| The first line of defense against viral infection is innate immune response that characterized with the interferon (IFN) production, which triggers the induction of a broad scope of antiviral proteins. Although some IFN-inducible proteins, for example protein kinase R (PKR), the GTPase Mxl, ribonuclease L (RNaseL), ISG15(IFN-stimulated protein of15kDa), APOBEC3G, Tetherin, and IFIT, have been functionally well characterized as antiviral effectors, the functions of most IFN-inducible proteins remain unknown. Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is an IFN-inducible protein that has recently received more and more attentions. It has a number of functions, from being an antiviral protein to modulating signaling events. Viperin inhibits the replication of a wide range of viruses, including influenza A virus, hepatitis C virus (HCV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Dengue virus (DENV). Intriguingly, Viperin also enhances human cytomegalovirus (HCMV) infectivity by remodeling the cellular actin cytoskeleton. The activity on lentiviruses, such as HIV-1, was appeared complicated. For instance, one of the studies showed that Viperin inhibited the replication of HIV-1via inhibiting the viral egressing in cultivated cells, but another one manifested that the restriction of Viperin only to certain specific strains of HIV-1. More investigations should be performed to have a better understanding on the interaction between Viperin and lentiviruses.Equine infectious anemia virus (EIAV) is a lentivirus similar to HIV-1and causes a persistent infection characterized by recurring febrile episodes associating viremia, fever, thrombocytopenia and wasting symptoms in equids. EIAV is macrophage tropic. Infected macrophages act as a reservoir of viral quasispecies and the source for spreading of virus throughout the course of EIAV infection. Because EIAV is one of the extensively studied lentiviruses, the antiviral activity of equine Viperin (eViperin) to EIAV infection and replication were examined and the molecular bases for these actions were investigated in a study presented here, which provided additional data for better understanding the biological behavior of Viperin.To characterize the restrictive activity of eViperin to lentivirus, the cDNA encoding this cellular factor was first cloned and its amino acid sequence was compared with sequences of other mammal Viperins, including human, rhesus macaque, mouse and swine. The first70residues are highly variable, which include the N-terminal a-helix domain. Of note, several leucine sites are conserved, which implicate that leucine are important for a-helix function. In addition, three cysteine residues (CxxxCxxC) in the radical SAM domain, which are important for antiviral activity, are conserved in the eViperin sequence. To examine if EIAV infection played a role in regulating eViperin expression in eMDM, a relative real-time reverse transcript (RT)-PCR analysis was performed. Results demonstrated that two up-regulated peaks appeared at24and96hours post infection and the latter peak was2.8-fold increased. The observation that eViperin expression was increased by EIAV infection prompted the investigation of interaction between this cellular factor and EIAV in the target cells. To this end, eViperin was over-expressed in eMDM to examine its effect on EIAV replication in these cells. Because the efficacy of transfecting expression plasmids to eMDM was low, an adenoviral vector was utilized to deliver eViperin cDNA expressing unit into eMDM. The EIAV produced in these cells was measured at48and72hours post infection. As indicated by viral reverse transcriptase (RT) activity, the replication of EIAV was reduced by approximate half by the recombinant eViperin72hours after the infection when compared with the virus grew in cells transfected with the empty vector. EIAV was inoculated to the cells transfected with specific siViperin to knockdown eViperin mRNA and then significantly enhanced the production of EIAVwhen compared with the controls of nonspecific siRNA and mock transfection.To determine the mechanism of eViperin for its inhibition of EIAV production, the Viperin expressing vector was co-transfected to293T with EIAV-CMV-3-8. The amounts of EIAV-CMV-3-8in the cell lysate and culture supernatant were measured for the protein levels of capsid (CA, p26) and its precursor Gag protein (p55) by Western blot at48post transfection. The viral structural proteins in the cell lysate were not affected by eViperin. However, the level of p26in virions budding to the culture supernatant was largely decreased by the expression of recombinant eViperin in a dose dependent manor. This result indicate that the EIAV budding was inhibited by eViperin. Furthermore, it was examined that if eViperin inhibited EIAV replication via interrupting the release of viral CA from cells. A codon optimized Gag expressing plasmid was co-transfected to HEK293T cells with increasing doses of the eViperin expression vector. The protein levels of Gag in the cell lysate and culture supernatant were examined by Western blot, which revealed that the EIAV Gag was progressively decreased in the supernatant, but not in the cell lysate, with the increasing expression levels of eViperin, indicating the impaired budding of Gag. To examine that if eViperin interrupted EIAV Gag budding by direct interaction with the viral protein, the intracellular co-localization of these two proteins was further examined. The intracellular eViperin and Gag were detected by specific antibodies labeled with fluorescents dyes and recorded by confocal microscopy. Images of fused dyes clearly indicated the co-localization of these molecules. To determine which domain(s) of eViperin was crucial for the suppression of EIAV Gag budding, the a-helix domain, radical SAM domain and conserved C-terminus were respectively deleted. Results remaining inhibitory activity of deletion mutated eViperin showed that both the a-helix domain and SAM domain, but not the C-terminus, were necessary for eViperin to reduce the EIAV Gag levels in culture supernatant. Because deletion of the radical SAM domain largely reversed eViperin inhibition to the Gag budding, but not the co-localization with the EIAV CA, it is presumable that the eViperin co-localized with EIAV Gag is necessary, but not sufficient, to inhibit the release of viral CA. The radical SAM domain was required for eViperin suppression of EIAV budding. In documented studies, mutation of cysteine residues in the first motif (S1) of radical SAM domain of human Viperin increased the infectivity of HCV, WNV and DENV in HEK293cells. The importance of three cysteine residues in this motif of eViperin in the antiviral activity against EIAV was examined in this study. Results of Western blot revealed that neither single nor all the three cysteine-residue mutations affected the eViperin activity against EIAV, which indicated that the anti-EIAV activity based on a molecular mechanism different from that of human Viperin against HCV.There are three SAM motifs in the human Viperin, each of which is essential for its anti-HIV-1activity investigated via a series of mutants of these three motifs of the SAM domains. Mutation of the first SAM motif (S1) did not affect the EIAV Gag budding, and the second one (S2) only partially reduced the density of the p55band. In contrast, the deletion of key residues in the third SAM motif (S3) abolished the restriction of eViperin to the EIAV Gag budding. In addition, combinatory deletion of S1and S2reversed the Viperin-mediated inhibition. Interestingly, although the mutant S1+S2+S3lost the inhibition to p55budding, the co-localization of eViperin with EIAV Gag was still observable, unchanged from that of the prototype Viperin, whereas S1+S2+S3mutations in human Viperin abolished its co-localization with HIV-1Gag. Studies on human Viperin revealed that this cellular factor effectively blocked the budding of HIV-1Gag, but no data for other HIV-1proteins have been documented. CA and Env are major structural proteins of HIV-1and EIAV, yet the maturation and packaging of these two proteins are associated with the cellular vascular membranes. The aforementioned results demonstrated that the eViperin inhibited the EIAV budding via impairing the production and secretion of the viral CA and its precursor Gag. It was therefore logical to investigate if eViperin also affect the production of EIAV Env. EIAV gp140is the precursor of the gp90surface protein and the gp45transmembrane protein. The effect of eViperin on the production and release of gp140was further investigated. EIAV gp140(Env) was co-expressed with increasing doses of eViperin in HEK293T cells, and the expressed gp140in the cell lysate and culture supernatant were examined using Western blot. Resultes showed that the intracellular level of Env was reduced with the increased expression of Viperin. Deletion of functional domains of eViperin revealed that the N-terminal a-helix domain, but not the SAM domain and conserved C-terminus domain, were essential for the inhibitory activity of eViperin to the Env expression and secretion. Here, the SAM domain appeared not needed for inhibition of the Env expression, but vital for the restriction of Gag, implicating a difference in the inhibitory mechanism. The EIAV non-structural proteins transactivator of transcription (Tat) and regulator of expression of viral proteins (Rev) are regulators of viral replication. To examine if eViperin also inhibited the production of these EIAV accessary proteins, HEK293T cell co-expressed either the EIAV Tat or Rev and increasing doses of eViperin. The protein levels of Tat and Rev in cell lysates were detected by Western blot, which showed no noticeable changes in the density of specific bands. A further analysis of mRNA by RT-PCR revealed that co-expression of eViperin did not influence the transcription of EIAV Env. The results imply that eViperin conduct restriction to EIAV structural proteins, but not non-structural accessary proteins, via a post-transcriptional mechanism, such as protein degradation. To investigated if ubiquitin-proteasome-dependant pathway or the lysosome-dependent pathway, the two major intracellular protein degradation pathways, were involved in the eViperin inhibition of EIAV structural proteins, these two pathways were inhibited by either MG132or chloroquine (inhibitors of ubiquitin-proteasome and lysosome pathway, respectively) in HEK293T cells that were co-expressing EIAV Env and eViperin. Results indicated that neither of these two inhibitors reversed the restriction of Viperin to EIAV Env expression, suggesting that translation or post-translational modification may be the step(s) that was susceptible to the eViperin inhibition. The appearance of condensed cytoplasmic vacuolar structures induced by the expression of recombinant eViperin was observed by confocal microscopy and this abnormal vacuolar structures disappeared when the a-helix domain of Viperin was deleted (which also abolished the inhibitory activity to EIAV Env and Gag). In contrast, deletions of either the SAM domain, which lost the inhibition to the Gag but not the Env expression, or the C-terminal domain, which did not impair the restriction to EIAV Env and Gag expression, did not affect the formation of condensed vacuolar structures. These results suggest that the eViperin anti-EIAV activity is associated with the plasma membrane, particularly the endomembrane system. It has been documented that equine lentivirus receptor-1(ELR1) is the sole receptor of EIAV, which locates on the cell membrane and mediates EIAV entry. Because aforementioned results revealed that eViperin inhibited the production and secretion of EIAV Gag and Env, and the procession and maturation of which were associated with the plasma membrane, it was of interest to investigate if eViperin also conducted inhibition on the EIAV entry via impairing ELR1production.To evaluate the effect of eViperin on EIAV entry, a HEK293T cell line (HEK293T/ELR1) contractually expressing ELR1was developed. An EIAV pseudotyped virus, which contained a luciferase reporter gene and packaged by EIAV CA and Env expressed by separated expression vectors, was generated to test the effect of eViperin on the EIAV entry. In addition, rhesus (rh) TRIM5a has been reported blocking EIAV entry in target cells, this restriction factor was applied here as a positive control. HEK293T/ELR1cells were transfected with either the expression vector for rhTRIM5a or eViperin, or an empty vector control. EIAV pseudotyped virus were inoculated24hours after the transfection. The luciferase activities detected24hours post infection demonstrated that both rhTRIM5a and eViperin significantly inhibited EIAV entry compared to the vector control. Next, the functional domains required for the eViperin inhibition of EIAV entry was examined. Consistent with the Viperin restriction of EIAV envelope production described previously, the a-helix domain was critical for EIAV entry as well. Because both the EIAV Env and ELR1are membrane-associated and Viperin appears impairing Env expression in a membrane-involved mechanism, we wondered if eViperin inhibited EIAV entry by decreasing ELR1production in the same way as to retard Env expression. To investigate, ELR1was co-expressed with the eViperin and protein levels of these two recombinant proteins were examined by Western blot. As expected, the expression of ELR1was largely impaired by the expression of eViperin in a dose-dependent manor. To further confirm that eViperin inhibition of EIAV infection was mediated by ELR1, a VSVG-pseudotyped EIAV was used to infect the HEK293T/ELR1cell line that was also transfected with either the eViperin or the rhTRIM5a positive control expression vectors. Titers of luciferase activity revealed that the infection of VSVG-pseudotyped EIAV was significantly inhibited by rhTRIM5α, but not by eViperin, indicating the importance of ELR1in the infection of EIAV reporter virus and the inhibitory effect of eViperin to EIAV infection was via the impairment of ELR1expression and therefore the retard of EIAV entry.Viruses have evolved to counteract host antiviral factors through various mechanisms. For example, the Vif protein of HIV-1antagonizes APOBEC3G via guiding ubiquitination on it, the Vpx protein encoded by HIV-2and related primate lentiviruses degrades SAMHD1by Vpx and Vpu protein of HIV-1antagonizes the restriction factor Tetherin by altering its normal cellular localization. It is interesting to examine if EIAV, a lentivirus similar to HIV and SIV, somehow counteracts the restriction of eViperin. In this study, HEK293T cells were co-transfected with eViperin expression vector and either a proviral DNA clone of EIAV or an empty vector. Results of Western blot showed that the co-expression of EIAV proteins dis not reduce the protein level of eViperin, indicating no direct degradation effect of EIAV proteins on eViperin. Proteins could be synthesized in cytoplasm or endoplasmic reticulum (ER) determined by where proteins were targeting. For EIAV Gag, Tat and Rev were synthesized in cytoplasm, so it expression were not affected by disruption of ER. However, for EIAV envelope and ELR1were membranes targeting proteins and were synthesized in endoplasmic reticulum, disruption of ER will affect the protein production. In previously study, influenza virus, HIV-1and HCMV replication was inhibited by viperin, we cannot rule out the possibility that viperin inhibit these viruses by down regulating it receptor. Interestingly, in HCMV infection, membrane associated protein (envelope glycoprotein, gB) synthesize also were inhibited by viperin. For α-helix was critical for decreasing envelope and ELR1production, and it was determinant for ER membrane morphology, these results are particularly consistent with previous studies that viperin can alter ER membranes and apparently induce ER membrane curvature. However, how was a-helix domain of viperin distort and curve ER membranes was unclear, whether this correlated with viperin could remodeling the cellular actin cytoskeleton need further investigation, and why distorted ER could decrease membrane associated protein synthesize. Resolution of these questions awaits additional experiments. Viruses have evolved various mechanisms to counteract antiviral factors. However, in our study, we did not find EIAV could counteract viperin. One can ask that why does EIAV directly induce a protein that would normally inhibit the virus replication? How does EIAV evade the effect of virus induced viperin? One possible explanation is that expression of viperin induced by virus up-regulated2.8fold, this may not sufficient to completed restriction of EIAV replication. For example, endogenous viperin may not enough to distort ER which critical for it antiviral activity. Another possible explanation is that EIAV perhaps could counteract viperin restriction other than degrade viperin. However, these need further exploration.In conclusion, our study revealed equine Viperin is an innate antiviral factor for EIAV. And we proposed a model that Viperin inhibit EIAV replication. First, Viperin inhibit EIAV Gag budding, both a-helix domain and radical SAM domain are necessary for this activity. However, inhibit Gag budding critical domain in SAM have some difference from inhibit HIV-1Gag report previously. Second, over-expression of Viperin inhibits synthesis of both EIAV envelope and EIAV receptor which inhibit virus production and virus entry, respectively. However, Viperin inhibits protein synthesis is a-helix domain dependent. These results indicated that Viperin may inhibit proteins expression that synthesized by endoplasmic reticulum (ER) pathway. |
