animals were mounted on 2% (vol/vol) agar pads on day 2 post the L1 larval stage and immobilized using 40 mM NaNP-values were determined by two-tailed unpaired Student’s t-test for pairwise comparison or Bonferroni multiple comparison with one-way ANOVA for multiple comparison using GraphPad Prism 7.0 software.XG and LM designed the experiments. XG performed the genetic, molecular, and transcriptome analyses with the assistance of JX, HC, DX, WP, and CZ. XG, YCM, and LM wrote the manuscript. LM managed the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Transient expression of SVA 3Cpro was associated with cleavage of NF-κB-p65 and Poly (ADP-ribose) polymerase (PARP), suggesting its involvement in virus-induced apoptosis. Most importantly, we showed that while cleavage of NF-κB-p65 is secondary to caspase activation, the proteolytic activity of SVA 3Cpro is essential for induction of apoptosis. Experiments using the pan-caspase inhibitor Z-VAD-FMK confirmed the relevance of late apoptosis for SVA infection, indicating that SVA induces apoptosis, presumably, as a mechanism to facilitate virus release and/or spread from infected cells. Together, these results suggest an important role of apoptosis for SVA infection biology.Senecavirus A (SVA), an oncolytic picornavirus used for cancer treatment in humans, has recently emerged as a vesicular disease (VD)-causing agent in swine worldwide. Notably, SVA-induced VD is indistinguishable from foot-and-mouth disease (FMD) and other high-consequence VDs of pigs. Here we investigated the role of apoptosis on infection and replication of SVA. Given the critical role of the nuclear factor-kappa B (NF-κB) signaling pathway on modulation of cell death, we first assessed activation of NF-κB during SVA infection. Results here show that while early during infection SVA induces activation of NF-κB, as evidenced by nuclear translocation of NF-κB-p65 and NF-κB-mediated transcription, late in infection a cleaved product corresponding to the C-terminus of NF-κB-p65 is detected in infected cells, resulting in lower NF-κB transcriptional activity. Additionally, we assessed the potential role of SVA 3C protease (3C Senecavirus, family Picornaviridae  is a non-enveloped single-stranded positive-sense RNA virus of the genus aviridae , 2. SVA aviridae , and subdiovirus . The SVAdiovirus . Processic triad , 4. Whilic triad  and playic triad –28.Since its identification, SVA has been associated with sporadic cases of vesicular disease in pigs in the US and Canada –31. HoweIn addition to its relevance to animal health, SVA has been tested as an oncolytic agent for cancer treatment in humans , 44–47. Picornaviruses modulate many host cellular pathways, including the host translation machinery, innate immune responses and cell survival or apoptosis. Foot-and-Mouth disease virus (FMDV), for example has been shown to inhibit nuclear factor kappa B- (NF-κB)  and inteWhile apoptosis usually functions as a host defense mechanism that ensures killing of infected cells , 57, sevNF-κB is a ubiquitous transcription factor that modulates not only cell death but also innate immunity and inflammatory responses. This pathway seems to play critical roles in the picornaviral life cycle , 27, 66.In the present study, we investigated the host apoptotic responses during SVA infection and dissected the interplay between the virus, host cell apoptosis and NF-κB signaling. Results here show that SVA induces apoptosis late in infection, which plays a critical role on the virus infection cycle, likely facilitating virus release from infected cells.2 in minimum essential medium  or RPMI 1640 medium (Corning®) supplemented with 20 or 10% fetal bovine serum , respectively. Cell culture media was supplemented with 2 mM L-glutamine (Corning®), penicillin , streptomycin , and gentamicin . Senecavirus A strain SD15-26 was isolated from a vesicular lesion from a finishing pig and has been previously characterized (Primary swine turbinate (STu) and NCI-H1299 non-small human lung carcinoma cell lines (ATCC® CRL-5803) were cultured at 37°C with 5% COcterized . Low paspro was fused with Flag-tag (N-terminus fusion), chemically synthesized (GenScript), and cloned into the pcDNA3.1 expression plasmid (pcDNA-Flag-3C). Additionally, SVA 3Cpro was amplified from SVA strain SD15-26 by RT-PCR and cloned into the eukaryotic expression plasmid pET28a as a His-tag fusion protein (pET28-SVA-3C). SVA strain SD15-26 VP1 expressing plasmid (pcDNA-HA-VP1) was kindly provided by Dr. Steve Lawson . Control plasmid pCMV-Flag-BAP is commercially available (Sigma). The plasmid pCMV-HA-NF-κB-p65 was constructed by standard cloning techniques. Briefly, porcine NF-κB-p65 coding sequence (GenBank NM_001114281) was PCR amplified from cDNA prepared from swine testicle (ST) cells and cloned in fusion with the HA epitope tag into the pCMV-HA-N expression vector (Clontech). Sequence identity of the expression plasmids generated here were confirmed by DNA sequencing.Luciferase reporter plasmids  are commercially available (Promega). The coding sequence of SVA 3CAntibodies against β-actin (clone C4), NF-κB-p65 , NF-κB-p65 (clone F-6), and IκBα (clone H4) were purchased from Santa Cruz Biotechnology. Antibodies against NF-κB-p65 , IKKα (clone3G12), IKKβ (D30C6), cleaved caspase-3 , and PARP (clone 46D11) were obtained from Cell Signaling. SVA VP1 and SVA VP2 mouse monoclonals and SVA whole virus antibodies were kindly provided by Dr. Steve Lawson (SDSU). Anti-Flag or anti-HA mouse monoclonal antibodies are commercially available . Anti-rabbit and/or anti-mouse secondary antibodies conjugated with Alexa Fluor® 594 and Alexa Fluor 488® were purchased from Life Technologies. IRDye 800CW-labeled anti mouse IgG and IRDye 680-labeledRD anti rabbit IgG secondary antibodies were purchased from Li-Cor Biosciences. Recombinant TNF-α was obtained from InvivoGen. Staurosporine was purchased from Cell Signaling and Z-VAD-FMK was obtained from Santa Cruz Biotechnology.pro was expressed using a prokaryotic expression system. SVA 3Cpro coding sequence (GenBank KX778101) was PCR amplified from cDNA of SVA SD15-26 and cloned into the bacterial expression plasmid pET-28a (EMD Millipore/Novagen). The recombinant protein was expressed in Escherichia coli strain BL-21 cells as a 6×His-tag fusion protein and purified using nickel-charged agarose resin (Qiagen) according to the manufacturer's instructions. Purified recombinant protein was used for polyclonal antibody production in rabbits.SVA 3Cad libitum throughout the study. The animal was immunized with 660 ng of SVA 3Cpro emulsified in water-in-oil (W/O) adjuvant . The antigen was administered by three 0.5 mL subcutaneous injections and one 0.5 mL intramuscular injection. Two weeks post-primary immunization the animal received a booster immunization as above. Two weeks after the booster immunization the rabbit was euthanized and exsanguinated, and polyclonal serum containing SVA 3Cpro-specific antibodies was isolated by centrifugation . All animal procedures and protocols for antibody production were reviewed and approved by the South Dakota State University Institutional Animal Care and Use Committee (IACUC) under approval number 15-095A.One adult 12-week old rabbit was kept in an individual cage with food and water in situ cell death detection kit  according to the manufacturer's instructions.Primary STu cells were cultured on glass cover slips and infected with SVA (MOI = 5) or mock-infected and then fixed at 12 h p.i. with 3.7% formaldehyde in PBS (pH 7.2) for 20 min. Positive control cells were treated with 0.1 μM staurosporine for 4 h and then fixed as above. Additionally, formaline-fixed paraffin-embedded (FFPE) tissue sections from a previous SVA pathogenesis experiment conducted in our laboratory  were subFormalin-fixed paraffin-embedded tissue sections (skin) from a previous SVA animal experiment were processed following standard histological procedures and stained with hematoxylin and eosin for histological examination. All animal procedures and protocols were reviewed and approved by the SDSU IACUC under approval number 16-002A.in situ hybridization (RNAScope®). ISH probe utilized was specific for the viral genome SVV . The ISH was performed as previous described  were used to perform escribed .Activation of caspase 3 and 7 (Casp-3/-7) was assessed by flow cytometry during SVA infection. Semi-confluent STu or H1299 cells were cultured in 12-well plates and infected with SVA (MOI = 5). At 2, 4, 5, 6, 7, 8, and 10 h p.i. the supernatant was removed and the cells trypsinized to dissociate the monolayer. Mock-infected cells and mock-infected cells treated with 0.3 μM staurosporine were used as negative and positive controls, respectively. After trypsinization, cells and supernatant were mixed and centrifuged at 1,500 × g for 5 min at 4°C. Activation of Casp-3/-7 was assessed using the CellEvent™ Caspase-3/7 Green Flow Cytometry Assay kit (Thermo Fisher Scientific) according to manufacturer's protocol. The flow cytometry data were acquired with an Attune NxT flow cytometer (Thermo Fisher Scientific) and analyzed using FlowJo software (TreeStar).To investigate activation of Casp-3/-7 in the context of NF-κB-p65 expression and SVA infection, STu cells were transfected with 1 μg of pCMV-HA-NF-κB-p65 or empty pCMV-HA plasmids (control) using Lipofectamine 3000 (Life Technologies) and subsequently infected with SVA (MOI = 5). Cells were collected at 4, 7, 8, and 10 h p.i. and processed for flow cytometry assessment of Casp-3/-7 activation as described above.50) per milliliter.SVA growth curves were performed in STu and H1299 cells. Cells were cultured in 12-well plates, inoculated with SVA (MOI = 5) and harvested at 2, 4, 8, 12, and 24 h p.i. Virus titers were determined on each time by limiting dilutions in H1299 cells. At 48 h p.i. cells were fixed , permeabilized (0.2% Triton-X), and stained with an anti-SVA whole virus rabbit polyclonal antibody. Viral titers were determined by the Spearman and Karber's method  and exprThe effect of NF-κB pathway activation during SVA replication was assessed by overexpression of NF-κB-p65 in STu cells. For this, semi-confluent STu cells were plated in 12-well plates and transfected with 1 μg of pCMV-HA-NF-κB-p65 or empty pCMV-HA (control) per well using Lipofectamine 3000 (Life Technologies) as recommended by the manufacturer. At 24 h post-transfection, cells were infected with SVA (MOI = 0.1). After 1 h adsorption, the inoculum was removed and fresh media was added. The cells were harvested at 2, 4, 8, 12, and 24 h p.i. and virus titers determined as described above.To assess the role of apoptosis on SVA infection cycle, multiple-step growth curves were performed in STu or H1299 cells. Cells were plated in 12-well plates and infected with SVA (MOI = 0.1). After 1 h adsorption, fresh media containing 30 μg/mL of Z-VAD-FMK, a pan-caspase inhibitor was added to the cells. Plain media was added to untreated control wells. Cells and supernatant were collected separately at 2, 4, 8, 12, and 24 h p.i. and subjected to virus titration. Additionally, the effect of apoptosis on SVA infection was assessed in STu cells infected with a high MOI. For this, cells were infected with SVA at an MOI = 5 and cells and supernatant collected separately at 8 h p.i. and subjected to virus titrations.To determine transcription levels of NF-κB target genes during SVA infection we performed RT-qPCR. STu cells cultured in 6-well plates were infected with SVA (MOI = 5), collected at 2, 4, 8, and 12 h p.i., and cellular RNA extracted using TRIzol reagent (Invitrogen) according to manufacturer's protocol. RNA samples were treated with DNase (Ambion) and further cleaned using the RNeasy® Mini kit (QIAGEN). Mock infected cells were used as a negative control. Transcription levels of TNF-α, Caspase 8, FADD, BAX, BAK, BCL-2, XIAP, CXCL8, PTGS2, IRF1, and NF-κBIA genes were determined using RT-qPCR and TaqMan gene expression assays (Thermo Scientific). The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. RT-qPCR reactions were performed using the RNA-to-Ct™ 1 Step-kit and TaqMan assays for each target gene. The amplification/detection reactions were performed in a 7500 Real Time PCR System (Applied Biosystems). Transcription of target gene was normalized to that of GAPDH and genome copy numbers were determined using the relative quantitation method. Data were analyzed and expressed as fold changes normalized to levels of RNA detected in mock infected cells.Expression and activation of NF-κB-p65 was assessed during early stages of SVA infection by IFA. STu cells cultured in 24-wells plates were infected with SVA (MOI = 5). At 0, 2, and 4 h p.i. cells were fixed using 3.7% formaldehyde in PBS (pH 7.2) for 20 min. The cells were washed three times with PBS and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature (RT). Plates were incubated for 1 h at RT with an antibody specific for the C-terminus-NF-κB-p65 (1:250 in PBS/1% BSA) and for SVA (1:250 in PBS/1% BSA). After primary antibody incubation, cells were washed as above and incubated for 1 h at RT with appropriate secondary antibodies conjugated with Alexa Fluor 594 and/or 488 (1:250 in PBS/1% BSA). Cells were washed three times with PBS and nuclear stain was performed with DAPI (Thermo Scientific). Cells were visualized using a fluorescence microscope .Expression and activation of NF-κB-p65 was also assessed during late stages of SVA infection by IFA. STu cells cultured in 24-well plates were infected with SVA (MOI = 1). At 7 and 8 h p.i. cells were fixed and IFA was performed as described above.pro on expression of NF-κB-p65 was assessed by IFA. H1299 cells were transfected with pcDNA-Flag-3C (100 μg) expression plasmid and subjected to IFA staining. C-terminus-NF-κB-p65 (1:250 in PBS/1% BSA) and Flag-Tag-specific antibodies (1:250 in PBS/1% BSA) were used as primary antibodies. All IFA steps were performed as described above.The effect of SVA 3CThe effect of SVA on apoptosis and NF-κB signaling pathway was investigated by western blots. Semi-confluent monolayers of STu cells cultured in 6-well plates were infected with SVA (MOI = 10) and harvested at 0.5, 1, 2, 3, 4, 8, and 12 h p.i. Mock-infected cells were used as negative controls. This experiment was also performed in H1299 cells, with samples collected at 4, 8, 12, and 24 h p.i. Mock-infected cells unstimulated or stimulated with 100 ng/mL of TNF-α were used as negative and positive controls, respectively. Cells were lysed with M-PER mammalian extraction reagent (Thermo Scientific) containing protease inhibitors (RPI). One hundred microgram whole cell protein extracts was resolved by SDS-PAGE in 10% acrylamide gels and transferred to nitrocellulose membranes. Blots were incubated with 5% non-fat dry milk in PBS overnight at 4°C and then probed with the antibodies indicated in the Figures, followed by incubation with appropriate Dye-light fluorescent conjugate secondary antibodies. The membranes were scanned with the Odyssey infrared imaging system (Li-Cor). Densitometric analysis was performed using ImageJ® with expression levels of selected target proteins being normalized to those of the housekeeping gene β-actin.pro on apoptotic pathways was assessed under transient expression experiments. H1299 cells cultured in 6-wells plates were transfected with 2 μg of pcDNA-Flag-3C and harvested at 18 h post-transfection. Positive control cells were stimulated with TNF-α (100 ng/mL) for 1 h. In the experiments with catalytic dead mutants of SVA 3Cpro, H1299 cells were transfected with 2 μg of pcDNA-Flag-3C, pcDNA-Flag-3C(H47D), or pcDNA-Flag-3C(C159R) and harvested 18 h post-transfection. Protein extracts and western blots were performed as described above.The effect of SVA 3Cpro during viral replication and its relationship with the cleavage of NF-κB-p65 and apoptosis, STu cells were infected with SVA (MOI = 10) and collected the total protein extract at 2, 4, 5, 6, 7, 8, and 12 h p.i. Western blot was performed as above, and the blots were probed with antibodies indicated in the Figures.To assess the expression of SVA 3CFigures 8B–D.To determine the cleavage site of NF-κB-p65, semi-confluent H1299 cells were cultured in 6-well plates and co-transfected with an empty plasmid (negative control) or pcDNA-Flag-3C (2 μg/well) and either pCMV-HA-NF-κB-p65, pCMV-HA-NF-κB-p65(478R/479D), pCMV-HA-NF-κB-p65(482R/483D), pCMV-HA-NF-κB-p65(478R-483D), pCMV-HA-NF-κB-p65(444L-450R), pCMV-HA-NF-κB-p65(464V-467E), pCMV-HA-NF-κB-p65(444L-450R 478R-483D), or pCMV-HA-NF-κB-p65(464V-467E 478R-483D) (1 μg of each). Cells were harvested at 18 h post-transfection and protein extracts and western blots performed as described above. Antibodies used are indicated in pro were responsible for NF-κB-p65 cleavage, semi-confluent H1299 cells were cultured in 6-well plates and co-transfected with an empty plasmid (negative control) or pcDNA-Flag-3C (1 μg/well) and pCMV-HA-NF-κB-p65 (2 μg/well). At 3 h post-transfection, fresh media was added to negative controls and fresh media containing or not 150 μg/mL of Z-VAD-FMK was added to the cells transfected with pcDNA-Flag-3C and pCMV-HA-NF-κB-p65. Cells were harvested at 18 h post-transfection and protein extracts and western blots performed as described above. Antibodies used are indicated in Figure 8E.To investigate if caspases or SVA 3CThe effect of SVA on NF-κB-mediated transcription was investigated by luciferase reporter assays. To detect the ability of SVA to inhibit NF-κB activation, semi-confluent H1299 cells cultured in 24-well plates were co-transfected with the NF-κB-luciferase reporter plasmid (315 ng/well) and the control plasmid pRL-TK (35 ng/well) using Lipofectamine 3000 (Life Technologies) and infected with SVA (MOI = 5). SVA-infected cells were stimulated or not with TNF-α (100 ng/mL) at 2, 4, 8, or 12 h p.i. and incubated for 12 h post-TNF-α stimulation. Mock-infected cells were also stimulated with TNF-α for 12 h. The dual-luciferase reporter system (Promega) was used to determine the luciferase activity in each treatment condition following the manufacturer's instructions. Firefly luciferase activity was normalized to renilla luciferase activity and fold-changes calculated based on the levels of luciferase detected in mock-infected/non-stimulated cells.pro on NF-κB-mediated transcription was investigated by luciferase assays. H1299 cells were co-transfected with pNF-κB-luciferase reporter plasmid (315 ng/well), pRL-TK (35 ng), and either pcDNA-Flag-3C (550 ng/well), or control plasmids pCMV-Flag-BAP (550 ng/well) or pcDNA-HA-VP1 (550 ng/well). At 14 h post-transfection cells were stimulated with TNF-α, harvested at 12 h post-TNF-α-stimulation and the luciferase activity was determined as described above.The effect of SVA 3Cpro is required for its effect on NF-κB-mediated transcription, H1299 cells were co-transfected with pNF-κB-luciferase reporter plasmid (315 ng/well), pRL-TK (35 ng), and either pcDNA-Flag-3C (550 ng/well), pcDNA-Flag-3C(H47D) (550 ng/well), or pcDNA-Flag-3C(C159R) (550 ng/well) expression plasmids. At 14 h post-transfection, cells were stimulated with TNF-α, harvested at 12 h post-TNF-α-stimulation and the luciferase activity was determined as described above.To assess whether the proteolytic activity of 3Cpro and in the porcine NF-κB-p65 were introduced in the SVA 3Cpro- and NF-κB-p65-encoding plasmids using the Q5® Site-Directed Mutagenesis Kit (New England BioLabs® Inc.) according to the manufacturer's instructions. Site directed mutagenesis primers containing nucleotide substitutions targeting amino acids H47 and C159 leading to substitutions of H to D, or C to R on SVA 3Cpro were designed and used with the site-direct mutagenesis protocol to generate the mutant plasmids pcDNA-Flag-3C(H47D) and pcDNA-Flag-3C(C159R). NF-κB-p65-specific primers containing nucleotide substitutions targeting the putative SVA 3Cpro cleavage sites individually (478QL and 482QG) or simultaneously 478QLLNQG were designed and used with the site-direct mutagenesis protocol, resulting in plasmids pCMV-HA-NF-κB-p65(478R/479D), pCMV-HA-NF-κB-p65(482R/483D), and pCMV-HA-NF-κB-p65(478R-483D), respectively. Primers containing nucleotide substitutions targeting putative caspase cleavage sites in NF-κB-p65 coding sequence (444LQFDTDED and 464VFTD) were designed and used with site directed mutagenesis kit, resulting in plasmids pCMV-HA-NF-κB-p65(444L-450R) and pCMV-HA-NF-κB-p65(464V-467E). Double caspase and SVA 3Cpro cleavage site-mutants were also generated, resulting in plasmids pCMV-HA-NF-κB-p65(444L-450R 478R-483D) and pCMV-HA-NF-κB-p65(464V-467E 478R-483D). The sequences of each NF-κB-p65 mutant are indicated in Figure 8E. Correct nucleotide substitutions were confirmed by DNA sequencing.Mutations in the catalytic triad of SVA 3Cpro template-based protein structure modeling was performed using RaptorX web server . After 1 h adsorption, the inoculum was removed and fresh 1%-agarose media containing or not 50 μg/mL of Z-VAD-FMK was used to overlay the cells. The agarose overlay was removed and cells were fixed at 24 h p.i. with 3.7% formaldehyde in PBS (pH 7.2) for 20 min. The cells were stained with 1% Crystal Violet solution for 20 min and washed four times with PBS. The area in mm2 of each viral plaque was calculated using ImageJ®.Confluent primary STu cells cultured in 6-well plates were infected with serial 10-fold dilutions of SVA . Statistical significance of the data was assessed using the Replication of SVA in primary STu cells induces cytopathic effect (CPE) characterized by cell rounding and plasma membrane blebbing . Therefoin vivo, paraffin-embedded skin sections collected from SVA-infected pigs on day 7 post-infection (p.i.), were subjected to TUNEL. Skin sections were obtained from characteristic SVA vesicular lesions  than those detected at 7 h p.i.  (p < 0.01 when compared to mock-infected cells). A significant increase in the frequency of cells presenting active Casp-3/-7 was also detected at 8–10 h p.i.  in SVA-infected H1299 cells  . Interes99 cells . Similar99 cells . Togethep < 0.05 when compared to mock-infected cells). Additionally, the pro-inflammatory NF-κB-regulated genes, including CXCL8, PTGS2, IRF1, NF-κBIA, and TNF-α were up-regulated during SVA infection (4–12 or 8–12 h p.i.) (K). These results suggest modulation of NF-κB-regulated gene expression during SVA infection in primary STu cells.NF-κB is one of the major players modulating host cell apoptosis and pro-inflammatory responses , 77. Thu h p.i.) –K. TheseGiven the transcriptional changes and modulation of apoptotic pathway observed in SVA infected cells  and 3, wp < 0.05; Interestingly, at later times p.i. (7–8 h p.i.) on a low MOI experiment, many of the SVA-infected cells presented a marked decrease in expression of NF-κB-p65 , suggestNF-κB transcriptional activity was assessed during SVA infection using an NF-κB-luciferase reporter assay. A significant decrease in TNF-α-induced NF-κB-mediated luciferase activity was observed between 8 and 12 h post-SVA infection . Collectpro, the main protease encoded by the virus   , a markeng cells . Accumulng cells , was alsF-κB-p65 . IFA expng cells . Similar of PARP , suggestpro expression on cleavage of NF-κB-p65 and activation of apoptotic pathways in the context of SVA infection were investigated by western blots. Expression of SVA 3Cpro was first detected using a SVA 3Cpro-specific rabbit polyclonal antibody at 5 h p.i., with increasing levels of the protein being detected up to 12 h p.i.  when compared to cells expressing either VP1- or BAP-control proteins  that is required for its protease activity (pro we introduced a mutation at His 47 (H47D) or Cys 159 (C159R) using site-directed mutagenesis (pro-expressing cells. H1299 cells were transfected with plasmids encoding the wild type or each of the 3Cpro mutants (H47D or C159R) and total protein extracts were subjected to western blot analysis. Notably, while expression of the wild type SVA 3Cpro resulted in activation of Casp-3 and cleavage of NF-κB-p65, expression of the 3Cpro catalytic dead mutants H47D or C159R did not lead to Casp-3 activation nor NF-κB-p65 cleavage , expression of the 3Cpro mutants (H47D and C159R) did not inhibit TNF-α-induced NF-κB-mediated luciferase activity   were co-expressed with 3Cpro, NF-κB-p65 464-467E mutant was cleaved, while no cleavage of NF-κB-p65 444L-450R mutant was observed (pro- and Casp-cleavage site mutations. As shown in pro cleavage site (478R-483D), NF-κB-p65 was not cleaved (pro (pro.To dissect whether cleavage of NF-κB-p65 is directly mediated by SVA 3Casp-3/-6  and/or f/-6 (pro . Two cled 482QG)  and two d 482QG) . These pd 482QG) . Cleavagobserved . This ph cleaved . Additioved   . Togethe, H1299)  and indu, H1299)  might be+).Given our results demonstrating that overexpression of NF-κB-65 prevents SVA-induced apoptosis and results in lower viral yields from infected cells, we investigate the direct effect of apoptosis on SVA infection. For this, replication kinetics experiments were performed in cells treated or not with Z-VAD-FMK to directly inhibit caspase-mediated apoptosis . As showp < 0.001). Together these results suggest that induction of apoptosis late in SVA infection may be important for SVA release and/or spread from infected cells.Next, viral yields were evaluated in the supernatant or cell fraction of cell cultures treated with Z-VAD-FMK following high MOI SVA infection. Cells were infected with SVA (MOI = 5) and treated or not with apoptosis inhibitor (Z-VAD-FMK). While no differences in viral yields were detected in the cell fraction at 8 h p.i. , signifiPicornaviridae family  and cleavage of PARP, a key marker of cell death, suggesting that this viral protease is involved on host cell apoptosis , aspartate 83 (D83), and cysteine 159 (C159). To assess the importance of the proteolytic function of SVA 3Cpro to its pro-apoptotic activity and inhibitory effect on the NF-κB pathway, we generated two 3C mutants containing single aa substitutions at H47 (H47D) and C159 (C159R) to disrupt the catalytic triad of the protein. Notably, while expression of wild type 3Cpro resulted in activation of caspases and cleavage of NF-κB-p65, mutations on the catalytic triad of 3Cpro (H47D and C159R) abolished these phenotypes, indicating that the protease activity of SVA 3Cpro is essential for its function on these pathways.The picornaviral 3Cic triad , 80. Thiic triad . The prepro or Lpro by these viruses, respectively. Initial results here, suggested that SVA 3Cpro could also be potentially be involved with this function. However, since cellular caspases are also able to cleave NF-κB-p65  Policy on Humane Care and Use of Laboratory Animals, and recommendations of the Guide for the Care and Use of Laboratory Animals. The protocols were approved by the SDSU IACUC (15-095A and 16-002A).All datasets generated for this study are included in the manuscript and/or the supplementary files.MF designed, performed, and analyzed the experiments and data. MM, JO, LJ, and SL developed reagents and helped with experiments. DD planned experiments, analyzed data, and secured funding. MF and DD wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Usp44-/- mice are more susceptible to HSV-1 infection as indicated by higher tissue viral titers, greater tissue damage and lower survival rate. These findings suggest that USP44 plays a specific and critical role in the regulation of innate immune response against DNA viruses.Mediator of IRF3 activation  senses the second messenger cyclic GMP-AMP (cGAMP) which is synthesized upon DNA virus infection and activates innate antiviral immune response. It has been demonstrated that the activity of MITA is delicately regulated by various post-translational modifications including polyubiquitination. In this study, we identified the deubiquitinating enzyme USP44 as a positive regulator of MITA. USP44 is recruited to MITA following DNA virus infection and removes K48-linked polyubiquitin moieties from MITA at K236, therefore prevents MITA from proteasome mediated degradation. USP44-deficiency results in acceleration of HSV-1-induced degradation of MITA and reduced induction of type I interferons (IFNs) and proinflammatory cytokines. Consistently,  Cyclic GMP-AMP synthase (cGAS) senses cytosolic dsDNA and initiates signal transductions, leading to activation of innate immune response. MITA is the key adaptor protein downstream of cGAS and plays a critical role in cGAS-mediated signaling. The activity of MITA is tightly regulated by various post-translational modifications including polyubiquitination and deubiquitination. Here we found that the deubiquitinating enzymes USP44 associates with MITA and removes the K48-linked polyubiquitin chains from MITA, therefore maintains the stability of MITA after DNA virus infection. Deficiency of USP44 results in accelerated degradation of MITA, impaired induction of type I IFNs and proinflammatory cytokines, and increased viral replication. These findings suggest that USP44 is a positive regulator of MITA and plays an important role in the regulation of innate immune response against DNA viruses. The innate immune response is the first line of host defense against pathogens. Germline-encoded pattern recognition receptors (PRRs) recognize conserved molecular motifs of pathogens called pathogen-associated molecular patterns (PAMPs) and trigger a series of signaling events, leading to induction of type I IFNs, proinflammatory cytokines and downstream antiviral effector proteins, which eventually inhibit the replication of pathogens and eliminate the infected cells –4.in vivo [Viral nucleic acids act as typical PAMPs that trigger innate immune response. Viral RNAs are recognized by endosomal Toll-like receptors (TLRs) and cytosolic RIG-I-like receptors (RLRs) such as retinoic acid-inducible gene-1 (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) . Meanwhiin vivo . In recein vivo –17. Uponin vivo . cGAMP tin vivo –23. Oncein vivo , 24, 25.in vivo , 24. IRFin vivo , 27.As a key adaptor protein in innate immune response against DNA viruses, the activity of MITA is delicately regulated. Several post-translational modifications, such as phosphorylation, sumoylation and polyubiquitination, have been reported to play important roles in regulation of MITA . For exaDeubiquitination is a reverse biochemical process of polyubiquitination, in which polyubiquitin chains previously added to target proteins are removed by deubiquitinating enzymes (DUBs). Recently, several DUBs have been reported to regulate the antiviral innate immune response. For example, USP14 removes K48-linked polyubiquitination of cGAS therefore impairs autophagic degradation of cGAS . It has In this study, we screened for DUBs that involve in DNA virus-triggered induction of type I IFNs and identified ubiquitin-specific protease 44 (USP44) as a positive regulator. USP44 consists of 712 amino acids, which contains a Zn-finger domain at the N-terminus and a USP domain at the C-terminus . USP44 hIFNB promoter in reporter assays. These efforts led to identification of USP44 as a candidate protein that could potentiate activation of the IFNB promoter triggered by the DNA virus herpes simplex virus 1 (HSV-1)  and NF-κB in a dose-dependent manner in HEK293 cells  . Overexp93 cells . Consistt manner . We nextto HSV-1 . In addito HSV-1 . In contto HSV-1 . Moreoveto HSV-1 . These rIFNB1, IL6 and CXCL10 induced by HSV-1 infection and transfected dsDNA, such as HSV120 and interferon stimulating DNA (ISD), was markedly reduced in USP44-RNAi expressing THP-1 cells compared with control cells , which are permissive to human cytomegalovirus (HCMV) infection. We found that knockdown of USP44 inhibited HCMV- and HSV120-induced transcription of 10 genes , but had10 genes . These rin vivo, we used Usp44 knockout mice (Usp44 mRNA was lowly expressed in wild-type bone marrow-derived macrophages (BMDMs) and murine lung fibroblasts (MLFs), but was up-regulated following HSV-1 infection. In these cells, Usp44 mRNA was not detectable either before or after HSV-1 infection in Usp44-/- cells  infection was significantly dampened in Usp44-/- compared with Usp44+/+ BMDMs (Usp44-/- BMDMs (Usp44-/- mouse lung fibroblasts (MLFs) (To further investigate the roles of USP44 out mice . Usp44 m/- cells . qPCR ex/+ BMDMs . In addi/+ BMDMs . In cont/+ BMDMs . Consist/- BMDMs . Similars (MLFs) .in vivo, wild-type and Usp44 knockout mice were intra-peritoneally infected with HSV-1 at lethal or non-lethal dose. While the concentrations of serum cytokines including IFN-β, IL-6 and CXCL10 were obviously lower in Usp44-/- than that in the wild type mice  (Usp44 in mouse BMDMs inhibited cGAMP-triggered transcription of downstream genes (Usp44-/- MLF cells (iMLFs) with Flag-tagged USP44 (USP44-Flag) by retrovirus-mediated transduction. Using this cell line, we found that USP44 was associated with MITA after HSV-1 infection  . Interesam genes . These ram genes . Unfortunfection . USP44 hnfection . Since MIFNB promoter (Usp44-/- iMLFs with wild-type USP44 or USP44(C282A) respectively. qPCR results showed that HSV-1-induced transcription of Ifnb1, Il6 and Cxcl10 genes was rescued by reconstitution of USP44 but not USP44(C282A) , a deubiquitinase inactive mutant of USP44, lost its ability to enhance cGAS-MITA-mediated activation of the promoter . qPCR expromoter . Consistpromoter . To furt4(C282A) . These rIn vitro experiments confirmed that USP44 but not USP44(C282A) directly deubiquitinated MITA (We next determined whether USP44 can deubiquitinate MITA. We found that USP44 but not USP44(C282A) caused deubiquitination of MITA . In vitrted MITA . Conversted MITA .Usp44-/- cells. The results showed that knockout of USP44 potentiated K48- but not K63-linked ubiquitination of endogenous MITA  of MITA was targeted by USP44. We performed deubiquitination assays with MITA and a series of its KR mutants where the lysine residues have been individually mutated to arginine. The results showed that among all the KR mutants of MITA, the K236R mutant was resistant to USP44-mediated deubiquitination while the K150R and K224R mutants also exhibited minimal resistance, suggesting that USP44 mainly deubiquitinates MITA at K236 . Taken tUsp44-/- MLFs compared with the wild-type cells. Importantly, such aggravation was rescued by reconstitution of Usp44-/- MLFs with USP44 but not USP44(C282A), or by proteasomal but not autophagic or lysosomal inhibitors  resisted USP44-mediated deubiquitination. Previous studies have reported that E3 ligases RNF5, TRIM30α and TRIM29 mediate K48-linked polyubiquitination of MITA at K150, K275, K288, K337 and K370 [Several lines of evidence suggest that USP44 deconjugates K48-linked polyubiquitin from K236 of MITA after viral infection, which in turn inhibits proteasomal degradation of MITA. First, USP44 deubiquitinated MITA both and K370 –47, howePreviously, it has been demonstrated that USP13 removes K27-linked polyubiquitin chains from MITA and impairs its recruitment of TBK1, contributing to inhibition of innate immune response to DNA viruses . It has Previous studies have shown that USP44 plays roles in tumorigenesis, cell cycle and DNA damage response , 43, 57.Dual-specific luciferase assay kit (Promega); RNase inhibitor, lipofectamine 2000 (Thermo); GeneMute siRNA Transfection Reagent (SignaGen Laboratories); SYBR (Bio-Rad); polybrene (Millipore); 2’3’-cGAMP (InvivoGen); recombinant IFN-β (R&D Systems); HSV120, ISD (Sangon Biotech); and ELISA kits for murine IFN-β and IL-6 (BioLegend) and CXCL10 (Boster) were purchased from the indicated manufacturers.Rabbit monoclonal antibodies against p-TBK1, p-IRF3, p-IKKβ, p-p65, MITA ; TBK1 (Abcam); β-actin ; mouse monoclonal antibodies against Flag (Sigma); HA (OriGene); and ubiquitin (Santa Cruz Biotehcnology) were purchased for the indicated manufacturers.HSV-1 (KOS strain) was purchased from China Center for Type Culture Collection. Human embryonic kidney (HEK293T) (Cat # CRL-11268) and human peripheral blood monocyte (THP-1) (Cat # TIB-202) cells were purchased from ATCC. Human foreskin fibroblasts (HFFs) were provided by Dr. Min-Hua Luo (Wuhan Institute of Virology).IFNB promoter, ISRE and NF-κB reporter were previously described [Expression plasmids for USP20-His, CYLD-His, USP44-His, USP20-Flag, CYLD-Flag, USP44-Flag, USP44-Myc and its mutant were constructed by standard molecular biology techniques. Expression plasmids for HA-tagged cGAS, MITA, TBK1, IRF3, ubiquitin and ubiquitin mutants, and Flag-tagged MITA, MITA mutants, and the escribed –63.HEK293T cells were transfected using a standard calcium phosphate precipitation protocol as previously described –66.The HEK293T cells were transfected with two packaging plasmids (pGAG-Pol and pVSV-G) together with empty vector, or the indicated plasmids respectively by calcium phosphate precipitation. Twenty-four hours later, medium was replaced. Fourty-eight hours later, the recombinant virus-containing medium was then filtered with 0.45 μm filter and added to THP-1 or iMLF cells in the presence of polybrene (8 μg/mL). Twenty-four hours post infection, cells were selected with puromycin (1 μg/mL) for 7 days before experiments.Total RNA was isolated for qPCR analysis to measure mRNA levels of the indicated genes as previously described , 67–69. Human GAPDH: Forward-GACAAGCTTCCCGTTCTCAG;Reverse-GAGTCAACGGATTTGGTGGT.Human USP44: Forward-CGTATGTGACCAGTGTAA;Reverse-GTGGCATATCATAAGTTGTT.Human IFNB1: Forward-TTGTTGAGAACCTCCTGGCT;Reverse-TGACTATGGTCCAGGCACAG.Human IFIT1: Forward-TCATCAGGTCAAGGATAGTC;Reverse-CCACACTGTATTTGGTGTCTACG.Human IL6: Forward-GCCGCATCGCCGTCTCCTAC;Reverse-CCTCAGCCCCCTCTGGGGTC.Human CXCL10: Forward-GGTGAGAAGAGATGTCTGAATCC;Reverse-GTCCATCCTTGGAAGCACTGCA.Murine Gapdh: Forward-ACGGCCGCATCTTCTTGTGCA;Reverse-ACGGCCAAATCCGTTCACACC.Murine Usp44: Forward-CCACTTCCCAAAGGAGACTTAT;Reverse-CACTGGTATCTCTCTGGAAACTC.Murine Ifnb1: Forward-TCCTGCTGTGCTTCTCCACCACA;Reverse-AAGTCCGCCCTGTAGGTGAGGTT.Murine Ifit1: Forward-ACAGCAACCATGGGAGAGAATGCTG;Reverse-ACGTAGGCCAGGAGGTTGTGCAT.Murine Il6: Forward-TCTGCAAGAGACTTCCATCCAGTTGC;Reverse-AGCCTCCGACTTGTGAAGTGGT.Murine Cxcl10: Forward-ATCATCCCTGCGAGCCTATCCT;Reverse-GACCTTTTTTGGCTAAACGCTTTC.HSV-1 ICP22: Forward-TGTTTGGAGACCAGACGGTA;Reverse-CATCGGAGATTTCATCATCG.HSV-1 ICP27: Forward-GGCCTGATCGAAATCCTAGA;Reverse-GTCAACTCGCAGACACGACT.HEK293T cells were lysed in 1mL NP-40 lysis buffer . For each immunoprecipitation, 0.4 ml aliquot of the lysate was incubated with control IgG or the indicated antibody (0.5 μg) and 15 μL of Protein G Sepharose  at 4°C for 3 h or overnight. The Protein G sepharose beads were washed three times with 1 mL of lysis buffer containing 0.5 M NaCl. Immunoblot analysis was performed by standard procedures.Double-stranded oligonucleotides corresponding to the targeting sequences were cloned into the pSuper-Retro-RNAi plasmid (Oligoengine) as previously described –72. The g for 5 min. The cells were then plated in culture medium , 50 U/ml penicillin, 50 μg/ml streptomycin, 15 mM HEPES, 2 mM L-glutamine). For generation of immortalized MLFs, MLFs were infected with SV40 in the presence of polybrene (8 μg/mL), 24 h later cells were cultured with fresh medium and immortalized cells were selected.Bone marrow cells were isolated from tibia and femur. For preparation of bone marrow-derived macrophages (BMDMs), bone marrow cells were cultured in 10% M-CSF-containing conditional medium for 5 days. For murine lungs fibroblasts (MLFs), lungs were minced and digested in calcium and magnesium free HBSS containing 10 μg/ml type II collagenase and 20 μg/ml DNase I for 1 h at 37°C with shaking. Cell suspension was centrifuged at 500 Usp44-/- mice was confirmed by sequencing PCR products amplified from the genomic DNA isolated from mouse tails using the following primers: #1: 5’- CTCCAATTCCGATCATATTCAATAAC-3’, #2: 5’-CCTCATCCCCACTTCCCAAAGGAG-3’ and #3: 5’-CTCTCTCCCCCATAAAGCCACTGC-3’.The genotype of the Usp44+/+ and Usp44-/- mice were intra-peritoneally (i.p.) or intra-nasally (i.n.) infected with HSV-1. The serum was collected at 6 h post infection for measurement of IFN-β, IL-6 and CXCL10 by ELISA. The viability of the infected mice was monitored for 9 days.Eight-week-old g for 30 min, and the supernatants were collected for plaque assays as previously described [The brain from HSV-1 infected mice were collected 5 days post infection. The brains were weighed and homogenized with PBS, followed by centrifugation at 1620 escribed , 62, 67.HEK293T cells were transfected with USP44-Flag for 24 h, nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo 78835).g for 10 min and the supernatant was centrifuged at 5000 g for 10 min. The supernatant from this step was further centrifuged at 20000 g for 30 min. The pellet was membraneous fraction and the supernatant was cytosol.For isolation of membraneous and cytosolic fractions, cells were washed with PBS and lysed by bouncing for 50 times in a homogenization buffer . The homogenate was centrifuged at 500 Cells were lysed with the lysis buffer (100 μl) and the supernatants were denatured at 95°C for 5 min in the presence of 1% SDS. The denatured lysates were diluted with NP-40 lysis buffer until the concentration of SDS was reduced < 0.1% followed by immunoprecipitation with the indicated antibodies.Denature-IP was performed to obtain ubiquitin-modified MITA from HEK293T cells co-transfected with Flag-tagged MITA and HA-Ubiquitin. The immunoprecipitates were eluted by Flag peptide . USP44 or USP44(C282A) protein was obtained utilizing an in vitro transcription and translation kit (Promega). The ubiquitin-modified Flag-MITA was incubated with USP44 or USP44(C282A) at 37°C for 2 h followed with overnight incubation at 16°C in the presence of ATP (1 μM). The mixtures were analyzed by immunoblots with the indicated antibodies.All mice were housed in the specific pathogen-free facility and viral infection experiments were carried out in an ABSL-2 facility at Wuhan Institute of Virology. The experimental protocol was adhered to the International Guiding Principles for Biomedical Involving Animals. The protocol for animal experiments were approved by the Institutional Animal Care and Use Committee of Wuhan Institute of Virology .Student’s t test was used for statistical analysis with Microsoft Excel and GraphPad Prism Software, P<0.05 was considered significant. For the mouse survival study, Kaplan-Meier survival curves were generated and analyzed by Log-Rank test.S1 Fig5) were co-transfected with the IFNB promoter (0.05 μg) and empty vector or independent human DUB cDNA expression plasmids (0.05 μg) for 24 hours. Cells were then left uninfected or infected with HSV-1 (MOI = 1) for 12 hours before luciferase assays were performed. Graphs show mean ± S.D. n = 3. *P < 0.05, **P < 0.01 (Student’s t-test).HEK293 cells (1 x 10(TIF)Click here for additional data file.S2 Fig5) stably expressing USP44-RNAi were transfected with ISD (2 μg/ml) for 12 hours before qPCR analysis. Graphs show mean ± S.D. n = 3. *P < 0.05, **P < 0.01 (Student’s t-test).THP-1 cells (4 x 10(TIF)Click here for additional data file.S3 Fig(A) Genotyping of Usp44 knockout mice. (B) BMDMs  or MLFs  were left uninfected or infected with HSV-1 (MOI = 1) for the indicated times, followed by qPCR analysis. (C) BMDMs (4 x 105) were left uninfected or infected with VACV for the indicated times before qPCR analysis. (D) BMDMs (4 x 105) were transfected with ISD (2 μg/ml) for the indicated times before qPCR analysis. (E) MLFs (4 x 105) were infected with HSV-1 (top) (MOI = 1) or transfected with HSV120 (middle) or ISD (bottom) (2 μg/ml) for the indicated times before qPCR analysis were performed. (F) MLFs (4 x 105) were left uninfected or infected with SeV (MOI = 1) for 6 h before qPCR analysis. (G) MLFs (4 x 105) were infected with HSV-1 (MOI = 1) for the indicated times, followed by immunoblot with the indicated antibodies. Graphs show mean ± S.D. n = 3. *P < 0.05, **P < 0.01 (Student’s t-test).(TIF)Click here for additional data file.S4 Fig6) were transfected with USP44-Flag plasmids for 24 h, followed by nuclear and cytoplasmic extraction.HEK293T cells (2 x 10(TIF)Click here for additional data file.S5 Fig(A) HEK293T cells (1 x 105) were co-transfected with empty vector or cGAS and MITA, IFN-β (0.05 μg), ISRE (0.05 μg) or NF-κB (0.01 μg) reporter plasmids, and the indicated plasmids for 24 hours before luciferase assays were performed. (B) HEK293T cells (1 x 106) were transfected with the indicated plasmids for 24 hours before deubiquitination assays were performed.(C) iMLF stable cell lines (4 x 105) were left uninfected or infected with HSV-1 (MOI = 1) for the indicated times before qPCR analysis. Graphs show mean ± S.D. n = 3. *P < 0.05, **P < 0.01 (Student’s t-test).(TIF)Click here for additional data file.


C. elegans histone methyltransferase responsible for H3K9me1 and me2 deposition. We used molecular, genetic, and biochemical methods to investigate the biological role of SMRC-1 and to explore its relationship with MET-2. SMRC-1, like its mammalian ortholog SMARCAL1, provides protection from DNA replication stress. SMRC-1 limits accumulation of DNA damage and promotes germline and embryonic viability. MET-2 and SMRC-1 localize to mitotic and meiotic germline nuclei, and SMRC-1 promotes an increase in MET-2 abundance in mitotic germline nuclei upon replication stress. In the absence of SMRC-1, germline H3K9me2 generally decreases after multiple generations at high culture temperature. Genetic data are consistent with MET-2 and SMRC-1 functioning together to limit replication stress in the germ line and in parallel to promote other germline processes. We hypothesize that loss of SMRC-1 activity causes chronic replication stress, in part because of insufficient recruitment of MET-2 to nuclei.Histone modifications regulate gene expression and chromosomal events, yet how histone-modifying enzymes are targeted is poorly understood. Here we report that a conserved DNA repair protein, SMRC-1, associates with MET-2, the  Caenorhabditis elegans, we show that a conserved histone-modifying enzyme, MET-2, associates with a conserved DNA repair protein, SMRC-1. In mammals, the SMRC-1 homolog, SMARCAL1, participates in repairing DNA that is damaged during replication. Focusing on the tissue responsible for production of sperm and eggs, the germ line, we find that SMRC-1 protects cells from DNA replication stress and promotes the accumulation of nuclear MET-2. Moreover, SMRC-1 affects MET-2 germline activity (as measured by histone modification state) in populations grown for multiple generations at stressful culture temperatures. Genetic analysis indicates that MET-2 and SMRC-1 participate in a common mechanism to limit DNA damage in the germ line. We propose that histone modifications are regulated to promote DNA replication and DNA repair.Post-translation modifications to histone proteins are known to regulate gene expression and chromosomal events such as recombination. Histone modifications are highly dynamic and are deposited by large number of histone-modifying enzymes. Little is known about how these enzymes are regulated. Using a model system, the nematode  Repetitive sequences pose challenges to genome integrity during DNA replication, DNA repair, and transcription. In eukaryotes, repetitive genomic regions typically adopt a condensed chromatin structure that is thought to limit potentially harmful consequences of repetitive sequences by limiting transcription, stabilizing DNA to promote correct repair of DNA breaks, and limiting formation of secondary structures that would otherwise impede DNA replication –4. InappC. elegans, different repetitive sequences accumulate H3K9me2 and/or H3K9me3  lines at 20°C. omIs1 rescues the met-2(n4256) brood size.(B) Wildtype (strain N2) tissue serves as a negative control for anti-FLAG immunolabeling. (C) Immunolabeling of H3K9me2 in N2 wildtype and CRISPR-tagged (TIF)Click here for additional data file.S2 Figsmrc-1 M-Z- population and a much larger 92% of the met-2 smrc-1 M-Z- population. *, Includes all individuals with somatic gonad defects. (B) Examples of adult mutant hermaphrodites labeled with the DNA dye, DAPI, to visualize germ cell morphology. Relevant germline features are labeled. *, distal end of gonad arm. (C) CED-1::GFP expression in adult smrc-1 met-2 M-Z- hermaphrodite germ lines. Images show representative examples of the three different CED-1::GFP expression patterns in smrc-1 met-2 M+Z- individuals raised at 25°C. Upper panels, differential contrast interference (DIC) images. Lower panels, GFP expression. Left, the gonad arm failed to extend, and a small cluster of germ cells is present adjacent to the vulva. Arrow, proximal germ cells undergoing engulfment. Middle, CED-1::GFP is present throughout the gonad arm indicating extensive apoptosis. Right, CED-1::GFP is not visible. N = 54.(A) Distribution of germline defects in F2 M-Z- mutants. N, number of sterile gonad arms evaluated. Note that sterile hermaphrodites represent only ~8% of the total (TIF)Click here for additional data file.S3 Figsmrc-1, spo-11 and smrc-1;spo-11 mutants. Diagram represents a hermaphrodite germline where the nuclei in leptotene–pachytene have been evenly divided into six zones based on cell row counts. The key indicates the percentage of total nuclei containing the indicated number of RAD-51 foci. (B) Recombination frequency was mapped in two genetic intervals in the chromosome I gene cluster defined by unc-11 dpy-5 (genetic map position -2.51 to 0.00) and dpy-5 unc-13 (genetic map position 0.00 to +2.07). Wildtype and smrc-1(om136) animals were assayed in parallel. Recombination frequency was calculated according to Brenner [smrc-1(-) relative to wildtype. (D) Overall crossover distribution in smrc-1 mutants resembles wildtype except in interval 4. * P<0.03. Data are presented as % (number of events).(A) SMRC-1 activity impacts the distribution of RAD-51 foci during meiotic prophase. Data are summarized for wildtype and  Brenner . (C) Who(TIF)Click here for additional data file.S4 FigGermline tissue co-labeled with anti-MYC and anti-FLAG, counterstained with DAPI, and visualized with confocal microscopy. Pairwise combinations of DNA, MET-2, and SMRC-1 labeling are shown for (A) pachytene and (B) proliferative germ cells. Note that (A) includes the same tissue shown without DNA labeling in (TIF)Click here for additional data file.S5 FigSMRC-1 abundance in distal germ cell nuclei increases upon exposure to hydroxyurea. Box-and-whisker plots represent the mean anti-FLAG immunolabeling intensity as normalized to (left) the mean DAPI fluorescence intensity and (right) the mean anti-H3 fluorescence intensity. These data complement and are consistent with normalization data presented in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 DataSpreadsheet contains the numeric data for graphs and statistics contained in Figs (XLSX)Click here for additional data file.