Viruses have developed a number of mechanisms to subvert or prevent IFN induction and response (7
). In the case of PRRSV, both North American and European PRRSV strains have been shown to be poor inducers of IFN in vitro
and in vivo
. Infection of PAMs with the North American prototype PRRSV VR-2332 significantly reduced their ability to produce type I IFN (36
). Furthermore, the absence of IFN-α production in TGEV-infected swine alveolar macrophages, which had been previously infected with PRRSV, implicates an active inhibition of IFN production by PRRSV. In summary, all of the studies are consistent with the fact that PRRSV exerts an active suppression of type I IFN response. In agreement with these, we have shown here that PRRSV infection of monocyte-derived macrophages showed inhibition of IFN-α and IFN-β production both at the mRNA and the protein levels. Our data show that five PRRSV NSPs—the NSP1α, NSP1β, NSP2, NSP4, and NSP11—have roles in IFN antagonism and inhibit activation of the IFN-β promoter.
In the present study we used TLR3- and RIG-I-mediated dsRNA signaling pathways as model systems to understand the mechanism of innate immune evasion by PRRSV. NSP1α, NSP1β, and NSP11, when stably expressed in HEK293-TLR3 cells, were able to inhibit upregulation of endogenous ISG56 by dsRNA. Moreover, stable expression of NSP1β inhibited endogenous ISG56 induction by SeV. Investigation of IRF3 phosphorylation status after dsRNA stimulation showed that NSP1β inhibits IRF3 (Ser396) phosphorylation and subsequent nuclear translocation. Although the biochemical nature of this inhibition by PRRSV NSP1β remains to be characterized, there are several mechanisms by which different viruses have been shown to inhibit IRF3 activation. Certain paramyxoviral V proteins interact with IKK
/TBK1 and function as a competitive inhibitor for IRF3 phosphorylation (33
). The W protein of Nipah virus specifically interacts with karyopherin α3 and α4 through its nuclear localization signal and prevents IRF3 translocation (47
). It has been reported that NSP2 of EAV, the prototypic member of Arteriviridae
family, antagonizes host innate immune response by deconjugating ubiquitin and ISG15 from their cellular substrates (15
). Therefore, it is possible that NSP1β, a member of cysteine protease family that includes most deubiquitinases, might also use a similar strategy of deconjugating important ubiquitin modifications of IRF3 or RIG-I (6
). All of these possible mechanisms of inhibition of dsRNA signaling by NSP1β are currently under investigation.
NSP1β and NSP11 expression also inhibited transcription from an NF-κB-responsive promoter, but NSP1α expression has no inhibitory effect. The suppressive role of NSP1β in NF-κB-mediated signaling was further evident from the low level of IL-8 induction in dsRNA-treated HEK293-TLR3 cells stably expressing NSP1β. This finding contradicts a previous report that found PRRSV infection of MARC-145 cells and alveolar macrophages resulting in the activation of the NF-κB pathway through the degradation of IκB (28
). The disagreement might be due to the fact that the NF-κB activation in that study was assayed at a late phase of virus infection (24 to 48 h after virus infection). The activation of NF-κB is an immediate-early event that occurs within minutes after exposure to a stimulus and results in a strong transcriptional stimulation of several cytokines (21
). Considering that the PRRSV replication cycle in macrophages is completed by 12 h, this effect may be a secondary effect of virus infection. Inhibition of NF-κB pathway by PRRSV NSPs, especially during the early infection phase, would help the virus not only to subvert the host innate immune response but also to produce enough progeny virus before being released by apoptosis as proposed by others (10
PRRSV NSP1 contributes two accessory proteinases NSP1α and NSP1β for the processing of replicase polyprotein. NSP1α and NSP1β both possess two papainlike cysteine protease (PCP) domains, PCPα and PCPβ, which mediate the release of NSP1α and NSP1β from the polyproteins (12
). Previous reverse genetics experiments showed that PCPα activity is essential for subgenomic mRNA synthesis but dispensable for genome replication. However, mutation of active-site residues of PCPβ resulted in no genome replication and a failure in virus recovery after electroporation of in vitro
-transcribed RNA, underscoring its important role (26
). Whether the NSP1β active site residues (Cys and His) are involved in its IFN antagonistic activity in the context of whole virus infection remains to be investigated. However, it may not be feasible to structurally separate proteolytic function of NSP1β from its IFN antagonistic function because of the general multifunctional nature of viral proteins. This multifunctional nature of NSP1 has been already established in EAV, and it has been found that the structural integrity of NSP1 is essential for transcription (54
). In such case, given the essential function of PRRSV NSP1β in virus viability, it may not be possible to recover by reverse genetics a replication-capable virus that lacks anti-IFN activity.
The inadequate immunological response to PRRSV infection is underscored by the fact that an infected animal exhibits prolonged viremia, persistent infection, and continuous virus shedding. The poor type I IFN production at the site of PRRSV infection (e.g., lungs) has long been considered the root cause behind defective or suboptimal initiation and elaboration of the antigen-specific adaptive immune response (1
). Influenza virus carrying truncated nonstructural 1 (NS1) protein, which counteracts the host type I IFN response, is highly attenuated in mice, swine, and chicken models (42
). Another important example of correlation between anti-IFN activity and virulence is Ebola virus VP35 protein, which inhibits IRF3 activation. Single amino acid mutation (R312A) of VP35 protein, which compromises its ability to inhibit IRF3 activation, renders the virus highly attenuated in mice (20
). It is possible that the strong anti-IFN activity of the NSPs of PRRSV may have a direct effect in the paradoxical immune response observed in PRRSV infection and would explain the inability of the host to clear PRRSV. Such an important immunomodulatory effect of these NSPs may also be the basis for the pathogenesis and virulence of this virus. On the other hand, pestiviruses, which include classical swine fever virus (CSFV) and bovine viral diarrhea virus, have been shown to induce proteasomal degradation of IRF3 by the viral autoprotease Npro, which subsequently inhibits IFN-α/β production (4
). A recent investigation into the correlation between the ability of CSFV for IRF3 degradation and viral virulence found that impairment of the IFN antagonistic activity did not give rise to an attenuated virus (43
). The authors of that study suggest that the inhibition of type I IFN may have a role in longer persistence of the virus in pigs. Previous results from our laboratory indicated that viral NSP3-8 might play a major role in PRRSV virulence (27
). Taken together with our findings reported here, this suggests that the pathogenesis of PRRSV may be a complex summation of several major effectors contributing to the overall pathogenesis of PRRSV. The NSP1β acts as a suppressor of innate immune response, and NSP3-8 determines the virulence and invasive capacity, which helps the virus to translocate and cross placenta and infect fetuses. Highly pathogenic viruses such as SARS-CoV and Ebola viruses encode multiple proteins targeting different steps in IFN signaling. This not only ensures complete inhibition of the IFN response but also provides a fail-safe mechanism for the virus in case a single protein became dysfunctional. Our current findings that five NSPs of PRRSV are involved in IFN antagonism, though of variable ability, point toward PRRSV's pathogenic nature.