COS-7 and HEK293T cells were cultured at 37°C in Dulbecco's modified MEM (DMEM. Gibco-BRL) supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco-BRL).

Mouse anti-Flag, anti-Myc, anti-hemagglutinin (HA), and anti-β-actin monoclonal antibodies (mAbs) were purchased from ABmart. Rabbit anti-Flag polyclonal antibody (pAb) was purchased from Proteintech. Mouse non-specific control IgG antibody was purchased from eBioscience Inc. Rabbit anti-gD pAb was gifted by Dr. Roselyn J. Eisenberg (School of Veterinary Medicine, University of Pennsylvania), and mouse anti-gD mAb was purchased from Santa Cruz Biotechnology.

The ORF of viperin was amplified by PCR using pViperin-Flag expression plasmid (provided by Dr. Yi-Ling Lin, Genomics Research Center, Academia Sinica, Taiwan) (18) as the template, which was then cloned into pEGFP-N1 (Clontech) to yield pEGFP-viperin. The US6 ORF of HSV-1 (F strain) glycoprotein D (gD) was also amplified from HSV-1 DNA pYEbac102 (37, 38), with forward primer 5′-AGG AAT TCA TGG GGG GGG CTG CCG CCA GG-3′ and reverse primer 5′-CGG GAT CCT TGT AAA ACA AGG GCT GGT G-3′. The purified PCR product was digested with EcoRI and BamHI and then inserted into the corresponding digested pEYFP-N1 (Clontech) to yield plasmid pgD-EYFP, as described previously (39–43). Reporter plasmids pNF-κB-Luc, pIFN-β-Luc and pRL-TK were offered by Dr. Chunfu Zheng (School of Basic Medical Sciences, Fujian Medical University) (40–43). Ubiquitin expression plasmids pEFIRES-HA-Ub, pEFIRES-HA-Ub (K48) and pEFIRES-HA-Ub (K63) were provided by Dr. Jun Cui (School of Life Sciences, Sun Yat-sen University) (44). Other expression plasmids including IRAK1-HA (Dr. Hongyan Wang, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) (45), pCMV-Flag-IRAK1 (Dr. Hongbin Shu, School of Life Sciences, Wuhan University), TRAF6-myc (Dr. Jiahuai Han, School of Life Sciences, Xiamen University) (46), Flag-tagged IRF3/5D (Dr. Rongtuan Lin, Department of Medicine, McGill University) (47), Flag-tagged IRF7/6D (Dr. John Hiscott, Lady Davis Institute, Jewish General Hospital) (48), pcDNA3.1-gD (Dr. Gary H. Cohen, University of Pennsylvania), pECFP-Golgi (Dr. Suzanne R. Pfeffer, Department of Biochemistry, Stanford University School of Medicine) (49), mCherry-KDEL (Dr. Lee H. Wong, Department of Biochemistry and Molecular Biology, Monash University; Dr. Philippe Collas, Institute of Basic Medical Sciences, University of Oslo) (50) and TOM70-CFP (Dr. Frits Kamp, Adolf-Butenandt-Institute, Ludwig-Maximilians-University) (51) were gifts from the providers shown as indicated.

COS-7 cells were grown overnight to 80% confluence on microscopy cover glass (NEST) placed in six well plate (Corning), then plasmid transfection and fluorescence microscopy experiments were carried out as described previously (40–43, 52). Briefly, COS-7 cells were transfected with the indicated plasmids DNA mixed with polyethylenimine (PEI) transfection reagent (Polysciences) according to the manufacturer's instructions. Twenty-four hours post-transfection, cells were fixed with 4% (v/v) paraformaldehyde (Beyotime Biotechnology) for 20 min at room temperature, washed for 3 times with PBS, and incubated with 0.2% Triton X-100 (Beyotime Biotechnology) for 30 min. Subsequently, cells were incubated with rabbit anti-gD pAb or mouse anti-Flag mAb, followed by incubation with tetramethyl rhodamine isocyanate (TRITC)-conjugated goat anti-rabbit IgG (Pierce) or fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Sigma-Aldrich), then stained with or without Nile Red (Sigma) for lipid droplets for 30 min, and finally stained with DAPI (4′6-diamidino-2-phe-nylindole) (Cell Signaling Technology) for 5 min when needed. Images were obtained with a confocal microscope (Axio-Imager-LSM-800, ZEISS, Germany) using a 600× oil-immersion objective. Each image represents a vast majority of the cells with similar subcellular distribution, and white color shows the co-localization of colors merged with green, blue and red, yellow color shows the co-localization of colors merged with green and red. All scale bars indicate 10 um.

The DLR assays were performed as described previously (38, 40–42, 53). In short, HEK293T cells were plated on 24 well dish (Corning) at a density of 1 × 10⁵ cells per well-overnight before transfection. Cells were then co-transfected with 100 ng of the indicated expression plasmid, 100 ng of IFN-β or NF-κB promoter reporter and 10 ng of pRL-TK (internal control) to normalize transfection efficiency. Twenty-four hours post-transfection, the luciferase activity was detected with a luciferase assay kit (Promega).

HSV-1 bacterial artificial chromosome (BAC) Luc (F strain, synchronally expressing firefly luciferase, and GFP fluorescent protein) was offered by Dr. Chunfu Zheng (54), which was reproduced and reposited in our lab. HEK293T cells were plated on 12 well-plate (Corning) overnight before infection, then HSV-1 BAC Luc was dissolved in DMEM medium and added to the cells at an MOI (multiplicity of infection) of 1. The virus was incubated for 1.5–2 h at 37°C in a 5% CO₂ incubator and replaced with medium supplemented with 2% FBS to continue culture for the indicated times, then cells were harvested for luciferase reporter assays to determine the replication kinetics of HSV-1 (33, 34). Here, all experiments related to HSV-1 infection were carried out in the Biosafety Level II laboratory, and all operations were strictly performed in accordance with the biosafety operation requirements of Guangzhou Medical University.

HEK293T cells cultured in 6 well plate were transfected with indicated amounts of expression plasmid. Twenty-four hours post-transfection, total RNA was extracted with TRIzol reagent (Invitrogen). Samples were then subjected to reverse transcription to cDNA with RT reagent (TSINGKE). The acquired cDNA was taken as a template for qPCR, to detect the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (internal control) and IFN-β, using a qPCR instrument (BIO-RAD, CFX96). Primers used for GAPDH (forward primer 5′-AGG TCG GTG TGA ACG GAT TTG-3′ and reverse primer 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′) and IFN-β (forward primer 5′-ATGACCAACAAGTGTCTCCTCC-3′ and reverse primer 5′- GGAATCCAAGCAAGTTGTAGCTC-3′) were referred to Bing Tian's report (55).

Co-IP assays were performed as previously described (40–42, 56–58). In brief, HEK293T cells were co-transfected with expression plasmids combination bearing EYFP, Flag, Myc or HA tag. Twenty-four hours post-transfection, transfected cells were infected with or without HSV-1 BAC Luc for 16 h, then cells were collected and lysed on ice with RIPA lysis buffer (Beyotime Biotechnology). For each immunoprecipitation (IP), an equivalent of lysate was incubated with mouse anti-Flag, anti-Myc or anti-HA mAb or non-specific control mouse antibody (IgG) and a 1:1 slurry of protein A/G PLUS-Agarose (Santa Cruz Biotechnology) at 4°C overnight. The Sepharose beads were then washed at least three times with lysis buffer added with 500 mM NaCl. Finally, immunoprecipitated proteins and cell lysates were subjected to immunoblotting (IB) assays with the indicated antibodies. The original IB results are shown in the Supplementary Material.

Statistical analyses were performed using Graphpad Prism 6 software. All data were normally distributed, and the homogeneity of variances was examined with Levene's test. As the samples were normally distributed and displayed homogenous variance, statistical analyses were performed using one-way ANOVA. In the event of a difference being present, Bonferroni-adjusted post hoc tests were performed to identify specific effect. Moreover, Student t test (unpaired two-tailed t-test) was used when needed. Data were expressed as means and standard deviations (mean ± SD) from three independent experiments, with significant differences marked on the figures. Significance levels were defined as ns, not significant, P > 0.05; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; and ^****P < 0.0001.

To find out which HSV-1 protein may interact with viperin, some HSV-1 encoded cytoplasmic localization proteins (2) were firstly screened, by co-transfection of viperin and HSV-1 protein expression plasmids and analyzing which HSV-1 protein can co-localize with viperin or alter its subcellular localization, and gD (US6), US4 (gG), and UL1 (gL) were identified. Our preliminary experiments found that there were significant differences in the interaction mechanisms among viperin-gD, viperin-US4 and viperin-UL1 (unpublished data). Therefore, the in-depth study of the interaction mechanisms between viperin and each protein of gD, US4, or UL1 would be an independent big project, and they need to be investigated separately. In addition, gD, US4 or UL1 encode glycoproteins, they (especially gD) play a very important role in the invasion of HSV-1. Accordingly, gD was firstly selected to investigate the potential interaction mechanism with viperin. To this end, pEGFP-viperin, pViperin-Flag, pgD-EYFP, or pcDNA3.1-gD expression plasmid was individually transfected into COS-7 cells to characterize their subcellular localizations in live cells or chemically fixed cells. As shown in Figure 1, viperin was absolutely distributed in the cytoplasm in cells transfected with EGFP-Viperin (Figure 1A) or Viperin-flag (Figure 1C) expression plasmid, and gD mainly exhibited nuclear membrane or cytoplasmic membrane localization in cells transfected with gD-EYFP (Figure 1B) or 3.1-gD (Figure 1D) expression plasmid, which are consistent with previous studies (59–61). In an attempt to pursue whether gD binds to viperin, EGFP-Viperin, and gD-EYFP expression plasmids were co-transfected into COS-7 cells to detect whether gD co-localizes with viperin, since co-localization experiment is one of the important and popular methods to detect the potential interaction between different proteins. As results, gD co-localized with viperin and predominantly accumulated at the perinuclear region (Figure 1E, yellow signal). Furthermore, IFA also proved the co-localization of gD and viperin at the perinuclear region (Figure 1F, yellow signal), confirming the potential interaction between gD and viperin.

To further prove the interaction between gD and viperin, Co-IP assays were carried out. HEK293T cells were co-transfected with pcDNA3.1-gD and pViperin-Flag expression plasmids, then cell lysates were immunoprecipitated with anti-Flag mAb or non-specific control mouse IgG. As a result, gD was immunoprecipitated by Viperin-Flag with anti-Flag mAb (Figure 2A), whereas no such protein was immunoprecipitated with the control mouse IgG (Figure 2A). As negative controls, HEK293T cells were co-transfected with plasmids combination pcDNA3.1-gD/Flag vector (Figure 2B) or pViperin-Flag/pcDNA3.1 vector (Figure 2C). Then, cell lysates were immunoprecipitated with anti-Flag mAb or mouse IgG. Similarly, no target protein was immunoprecipitated by Flag vector (Figure 2B) or Viperin-Flag (Figure 2C), indicating gD could interact with viperin.

To continue determine the interaction between gD and viperin in the context of viral infection, HEK293T cells were transfected with pViperin-Flag expression plasmid, then infected with HSV-1 at an MOI of 1. Subsequently, cells were collected and Co-IP assays were performed. As shown in Figure 2D, gD again was immunoprecipitated by Viperin-Flag with anti-Flag mAb, whereas no such protein was immunoprecipitated with control mouse IgG, confirming gD could interact with viperin under physiological condition.

It is known that the N-terminal amphipathic a-helix is important for viperin to target to ER (60) and lipid droplets (62), and this subcellular localization is essential for suppressing viral replication (63). However, the vMIA-mediated mitochondria localization of viperin is favorable for HCMV replication (13). In order to probe the underlying mechanism of gD and viperin interaction, we continued to analyze whether gD can alter the normal localization of viperin. As control, GFP-Viperin or gD-EYFP was transiently co-transfected with the subcellular marker expression plasmid of TOM70-CFP (Mitochondrial marker) (Figures 3A,E), ECFP-Glogi (Golgi marker) (Figures 3B,F) or mCherry-KDEL (ER marker) (Figures 3C,G) into COS-7 cells, or cells were stained with Nile Red for lipid droplets (Figures 3D,H), and the cells were subsequently examined by confocal microscopy, to test the normal subcellular localizations of viperin and gD. As expected, viperin could co-localize with mCherry-KDEL (Figure 3C, yellow signal) and lipid droplets (Figure 3D, yellow signal), but not TOM70-CFP (Figure 3A) or ECFP-Glogi (Figure 3B) (13, 60, 62). However, gD-EYFP could not co-localize with all of the mentioned subcellular markers (Figures 3E–H). Then, expression plasmids combination of gD-EYFP/EGFP-viperin were transiently co-transfected with TOM70-CFP (Figure 3I), ECFP-Golgi (Figure 3J), or mCherry-KDEL (Figure 3K) into COS-7 cells, or cells were stained with Nile Red for lipid droplets. As results, co-expression of gD and viperin resulted in a pronounced co-localization with Golgi and lipid droplets markers (Figures 3J,L, white signal). Nevertheless, no obvious overlap area could be detected when gD and viperin were co-transfected with expression plasmid TOM70-CFP (Figure 3I) or mCherry-KDEL (Figure 3K). Therefore, viperin could accumulate at the Golgi apparatus and lipid droplets in the presence of gD.

It is reported that viperin can enhance TLR7/9-dependent production of IFN-I (29). To examine whether gD and viperin interaction is involved in the regulation of IFN-β expression, expression plasmid of gD-EYFP or pViperin-Flag or plasmids combination of gD-EYFP and pViperin-Flag were co-transfected with or without expression plasmid IRF3/5D or IRF7/6D into HEK293T cells, along with pIFN-β-Luc and pRL-TK reporter plasmids. As shown in Figure 4, both IRF3/5D and IRF7/6D alone could activate IFN-β expression, but no IFN-β activity was detected when HEK293T cells were only co-transfected with gD-EYFP and pViperin-Flag (Figures 4A–C). gD or viperin alone or combination of gD and viperin did not affect IRF3/5D-induced IFN-β activity (Figure 4A), however, gD or viperin alone could enhance IRF7/6D-induced IFN-β activity (Figure 4C). More importantly, the co-existence of gD and viperin activated a higher IFN-β promoter activity than that of gD or viperin (~2-fold) (Figure 4C). To further explore whether gD facilitates IFN-β activity through IRF7 in the presence of viperin, experiments were carried out as described in Figure 4C except for the reporter plasmids, and IFN-β mRNA accumulation was measured by RT-qPCR. As a result (Figure 4D), the change tendency of IFN-β mRNA was consistent with the DLR result shown in Figure 4C, suggesting the gD and viperin interaction could promote IRF7 mediated interferon expression.

Saitoh's study shows that viperin can interact with the signal mediators IRAK1 and TRAF6, so as to recruit them to the lipid bodies, which can regulate TLR7- and TLR9-IRAK1 signaling axis to mediate the expression of functionally important immune molecules in plasmacytoid dendritic cell (29). To determine whether the gD and viperin interaction can affect the interaction between viperin and IRAK1, pViperin-Flag, and IRAK-HA expression plasmids were co-transfected with gD-EYFP or EYFP control vector into HEK293T cells, then cells were harvested and analyzed by Co-IP assays. In constrast to the EYFP control, the association of viperin and IRAK1 was enhanced in the presence of gD (Figures 4E,F).

It is shown that the viperin-related K63-linked polyubiquitination of IRAK1 is crucial for the TLR7- and TLR9-dependent IFN-β production (29). To probe whether gD and viperin interaction participates in the polyubiquitination of IRAK1, HA-tagged Ub (WT), Ub (K48), or Ub (K63) expression plasmid was co-transfected with plasmids pCMV-Flag-IRAK1 and pViperin-Flag into HEK293T cells, with or without the presence of gD-EYFP. Then, Co-IP assays were performed. As results, polyubiquitinated forms of IRAK1 were detected when cells were co-expressed with viperin and Ub (WT) (Figure 4G, lane 1, Figure 4H), and this polyubiquitination was reinforced in the presence of gD (Figure 4G, lane 2, Figure 4H). Notably, gD catalyzed IRAK1 polyubiquitination with the expression of Ub (K63) (Figure 4G, lane 4, Figure 4H), but not Ub (K48) (Figure 4G, lane 3, Figure 4H), indicating gD interacted with viperin to promote K63-linked IRAK1 polyubiquitination. In short, we demonstrated that gD could facilitate IFN-β production through enhancing the interaction of viperin with IRAK1 and increasing K63-linked polyubiquitination of IRAK1.

Viperin is proved to be involved in the activation of NF-κB and AP-1 in T cells (64). To detect whether gD and viperin interaction also can modulate the NF-κB activity mediated by the key regulatory component of NF-κB signaling pathway, TRAF6 (11), expression plasmid gD-EYFP or pViperin-Flag or plasmids combination of gD-EYFP and pViperin-Flag were co-transfected with or without TRAF6-myc expression plasmid into HEK293T cells, along with pNF-κB-Luc and pRL-TK reporter plasmids. As results, overexpression of TRAF6 efficiently activated the NF-κB reporter, but no NF-κB activity was tested when HEK293T cells were only co-transfected with plasmids gD-EYFP and pViperin-Flag (Figure 5A). The expression of gD or viperin alone did not affect TRAF6-induced NF-κB reporter activity, however, gD and viperin combination significantly inhibited TRAF6-induced NF-κB reporter activity. Additionally, gD and viperin interaction constrained TRAF6-induced NF-κB promoter activity in a gD dose-dependent manner (Figure 5B), suggesting gD could modulate TRAF6-mediated NF-κB activity through viperin.

Polyubiquitination has emerged as an important regulatory mechanism in NF-κB signaling, and TRAF6 acts as a key substrate of K63-linked polyubiquitin chains in TNFR pathway, which serves as a mechanism to recruit TAK1 and IKK kinases and finally stimulate downstream NF-κB activation (65). To investigate whether gD and viperin interaction can affect the polyubiquitination of TRAF6, HEK293T cells were co-transfected with TRAF6-myc, pViperin-Flag and HA-Ub (WT) or HA-Ub (K63) constructs, along with or without gD-EYFP expression plasmid. Then, Co-IP assays were performed. As shown in Figure 5, no apparent difference of the TRAF6 polyubiquitination in the presence of gD or gD and viperin combination (Figures 5C,D), indicating gD and viperin interaction could not inhibit the polyubiquitination of TRAF6.

To further elucidate a clear molecular mechanism of how gD and viperin interaction inhibits NF-κB activity, pViperin-Flag, and TRAF6-myc expression plasmids were co-transfected into HEK293T cells, along with gD-EYFP or EYFP control plasmid. Then, Co-IP assays were carried out. In contrast to the EYFP control, gD significantly reduced the interaction between TRAF6 and viperin, suggesting gD could inhibit TRAF6-mediated NF-κB activity through competive binding viperin with TRAF6 (Figures 5E,F). Taken together, these results support that gD downregulated NF-κB activity by reducing the interaction between TRAF6 and viperin, but not affecting the polyubiquitination of TRAF6.

It is documented that the signal mediators IRAK1 and TRAF6 can interact with each other at the lipid bodies (29). To test whether gD and viperin interaction affects the interaction between IRAK1 and TRAF6, IRAK1-HA and TRAF6-myc expression plasmids were co-transfected into HEK293T cells, along with pViperin-Flag or expression plasmids combination of gD-EYFP and pViperin-Flag. Then, Co-IP assays were performed. As results, overexpression of viperin alone indeed induced significantly stronger interaction of IRAK1 and TRAF6 (Figure 6A, lane 2, Figure 6B), since viperin is critical for the recruitment of IRAK1 and TRAF6 to lipid bodies, which are the transfer points of TLR7 and TLR9 signaling pathways (29). However, the IRAK1 and TRAF6 interaction became weaker in the presence of gD and viperin (Figure 6A, lane 3, Figure 6B), which was similar to that of the negative control (Figure 6A, lane 1, Figure 6B), indicating gD and viperin interaction could impede the interaction between IRAK1 and TRAF6.

In order to investigate the physiological significance of gD and viperin interaction during HSV-1 infection, HEK293T cells were mock-transfected or transfected with plasmid pViperin-Flag or plasmids combination of 3.1-gD and pViperin-Flag. Twelve hours post-transfection, cells were infected with HSV-1 BAC Luc at an MOI of 1 for 6, 12, or 24 h. Then, luciferase activity assays were performed to determine the replication kinetics of HSV-1. As shown in Figure 7, the luciferase activity gradually increased with the time extension of HSV-1 infection, and transfection with plasmid pViperin-Flag alone had no inhibitory effect on the HSV-1 propagation, which is consistent with our previous study (36). However, the HSV-1 proliferation was remarkably impaired when cells were co-transfected with 3.1-gD and pViperin-Flag expression plasmids. More importantly, this inhibitory trend was consistent at different time points of the infection (Figures 7A–C). Accordingly, these results indicated that the interaction between gD and viperin indeed could obstruct the reproduction of HSV-1.

TP was employed by company South China Vaccine Corporation Limited. The remaining 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.