Vaccinia Virus E3 Protein Prevents the Antiviral Action of ISG15

The ubiquitin-like modifier ISG15 is one of the most predominant proteins induced by type I interferons (IFN). In this study, murine embryo fibroblast (MEFs) and mice lacking the gene were used to demonstrate a novel role of ISG15 as a host defense molecule against vaccinia virus (VACV) infection. In MEFs, the growth of replication competent Western Reserve (WR) VACV strain was affected by the absence of ISG15, but in addition, virus lacking E3 protein (VVΔE3L) that is unable to grow in ISG15+/+ cells replicated in ISG15-deficient cells. Inhibiting ISG15 with siRNA or promoting its expression in ISG15−/− cells with a lentivirus vector showed that VACV replication was controlled by ISG15. Immunoprecipitation analysis revealed that E3 binds ISG15 through its C-terminal domain. The VACV antiviral action of ISG15 and its interaction with E3 are events independent of PKR (double-stranded RNA-dependent protein kinase). In mice lacking ISG15, infection with VVΔE3L caused significant disease and mortality, an effect not observed in VVΔE3L-infected ISG15+/+ mice. Pathogenesis in ISG15-deficient mice infected with VVΔE3L or with an E3L deletion mutant virus lacking the C-terminal domain triggered an enhanced inflammatory response in the lungs compared with ISG15+/+-infected mice. These findings showed an anti-VACV function of ISG15, with the virus E3 protein suppressing the action of the ISG15 antiviral factor.


Introduction
Type I interferons (IFN-a and -b) serve a critical role in antiviral innate immunity and in modulating the adaptive immune response to infection and tumor development [1].In response to infection or Toll-like receptor agonists, IFN is produced and consequently leads to the up-regulation of hundreds of IFN-stimulated genes (ISG) [2,3].One of the most highly induced genes is ISG15 that encodes a small UBL protein of 17 kDa that forms covalent conjugates with cellular proteins [4].ISG15 is composed of two domains, each of which carries high sequence and structural similarity to UB (33 and 32% for the N-and C-terminal domains, respectively) [5,6].
ISG15 conjugation (ISGylation) to substrate proteins occurs in a manner similar to UB conjugation by utilizing activating, conjugating and ligating enzymes to facilitate the addition of ISG15 to specific lysine residues [7].The ISG15 activating enzyme is ubiquitin E1 like protein (UBE1L), and the E2 enzyme for UB conjugation, UbcH8, also recognizes ISG15 [8,9,10].ISG15 is removed from conjugated proteins by an ISG15-specific protease, UBP43 (USP18 (UB-specific protease 18)) [11,12,13].UB as a central cellular regulator and UB-mediated proteolysis also plays a regulatory role in the immune system [14,15].While the degradation by the proteosome generally depends on poly-UB conjugation, protein modification by ISG15 does not typically cause substrate degradation [16].Instead it may alter the subcellular localization, structure, stability or activity of targeted proteins [17].A large number of cellular proteins that are associated with cellular cytoskeleton, stress response and chroma-tin remodelling were identified as ISG15 targets.ISG15 also targets proteins that play a role in the innate antiviral response, including: PKR, MXA, STAT1, JAK1 and RIG-I [18].ISGylation of these antiviral molecules may regulate their activity during viral infection.
ISG15 expression is almost undetectable under normal conditions but is strongly up-regulated during viral infections such as human cytomegalovirus (HCMV), herpes simplex virus (HSV), Sindbis virus (SV) and hepatitis C virus (HCV) [19,20,21,22,23,24].It has been speculated that the ISG15 up-regulation following viral infection is involved in different strategies of the antiviral response [25,26].Some viruses have developed specific strategies to counteract the activity of the IFN-stimulated genes (ISGs).The influenza B virus protein NS1B binds ISG15 and blocks protein ISGylation [27].Furthermore, constitutive expression of ISG15 in type I IFN receptor knockout (KO) mice confers potent antiviral activity against SV.This evidence suggests that ISGylation is important for protecting cells from viral infection [21].
Previously, using cDNA microarrays we described up-regulation of ISG15 after infection of HeLa cells with the attenuated VACV strains MVA and NYVAC, an effect not observed after infection with the virulent strain WR [28,29,30].Furthermore, the attenuated mutant VVDE3L that lacks the viral early protein E3 also produces an increase in the ISG15 mRNA levels [31].VVDE3L is a virus that only replicates in IFN-incompetent systems exerting IFN antagonist activity [32], is nonpathogenic in the mouse model, and provides protection against a wild-type virus challenge [33,34].The E3 protein represses the host cell antiviral response by multiple mechanisms, including inhibition of both PKR and RNase L, two enzymes induced by IFN and whose activation triggers a global inhibition of protein synthesis and virus replication [35,36,37] through the phosphorylation of eIF-2a (for PKR) and breakdown of RNA (for RNase L).Significantly, once activated both PKR and RNase L produced upregulation of ISG15 messenger levels [38,39].E3 also blocks induction of IFN-a/b through inhibition of phosphorylation of the transcription factors IRF3 and IRF7 [40,41] and prevention of NF-kB activation [42].
The biological significance of ISG15 mRNA induction in cultured cells after infection with the VACV mutants and its repression by the virulent WR is not known.Here we have investigated the role of ISG15 as an anti-VACV immunity factor using in vitro and in vivo systems based on MEFs and mice lacking ISG15.While in MEFs the yields of WR were slightly different between ISG15+/+ and ISG152/2 infected cells, the nonreplicating VVDE3L in ISG15+/+ cells grew more than one log better in ISG152/2 cells.Biochemical analyses showed that the E3 protein interacts with ISG15 through its carboxyl terminal domain.Repression of ISG15 with siRNAs or expression of ISG15 by a lentivirus vector in ISG15 null cells indicate that VACV replication can be controlled by ISG15 and that E3/ISG15 protein interaction is independent of the presence of PKR.In mice lacking ISG15, VVDE3L induced stronger pathogenesis than in ISG15+/+, an effect similarly triggered by a C-terminal deletion mutant (VVE3LD26C).Our findings reveal a novel VACV strategy to counteract the IFN antiviral response through interaction of the virus E3 protein with ISG15.

Expression of ISG15 is upregulated during infection of human HeLa and murine embryonic fibroblast cells with attenuated VACV strains
We and others have previously described upregulation of ISG15 transcript from gene expression profiles of HeLa cells infected with the attenuated VACV strains, VVDE3L [31], MVA [28] and NYVAC [30].This up-regulation was not observed in HeLa cells infected with the virulent WR [29].Here, we have validated the transcriptional changes in ISG15 mRNA levels after VACV infection by real-time RT-PCR.As shown in Table S1, ISG15 mRNA levels at different times postinfection (p.i.) were enhanced in HeLa cells infected with the mutant viruses compared to the virulent WR, in agreement with the microarray data (not shown).
To correlate changes in ISG15 protein levels, we analyzed by immunoblot the levels of ISG15 in WR-or MVA-or NYVAC-or -VVDE3L or uninfected MEFs.In agreement with the results of real time RT-PCR obtained in Hela cells, a clear increase in ISG15 protein levels was also observed in VVDE3L-or MVAinfected MEFs cells at 6 and 16 hpi (Fig. 1A).The increase was less apparent after NYVAC infection probably because overall protein synthesis is more severely inhibited by NYVAC than MVA [30].Moreover, the increase of ISG15 protein levels after VVDE3L or MVA infection required de novo protein synthesis as its accumulation was prevented by cycloheximide treatment discarding the possibility that infection might increase ISG15 protein levels by enhancing protein stability (not shown).It should be noted that there is an increase in the conjugation of ISG15 to its target proteins after VVDE3L, but reduced in levels after MVA infection (Fig. 1A).The findings of Fig. 1 establish a clear upregulation of ISG15 by the attenuated VACV mutants.

ISG15 has an anti-VACV role and modulates VVDE3L replication in cultured cells
Since the increase in ISG15 in Hela cells correlated with the attenuated phenotype of several VACV strains, we next examined the role of ISG15 in VACV replication using primary MEFs derived from ISG15+/+ and ISG152/2 mice.While the cytopathic effect (CPE) observed in ISG152/2 after WR infection (0.1 PFU/cell, 24 h) was similar to ISG15+/+ cells (Fig. 2A, upper panels), the CPE in ISG152/2 cells after VVDE3L infection was markedly increased with respect to that observed in ISG15+/+ cells (Fig. 2A, lower panels).The virus titers for WR were slightly increased in ISG152/2 compared to ISG15+/+ cells (Fig. 2B), while the yields of VVDE3L were increased in the ISG152/2 compared to ISG15+/+ cells (about 25-fold higher).The increase in virus titers correlated with increase in cellular mortality, as shown in Fig. 2A.The findings of Fig. 2 suggest that E3 expression might be suppressing ISG15 function.To define the breath of the E3 anti-ISG15 activity we analyzed the role of another antiviral factor, PKR, using MEFs derived from PKR2/2 mice.Both the difference in CPE and virus yields between VVDE3L infected ISG152/2 and PKR2/2 cells were clearly distinct (Fig. 1A), indicating that the in vitro replication of VVDE3L in ISG152/2 is a process independent of PKR.With PKR+/+ cells the CPE and virus yields were similar as for ISG15+/+ cells (not shown).
To provide further evidence for a VACV antiviral role of ISG15, we used siRNA to specifically block ISG15 mRNA production.Using siPORT Amine as a transfection reagent, MEFs were transfected with two specific ISG15 siRNAs (siRNA1 or siRNA2), or with a specific GAPDH siRNA (positive control) or with a scrambled siRNA (negative control).Twenty four hours after transfection cells were infected with WR or VVDE3L (0.1 PFU/cell), and ISG15 expression, CPE and virus titers were evaluated during the course of infection.As shown in Fig. 3A, the two ISG15 siRNAs decreased the expression of ISG15 by over 80% after 24 h of transfection (Fig. 3A).The decrease in ISG15 protein levels was accompanied by an enhanced CPE in VVDE3L infected ISG15+/+ cells (Fig. 3B); the difference in CPE was less clear in WR-infected cells.In addition, viral titers were enhanced in silenced ISG15+/+ cells infected with WR or VVDE3L (Fig. 3C).We also performed ISG15 mRNA inhibition with ISG15 siRNAs in PKR2/2 cells and found no changes in CPE and virus yields for WR and VVDE3L infected cells in comparison to the results obtained in PKR+/+ cells (not shown), indicating that the function of E3 protein is independent of PKR activity.

Author Summary
Modification of proteins by ubiquitin (UB) and ubiquitinlike proteins (UBL) represents a key regulatory process of innate and adaptive immune responses.Interferon-stimulated gene product 15 (ISG15) is a member of UBL molecules that can reversibly be conjugated to proteins mediating considerable antiviral response.In turn, many viruses, including poxviruses, have evolved strategies to block the antiviral and inflammatory effects of innate immune responses to keep cells alive until virus replication is completed.Here, a novel viral immune evasion mechanism that inhibits ISG15-dependent antiviral pathway is described.Vaccinia virus (VACV) pathogenesis in ISG15+/+ versus ISG152/2 mice is linked to the virus E3 protein, blocking the activity of ISG15 through its Cterminal domain.This effect was independent of PKR activation.ISG15 controls the inflammatory response regulating cytokine levels.Our findings support a new strategy for poxviruses to evade the host antiviral response through interaction of the virus E3 protein with ISG15.
Transfection of GAPDH siRNA or a scrambled siRNA followed by infection with WR or VVDE3L had no significant effect in either ISG15 protein level, CPE or virus production, indicating the specificity of ISG15 function.We checked that GAPDH protein levels were decreased only in the siRNA-GAPDH transfected cells, as measured by Western blot analysis (not shown).The results of Fig. 3 revealed that suppression of ISG15 protein levels leads to enhanced replication of WR and of VVDE3L, further supporting an anti VACV role of ISG15.

Absence of ISG15 expression is required for efficient VVDE3L infection in murine cultured cells
To further extend the role of ISG15 expression in VACV replication, we tested whether ectopic ISG15 expression in ISG152/2 cells leads to inhibition of WR or VVDE3L viral growth.Using pRVISG15-ires-GFP, an optimized retroviral vector that expresses efficiently ISG15 in transduced cells, we evaluated the CPE (see Fig. S1) and viral growth of WR or VVDE3L.Viral titrations showed that retroviral transduction of the ISG15 gene in ISG152/2 cells result in inefficient VVDE3L viral production, whereas non-transduced ISG152/2 cells infected with VVDE3L were able to produce infectious viral particles (Fig. 4C, left panel).Furthermore, yields of VACV infectious virus decreased in the ISG15-transduced cells in comparison to those that do not express ISG15 (Fig. 4C, right panel).These findings demonstrate that the absence of ISG15 is essential for the productive infection of VVDE3L and the increase in VACV production in murine cultured cells.
To characterize the effect of ISG15 over-expression in WR or VVDE3L replication in cells with endogenous ISG15, a similar approach was carried out as before but with retroviral transduction of ISG15+/+ cells, followed by measurements of WR or VVDE3L viral growth.As shown in Fig. 4, panels C-D, both viruses showed a decrease in infectious virus production in correlation with higher ISG15 expression levels.This experiment supports that ISG15 is a negative regulator of VACV replication.

E3 interacts with ISG15 through its carboxy-terminal domain
It has been described that the influenza B virus protein NS1B inhibits ISGylation after binding through its amino terminal domain to ISG15 [27,43].To test whether E3 protein, that contains a similar domain to NS1, is able to bind to ISG15 protein, we performed immunoprecipitation (IP) assays in PKR+/ + cells using the following different viruses: WR, MVA, VVDE3L lacking the entire E3L gene, and two deletion mutants VVE3LD83N and VVE3LD26C with truncated versions of E3L gene at the N and C-terminus [44].After ISG15 IP, the entire E3 protein binds efficiently to ISG15 and the N-terminal mutant D83N, that lacks 83 aminoacids and the PKR binding domain, binds efficiently to ISG15 (Fig. 5A).In contrast, the C-terminal mutant D26C, that lacks 26 aminoacids and the ability to bind dsRNA, does not bind ISG15 (Fig. 5B).We also performed the reverse IP using an anti-E3 antibody and only the E3 protein that lacks the C-terminus fails to be immunoprecipitated (Fig. 5B, left panel).When IP was performed without antibody or using a preimmune serum as a control, no interaction was observed (Fig. 5B, central panels).To analyze if RNA was involved in E3/ISG15 interaction, we treated the IP complex with RNase just before its loading in the SDS-PAGE, and found that the complex was destroyed, as no interaction was observed with any of the E3 proteins from the different viruses (Fig. 5B, left panel).This result indicate that RNA, and probably dsRNA, has a role as a linking component in the interaction between E3 and ISG15 proteins as its degradation abolishes the binding of both proteins.ISG15/E3 protein interaction was confirmed by confocal microscopy, as WR-, or MVA-or VVE3LD83N-infected MEFs showed co-localization between ISG15 and E3, while VVDE3L-or VVE3LD26C- infected MEFs did not (Fig. 5C).In addition, we also studied if the presence of PKR was relevant for this interaction by performing both IP and confocal experiments in PKR2/2 cells.Both approaches indicate that the interaction between ISG15 and E3 is independent of PKR, as in its absence the entire E3 and the protein that lacks the amino terminus are able to interact with ISG15 (Fig. 5D).The findings of Fig. 5 reveal that ISG15 binds the E3 protein in a PKR-independent manner and that binding requires the C-terminal domain of E3 spanning the RNA-binding site, which suggests that dsRNA acts as a linker.

Enhanced susceptibility of ISG15 KO mice to VVDE3L infection
To further show that ISG15 is a biological relevant antiviral molecule against VACV infection, we next evaluated the susceptibility of ISG152/2 mice to the virus.Thus, we infected by the intraperitoneal (i.p) route ISG152/2 or ISG15+/+ mice with WR at 2610 7 or with the attenuated VVDE3L at 10 8 PFU/ mouse and scored for prominent indicators of viral pathogenesis (weight loss and mortality).While there was reduced weight loss in ISG152/2 mice, survival was similar between both groups (Fig. 6, panel A and B).However, after VVDE3L infection the ISG152/ 2 mice displayed signs of disease within 2 days, characterized by ruffled fur and lack of activity, and 25% of the animals died within 1 to 2 days.Half of the mice infected with VVDE3L appeared sick at 4 days p.i., and 75% recovered after 7 to 8 days p.i. (Fig. 6C-D).We did not observe virus yields for ISG152/2 or ISG15+/+ mice infected with VVDE3L in liver or spleen, while virus titers were easily obtained in WR infected mice (not shown).
Since the inflammatory response might explain the rapid signs of illness in VVDE3L infected ISG15 KO mice, we measured serum cytokine levels (IL-6, TNF-a, IL-10, MCP-1, IFN-c, and IL-12 p 70) at early times post infection.In ISG15+/+ mice, IL-6 levels were similar in serum from WR-or VVDE3L-or mockinfected mice (Fig. 6E).In contrast, ISG152/2 mice infected with VVDE3L had an 8-fold increase in serum levels of IL-6 compared with those infected with WR (P,0.01;Fig. 6E).There were no changes in levels of other cytokines analyzed between the groups (not shown).
We also examined the extent of protection conferred in animals pre-immunized with WR or VVDE3L by i.p route.Thus, preimmunized mice (as in Fig. 6) were challenged by i.n route with WR at 2610 7 PFU/mouse.In the case of ISG15+/+ and ISG152/2 mice pre-immunized mice with WR, the challenge had little effect on weight loss and signs of illness, clear signs of protection (Fig. 7A-B).However after WR challenge, VVDE3L pre-immunized ISG152/2 mice developed weight loss and signs of sickness which was not observed in ISG15+/+ mice (Fig. 7C-D).These findings revealed a reduced protection to challenge with WR conferred by VVDE3L pre-immunized KO mice, indicating limited adaptive immune response triggered by VVDE3L infection of ISG15 2/2 mice.

The antiviral effect of ISG15 correlates with control of the inflammatory response after VACV infection
Since VVDE3L pre-immunized ISG15 KO mice developed disease transiently after i.n WR challenge and the upper respiratory tract is a natural route for variola virus infection, we next evaluated disease progression in ISG15+/+ and ISG152/2 mice after i.n inoculation with WR and several E3L deletion mutants (VVDE3L; VVE3LD83N and VVE3LD26C).attenuated MVA strain was included as control.Infected mice were scored for prominent indicators of viral pathogenesis (weight loss, signs of illness and mortality).After WR or VVE3LD83N infection, no significant differences in weigh loss and signs of illness were observed between ISG152/2 and ISG15+/+ mice, although slight differences in weight loss were observed between both groups of mice when inoculated with a lower dose of WR (Fig. 8A, upper panel).Mortality was higher in mice infected with WR, as all mice died within 7 days in the case of WR, while infection with VVE3LD83N caused 25% mortality (Fig. 8A-B).However, clear differences were observed after i.n.inoculation of VVDE3L or VVE3LD26C.While ISG15+/+ mice did not show signs of illness at any times p.i, ISG152/2 mice infected with VVDE3L or VVE3LD26C showed disease as revealed by clear signs of illness as soon as 2 days and 25% of the animals died.About half of the mice infected with VVDE3L or VVE3LD26C appeared sick at 4 days p.i, but 75% of them recovered after 7 to 8 days p.i, (Fig. 8C).
To analyze the status of ISGylation in the infected mice, lungs were homogenized and conjugation of ISG15 to its target proteins was determined by Western blot.While, ISG152/2 mice do not express ISG15 (Fig. 7A) and lungs homogenates from ISG15+/+ mice had similar amounts of the ISG15 protein, conjugation of ISG15 to its targets proteins is enhanced in lung extracts from mice infected with VVDE3L or VVE3LD26C (Fig. 9A).Similar result was also observed in MEFs infected in vitro with VVDE3L where high levels of ISG15 conjugates were clearly observed (Fig. 1A).These findings suggest that E3 blocks conjugation of ISG15 to its target proteins by its carboxy-terminal domain.
The presence of VACV proteins, as determined by Western blot, was more evident in lungs of ISG152/2 in comparison to ISG15+/+ mice infected with WR or the deletion mutant viruses (Fig. 9B).As expected, appearance of virus in lungs correlated with the presence of viral proteins in these tissue extracts (Fig. 9B-C).These results indicate that although the absence of ISG15 has no effect in the mortality of the mice at a high dose of WR inoculation (5610 6 PFU/mice; Fig. 8A, middle and lower panel), it has an effect in the replication of the WR and E3L mutant viruses, as seen by the different amount of viral protein and virus titers in lung tissues of ISG152/2 versus ISG15+/+ mice (Fig. 9B-C).
Histological examination of lung tissue showed that ISG15+/+ animals infected with the different mutant viruses had no inflammatory cells infiltrating the lung parenchyma.In contrast, lung sections obtained from ISG152/2 mice infected with VVDE3L or VVE3LD26C presented severe inflammation with alveolar wall thickening and infiltration of inflammatory cells (see enlarged sections in Fig. 10).This phenotype was not observed in WR-or VVE3LD83N-infected ISG152/2 mice (Fig. 10).This result indicates that in ISG152/2 mice, pathogenesis and development of an inflammatory response is mediated by the absence of E3 virus expression.This phenotype was maintained after VVDE3L and VVE3LD26C pointing to E3 as a major VACV molecule involved in virus evasion of the IFN-defense ISG15 antiviral protein.

Discussion
Pro-inflammatory and IFN-stimulated genes (ISGs) represent essential components of the innate immune response to viral infection (40).Upon viral entry into cells, ISG induction occurs in two waves: acute, IFN-independent induction of a subset of ISGs  modifies several important molecules and affects type I IFN signal transduction; ISG15 expression is markedly increased following viral infection (14,30,49), and many viruses encode inhibitors of the IFN-transduction pathway or specific inhibitors of ISG to avoid deleterious effects triggered by these cytokines.Among animal viruses, the poxvirus family contains a large array of genes which are used by the virus to evade host immune responses.VACV encodes multiple proteins that interfere with complement regulatory proteins, with many cytokines and chemokines, with TLRs (Toll like receptors) and signal transduction pathways, with apoptosis, and others [46].One of the VACV proteins with strong inhibitory activity of IFN-induced pathways is E3 [47].E3 represses the host cell antiviral response inhibiting both PKR and RNaseL, which trigger global inhibition of protein synthesis and virus replication [35,36,37].In addition, E3 blocks the activation of IRF3 [40,41], and effectively prevents the first wave of type I IFN synthesis.E3 has two domains, an N-terminal involved in the direct inhibition of PKR, its nuclear localization, and Z-DNA binding [34,48,49,50], and the C-terminal that contains the dsRNA-binding domain required for IFN-resistance and for the broad host range phenotype of the virus [44,51].It has been described that VACV lacking E3 (VVDE3L) replicates in PKR or RNaseL deficient cells [40].
Through the use of microarrays we identified the gene ISG15 as being induced in the course of infection of human cells with different strains of VACV [28,29,30].The attenuated mutant VVDE3L also produces an increase in ISG15 messenger levels [31].The reason for the induction of ISG15 mRNA levels by attenuated viruses (Fig. 1) is probably due to the activation of several cellular signal transduction cascades and of host transcription factors [28,30,31].Since MVA and NYVAC strains contain the E3L gene, this upregulation may be independent of E3 expression with induction being likely due to the increase in IFN-b levels.
In this study we showed that a VACV mutant lacking E3, which cannot grow in ISG15 WT cells, is able to replicate both in MEFs cells derived from ISG15 KO mice or in ISG15 silenced cells.In addition viral titers also increase in the absence of ISG15 indicating that ISG15 has an essential role against infection of VACV.During infection of MEFs from ISG15 KO or ISG15 depleted cells, the presence of E3 enhances viral production since the WR titers are greater than those after infection with the VVDE3L mutant virus.One explanation of this phenotype is that the mutant virus lacking E3 triggers apoptosis through PKR activation which, in turn, reduces virus production as previously described [52].The role of ISG15 in VACV replication was also supported by the more abundant VACV infectious virus and viral proteins produced in lungs of ISG152/2 mice compared with lungs of ISG15+/+ mice after infection with WR or with the E3L deletion mutant viruses (Fig. 9B-C).
While the depletion of ISG15 has an effect on VVDE3L mutant phenotype and restores virus growth, over-expression of ISG15 in ISG152/2 murine cells using a retroviral transduction system revert the restricted VVDE3L viral growth.Over-expression of ISG15 also reduces markedly WR titers reinforcing the idea that ISG15 plays a role in the control of VACV replication.
Inhibition of ISG15 function by VACV is likely due to its interaction with VACV E3, as shown by IP and confocal analyses.This interaction with ISG15 occurs independently of PKR, through the C-terminal region of E3 and requires RNA.We have shown that ISG15 controls the in vitro replication of VACV in a PKR-independent manner, as WR and VVDE3L titers do not increase in murine PKR2/2 cells in comparison to those observed in PKR+/+.While in murine cells VVDE3L is able to replicate in a PKR-independent manner, as also described in MEFs lacking RNase L [53], in human HeLa cells with PKR expression suppressed by siRNA, the mutant virus is able to grow [40].The differences in cell origin might explain the distinct effect of the IFN system in the control of VACV replication.
The mutant VVDE3L virus that was able to replicate in ISG152/2 MEFs (Fig. 2) did not replicate in ISG15 KO mice (Fig. 7), but surprisingly infection with VVDE3L provokes sickness and mortality only in ISG152/2 mice.This was probably related to the strong inflammatory response triggered by the mutant in ISG15 KO mice, as observed by the increased levels of IL-6 in serum (Fig. 4E).Although the biological relevance of this observation remains to be established, it can be suggested, in view of the functions assigned to ISG15 in the innate immune response [54], that this molecule plays a role as regulator of IFNtriggered innate responses during VACV infection.It will be of interest to know the type of innate response triggered in ISG15 KO mice infected with VVDE3L.
The inability of VVDE3L to cause significant disease in WT mice is presumably due, at least in part, to induction of type I IFN that, in turn, leads to up-regulation of antiviral proteins, such as PKR and 2-5 OAS/RNaseL system.The mutant virus that lacks the C terminus of E3L gene involved in the dsRNA sequestration (VVE3LD26C) is completely attenuated in WT mice; however deletion of the N terminus (VVE3LD83N) reduces pathogenesis 500-to 5,000-fold [33,48].We extended these in vivo studies using the i.n route and compared WR and E3L mutant viruses in WT and ISG15 KO mice.We observed that only after VVDE3L or VVE3LD26C inoculation (i.n), the mortality of mice was increased by the absence of ISG15 (Fig. 6).In the case of WR or VVE3LD83N, there were no differences in mortality of both viruses in ISG15 KO in comparison to WT mice although viral replication was enhanced in the lungs of ISG152/2 in comparison to ISG15+/+ mice.One explanation is that ISG15 is made non functional after infection with these viruses, because the carboxy-terminal domain of E3 binds to ISG15 and blocks its activity.These observations are in correlation with the reduced presence of conjugates in lungs of WT mice infected with WR or VVE3LD83N, compared with infection by VVDE3L or VVE3LD26C.Although lung homogenates presented similar amounts of ISG15 protein, the conjugation of ISG15 to its targets proteins, was greatly enhanced after infection with VVDE3L or VVE3LD26C (Fig. 7).This evidence suggests that inhibition of conjugation of ISG15 is mediated by E3 and this inhibition requires the presence of the dsRNA binding domain.Similar result was also observed in MEFs infected in vitro with VVDE3L where high levels of ISG15 conjugates were clearly observed (Fig. 1A).
The cause of mortality of ISG15 KO mice after infection with VVDE3L or VVE3LD26C was a massive inflammation of lungs with alveolar wall thickening and infiltration of cells (Fig. 8).These results indicate a role of ISG15 in the control of an inflammatory response by regulating cytokine levels.
Cytokine and chemokine release occurs rapidly in response to virus infection, with the aim of recruiting inflammatory leukocytes in order to limit virus replication and spread, and to induce adaptive immunity.However, prolonged expression of chemokines in the context of viral infections may be detrimental to the host.We find that in the absence of ISG15, infection with VVDE3L produces an increase of IL-6 that correlates with short-term morbidity and complications that include pulmonary function abnormalities.Although the mechanisms of this up-regulation remains to be established, it can be speculated in view of the functions assigned to ISG15 that it might be involved in the regulation of cytokine signal transduction, through the stabilization of specific signalling components that facilitate the development of a correct innate immune response.In this sense a family of intracellular proteins called suppressors of cytokine signalling (SOCS) are essential for the regulation of cytokine expression having a critical role in the regulation of the innate response.Considering that SOCS-1 and SOCS-3 negatively regulate the IFN-induced signal cascade, and VACV E3 protein inhibits the type I IFN response, it is possible that E3 or other viral proteins may regulate the IFN response by affecting SOCS protein expression regulating the ISG15 activity by an unknown mechanism.We have previously demonstrated that although WR provokes a general downregulation of cellular mRNAs, there are a discrete number of human genes that are induced selectively during the course of VACV infection.A variety of these upregulated genes encode different members of the SOCS family [29] indicating that probably VACV may modify SOCS protein expression to manipulate the cytokine pathway and the antiviral host response.This strategy may be used to reduce the efficacy of innate and acquired immune responses to infection.However, WR modification of cytokine or chemokine responses may also be a mechanism to recruit new targets for infection, or provide new niches for infection.It has been described that over-expression of HCV core protein inhibits IFN signalling and induces SOCS-3 expression.SOCS-1 and SOCS-3 proteins have been reported to inhibit IFN-induced activation of the JAK-STAT pathway and expression of antiviral proteins, such as M6A [55].
There are similarities between the functions of VACV E3 and the NS1 dsRNA-binding protein of influenza virus.NS1 blocks IRF3 phosphorylation and IFNb mRNA induction [56].In addition, NS1 is an inhibitor of PKR, suggesting that dsRNA sequestration is a strategy used by both RNA and DNA viruses to evade the IFN induction and action [57].Furthermore NS1B binds and blocks ISG15 protein inhibiting the ISGylation.The region of the NS1B protein that is required for this inhibition includes the domain that binds dsRNA.VACV may have a similar mechanism of influenza NS1 to evade ISG15 action as well.
We conclude that the cellular ISG15 protein has an essential role in VACV replication, acting as a negative feedback regulator of the cytokine signalling pathway and regulating in this way the innate response.VACV has therefore developed a mechanism to counteract this antiviral host response through E3.Because VVDE3L is not lethal to ISG15+/+ mice lacking PKR, RNase L, and M61 [40], there must be an additional IFN-induced antiviral pathway(s) effective against viruses, in which ISG15 should play an essential role.Understanding the host responses triggered by ISG15 and virus mechanisms of escape is necessary for development of therapies against important human pathogens.ISG15+/+ and ISG152/2 MEFs were transduced with hightiter viral supernatants corresponding to the pISG15-ires-GFP retroviral vector obtained as described [63].Supernatants were collected at 48 h after transfection, filtered through a 0.45-mmpore-size filter, and supplemented with complete DMEM medium +10% FCS before addition to growing MEFs.This protocol was repeated each 12 hours three times in presence of polybrene.The transduction efficiency was evaluated by Western-blot.Twentyfour hours after retroviral infection treated and non-treated control cells were mock-infected or infected with different VACV or VVDE3L viruses at 0.1 PFU/cell and and CPE were visualized by phase-contrast microscopy at the indicated times p.i.

Immunohistochemistry
Formalin-fixed lung from mice mock-infected or infected with WR, VVDE3L, VVE3LN83N or VVE3LN26 was resected, sectioned and stained with both hematoxilin and eosin as previously described [64].

Figure 1 .
Figure1.ISG15 protein levels are upregulated during infection of MEFs with the attenuated mutants of vaccinia virus.A. ISG15 protein levels after WR or VVDE3L or MVA or NYVAC infection.MEFs were mock infected (M) or infected at 5 PFU/cell with WR or MVA or NYVAC or VVDE3L and at the indicated times p.i, cell extracts were analyzed by Western blotting.Equal amounts of proteins were fractionated by SDS-PAGE, transferred to nitrocellulose paper, and reacted with an antibody that recognizes murine ISG15 protein.On the right, the molecular weight of the proteins in kilodaltons is indicated.Actin levels showed that the same amount of protein was loaded on the gel.Uninfected cells (M) served as control.B. Densitometric quantification of ISG15 protein in arbitrary units is indicated.The graphic represents these measurements in three independent experiments.doi:10.1371/journal.ppat.1000096.g001

Figure 2 .Figure 3 .WR
Figure 2. Effect of ISG15 on cytotoxicity and virus growth after infection of MEFs with virulent and E3L deletion VACV mutant viruses.A. ISG152/2, PKR2/2 or wild type cells were mock-infected or infected at 0.1 PFU/cell with WR or VVDE3L.At different times p.i, the CPE in the cells was examined by phase-contrast microscopy.B. Virus growth of WR and VVDE3L infected (0.1 PFU/cell) ISG15+/+, or ISG15 2/2, or PKR2/2 cells.At different times cells were harvested and virus yields were determined by plaque assay for WR or by immunostaining for VVDE3L.doi:10.1371/journal.ppat.1000096.g002

Figure 5 .
Figure 5. E3 interacts with ISG15 protein.A. PKR+/+ MEFs were treated with mouse IFN-a (100 units/ml) during 10 hrs and then infected with 3 PFU/cell of WR, or MVA or VVDE3L or VVE3LD83N or VVE3LD26C for 16 hours.Cell extracts were collected at 16 hpi and immunoprecipitated using anti-ISG15 serum, thoroughly washed and immunocomplexes analysed by SDS-PAGE and subjected to immunoblotting with antiserum to E3 or ISG15.B. PKR+/+ cells were treated as above and cell extracts were collected at 16 hpi and immunoprecipitated using anti-E3 (with or without RNase treatment (10 mg for 15 min at room temperature); or without antibody; or using a pre-immune serum, thoroughly washed and immunocomplexes analysed by SDS-PAGE and subjected to immunoblotting with antiserum to E3 or ISG15.C. PKR+/+ MEFs treated as in A were fixed at 16 h.p.i. and processed for immunofluorescence analysis by confocal microscopy using antibodies directed against ISG15 (red), E3 (green), and TOPRO for staining nuclei (blue).Merged images are presented in the lower panels.Cells were visualized by confocal immunofluorescence microscopy.D. Immunoprecipitation and immunoflurescence of PKR2/2 MEFs was performed as in A or in C. doi:10.1371/journal.ppat.1000096.g005

Figure 4 .
Figure 4. Effect of ISG15 overexpresion on virus growth after infection of MEFs with virulent and E3L deletion VACV mutant viruses.A-B.ISG152/2 MEFs were transduced with high-titer viral supernatants corresponding to the pISG15-ires-GFP retroviral vector.Levels of ISG15 were measured by Western blot using a specific murine anti-ISG15 antibody.Viral protein E3 levels were measured as control of infection and levels of eIF2a were used as a loading control.Virus growth of WR and VVDE3L were determined by plaque assay or by immunostaining.C-D.ISG15+/+ MEFs were transduced as above.Levels of ISG15 were measured by Western blot using a specific murine anti-ISG15 antibody.Viral protein E3 levels were measured as control of infection and levels of eIF2a were used as a loading control.Virus growth of WR and VVDE3L were determined by plaque assay or by immunostaining.Control indicates the non-transduced MEFs.doi:10.1371/journal.ppat.1000096.g004

Figure 6 .
Figure 6.Virulence of WR or VVDE3L after infection of ISG15+/+ and ISG152/2 mice.A-B.Mice were inoculated i.p with purified VACV (2610 7 PFU/mouse) or VVDE3L (10 8 PFU/mouse).Mice were individually weighed daily, and mean percentage weight loss of each group (n = 12) was compared with the weight immediately prior to infection.C-D.Survival rate after i.p inoculation with both viruses.Dead animals were scored daily and represented as the percentage of surviving animals.P#0.01 in all experiments.E. IL-6 measured by ELISA from serum collected at 3 hpi.Results represent the mean6SD of pooled samples from 6 mice.doi:10.1371/journal.ppat.1000096.g006

Figure 7 .Figure 8 .Figure 9 .
Figure 7. Evaluation of extent of protection of WR or VVDE3L pre-immunized ISG15+/+ and ISG152/2 mice after challenge with WR.Four mice per group were first i.p, immunized with purified WR (2610 7 PFU/mouse) or VVDE3L (10 8 PFU/mouse), and 30 days later animals were i.n.challenged with 2610 7 PFU/mouse of purified WR.A-B.Mice were individually weighed daily, and mean percentage weight loss of each group (n = 4) was compared with the weight of mice taken prior to the booster.C-D.For each mouse signs of illness, such as ruffled fur and lack of activity, were monitored with the time and are given in arbitrary units.doi:10.1371/journal.ppat.1000096.g007