In this paper, using both in vitro
and cell-based assays, we document for the first time that the nsp2 replicase subunits of all arteriviruses harbor a DUB activity that is likely used to counter RLR-mediated innate immune signaling. In addition, we established that the DUB domains of both arteri- and nairoviruses are capable of inhibiting RIG-I ubiquitination, suggesting that the deconjugation of ubiquitin from this PRR constitutes one of the mechanisms by which DUB-containing proteins from these viruses interfere with the innate immune response. The recent finding that a DUB encoded by Kaposi's sarcoma-associated herpesvirus, a DNA virus, also reduced IFN-β expression by inhibiting RIG-I ubiquitination highlights the potential benefits of this strategy for viruses at large (25
). Since the viral DUBs studied here were also capable of inhibiting MAVS-mediated IFN-β induction, it stands to reason that the deubiquitination of MAVS or other signaling molecules downstream of this factor also plays a role in the immune evasion mediated by these viral proteins.
To date, our knowledge concerning the role of innate PRRs in the recognition of arterivirus infection is limited. There is some evidence to suggest that TLR3 is important (53
), but our study yielded the first indication for the involvement of the RLRs in the recognition of arteriviral RNA. By transfecting MEFs with total RNA isolated from EAV-infected Vero cells, we have shown that the absence of MAVS, a central factor in RLR signaling, almost completely abrogated IFN-β expression, indicating that in these cells MAVS-mediated signaling is pivotal in the response to EAV infection (A). In addition, the absence of MDA5 strongly attenuated IFN-β induction compared to that in wild-type MEFs (A), suggesting that this sensor is the main RLR responsible for the recognition of arteriviral RNA. Since arteriviruses infect numerous different tissues and cell types during infection in vivo
), it is likely that many of these indeed rely on the RLR-mediated antiviral response, supporting the relevance of our data acquired in MEFs. Considering our findings, it seems counterintuitive that an arteriviral protein would target RIG-I in order to evade host immune signaling. However, in contrast to what was observed for mengovirus, the absence of MDA5 reproducibly failed to cause a complete loss of EAV-induced IFN-β expression (compare A and B). This suggests that another PRR, likely RIG-I, can mount an immune response against EAV infection in the absence of MDA5. In fact, West Nile virus (14
), dengue virus (40
), and the coronavirus murine hepatitis virus (34
) were previously found to be recognized by both MDA5 and RIG-I. Alternatively, not RIG-I but another PRR might be responsible for the observed residual level of IFN-β expression in the absence of MDA5. For example, recent work by Sabbah et al. (2009) showed that the NLR Nod2 detected viral RNA and that subsequent signaling by this molecule was also MAVS dependent (50
). Nevertheless, the fact that both MDA5 and RIG-I are ISGs suggests a role for these proteins during later stages of infection which might be distinct from their initial activities as PRRs and would explain why a virus that is not primarily detected by RIG-I would still benefit from inhibiting its activation. This hypothesis is further supported by the finding that several picornaviruses induce RIG-I degradation, despite the fact that they are commonly believed to be recognized solely by MDA5 (2
As in the case of arteriviruses, little is known about the innate immunity signaling pathways involved in the recognition of nairoviruses. Nevertheless, Habjan et al. (2008) showed that CCHFV and the related bunyavirus Hantaan virus process the 5′ end of their genomes, likely to prevent recognition by RIG-I (20
). In addition, there is recent evidence that infection with Hantaan virus can indeed be sensed by RIG-I (32
). Since viruses belonging to the same family are often recognized by the same PRRs, these findings suggest that CCHFV is also recognized by RIG-I. In nairoviruses, the deubiquitination of RIG-I might therefore be an additional mechanism to further inhibit RLR-mediated signaling.
Notably, our results showed that inhibition of the innate immune response by EAV nsp2-3, in contrast to EAV nsp2(N), is not strictly dependent on its DUB activity. Although the inhibitory activity of EAV nsp2-3 is clearly reduced upon mutagenesis of the protease's active-site residues, it is not completely abolished (B). In contrast, mutagenesis of the same catalytic residues in EAV nsp2(N) almost completely abrogated its inhibitory activity (A).
These observations are consistent with results previously obtained by others for coronavirus DUBs (8
). Both arteri- and coronaviruses belong to the order Nidovirales
and share a similar genome organization and expression strategy. Coronavirus nsp3, which can to a certain extent be considered the functional equivalent of arterivirus nsp2, contains two papain-like protease domains (PLP1 and PLP2). In some coronavirus species, however, PLP1 has lost its proteolytic activity or the entire PLP1 domain is missing. In these cases, PLP2 is the only active protease domain in nsp3 and is therefore referred to as PLpro. PLP2/PLpro of severe acute respiratory syndrome-associated coronavirus (SARS-CoV), murine hepatitis virus A59 (MHV-A59), human coronavirus NL63 (HCoV-NL63), and PLP1 of transmissible gastroenteritis virus (TGEV) were all shown to possess DUB activity, in addition to their role in the autoproteolytic maturation of the replicase polyproteins (3
). Notably, the coronavirus DUBs are of the USP subclass instead of the OTU subclass of DUBs. Like the arterivirus PLP2-DUB, the PLP2/PLpro domains of various coronaviruses were found to inhibit the innate immune response (8
). While the immune inhibitory activity of MHV-A59 PLP2 was claimed to be completely dependent on its DUB activity, that of HCoV-NL63 and SARS-CoV PLP2/PLpro was shown to only partially depend on its proteolytic activity. These observations suggest that both arteri- and coronavirus replicase proteins harbor additional immune evasive features that may be linked to but are not strictly dependent on the DUB activity present in their nsp2 or nsp3 subunit, respectively. It is conceivable that a catalytically inactive DUB would still be able to bind ubiquitin or ubiquitinated proteins, thereby hindering interactions necessary for signal transduction. In addition, our results suggest that expression of both wild-type and catalytically inactive EAV nsp2-3 decreases the overall amount of RIG-I(2CARD)
, which may explain the mutant's inhibitory potential (B, 3rd panel from the top, lanes 4 and 5). Further research is needed to elucidate whether these DUB-containing viral proteins possess additional immune evasive properties.
Contrary to our expectations, we found that arterivirus nsp2(N)
, which localizes to the cytosol, inhibited the IRF3-mediated activation of IFN-β expression (C). In contrast, IRF3-mediated IFN-β induction was hardly affected by the expression of EAV nsp2-3, which is membrane anchored and localizes to the perinuclear region of the cell, while EAV nsp2(N)
and nsp2-3 showed comparable inhibitory potential upon activation of signaling by RIG-I(2CARD)
or MAVS (compare A to C). Similar results were previously obtained for SARS-CoV PLpro, of which a soluble form inhibited IRF3-mediated activation of IFN-β expression (16
), while a membrane-anchored form did not (9
). Taken together, these results suggest that the subcellular localization of these proteins is an important determinant of the range of substrates that is accessible to them. Since expression of the full-length nsp2 protein together with nsp3 more accurately reflects the situation in the infected cell, it is likely that during infection inhibition also takes place upstream of IRF3. This would be consistent with previous observations by Luo et al. (2008), who showed that inhibition of the RLR pathway during PRRSV infection occurred at the level of MAVS or upstream of this key factor (38
Still, the question of the mechanism, albeit artificial, by which cytosolic EAV nsp2(N)
is able to inhibit IRF3 signaling remains, since to date there is no evidence for activation of this transcription factor by Lys63-linked polyubiquitination. On the contrary, it is well established that IRF3 is negatively regulated by Lys48-linked polyubiquitination, the removal of which would appear to be counterproductive in view of controlling IFN signaling (24
). Interestingly, Shi et al. (2010) recently showed that ISG-ylation, i.e., the conjugation of ISG15 to a target protein, of IRF3 positively regulated its activation by preventing Lys48-linked polyubiquitination (58
). In light of previous findings by Frias-Staheli et al. (2007) that arteri- and nairovirus DUBs can deconjugate ISG15 (15
), it is tempting to speculate that cytosolic nsp2(N)
, in contrast to membrane-anchored full-length nsp2, is able to de-ISGylate IRF3. However, IRF3 ISGylation per se
was shown not to be activating but merely increased the signaling potential of IRF3 by preventing its proteasomal degradation. Consequently, if a viral DUB would indeed de-ISGylate IRF3 to promote its degradation, it would at the same time need to refrain from removing any Lys48-linked polyubiquitin chains. This problem emphasizes that viral DUBs are likely able to distinguish between activating and inactivating Ub(-like) modifications. How viral DUBs make this distinction and how they would achieve a balance between the removal of the right and wrong modifiers pose interesting questions for future research.
Another interesting feature of viral DUBs is their apparent tendency to deubiquitinate all cellular proteins in a seemingly random fashion (B and C). In this respect, they differ from their mammalian counterparts, which generally have a more narrow specificity (15
). A possible explanation for this observed promiscuity, which is seen for arteri-, nairo-, and coronavirus DUBs (15
), is the fact that the enzymes were often studied only as isolated domains taken out of their natural full-length-protein context. For this reason, we have also studied PLP2-DUB in the context of EAV nsp2-3, of which the membrane-associated subcellular localization is similar to that observed during EAV infection. Although EAV nsp2-3 did seem to be more restricted than nsp2(N)
in the inhibition of IRF3-mediated IFN-β induction (C), PLP2-DUB also showed general DUB activity when overexpressed as part of full-length nsp2 (C). This suggests that the observed promiscuity is indeed an intrinsic property of these viral DUBs, although on the other hand, it should be noted that most of our experiments involved systems in which the DUBs were overexpressed. Future studies will aim to elucidate the role of viral DUB activity during the course of infection, for example, by using reverse genetics to engineer a virus that lacks this activity. Unfortunately, we have thus far been unable to create a viable mutant with this phenotype, mainly due to the intimate link between the DUB activity and polyprotein processing functions of PLP2.
Taken together, our results strongly suggest that arteriviruses as well as nairoviruses encode DUBs that are used to inactivate cellular proteins involved in innate immune signaling, as exemplified by the deubiquitination of RIG-I documented here. Strikingly, related DUBs from the OTU family seem to have been acquired and adapted for this purpose by apparently unrelated RNA viruses, the positive-stranded arteriviruses and the negative-stranded nairoviruses, thereby highlighting the selective advantages that must be linked to OTU DUB acquisition and the general plasticity of RNA virus genomes.