Porcine Reproductive And Respiratory Syndrome Virus Nsp11 Research Articles

Innate Immune Evasion of PRRSV nsp11 through Degradation of the HDAC2 by Its Endoribonuclease Activity.

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, represents a persistent menace to the global pig industry, causing reproductive failure and respiratory disease in pigs. In this study, we delved into the role of histone deacetylases (HDAC2) during PRRSV infection. Our findings revealed that HDAC2 expression is downregulated upon PRRSV infection. Notably, suppressing HDAC2 activity through specific small interfering RNA led to an increase in virus production, whereas overexpressing HDAC2 effectively inhibited PRRSV replication by boosting the expression of IFN-regulated antiviral molecules. Furthermore, we identified the virus's nonstructural protein 11 (nsp11) as a key player in reducing HDAC2 levels. Mutagenic analyses of PRRSV nsp11 revealed that its antagonistic effect on the antiviral activity of HDAC2 is dependent on its endonuclease activity. In summary, our research uncovered a novel immune evasion mechanism employed by PRRSV, providing crucial insights into the pathogenesis of this virus and guiding the development of innovative prevention strategies against PRRSV infection.

Porcine reproductive and respiratory syndrome virus infection inhibits NF-κB signaling pathway through cleavage of IKKβ by Nsp4

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious porcine pathogen that causes serious economic losses to the world swine industry. The inhibitor kappa B kinase β (IKKβ), a catalytic subunit of the IKK complex, plays multiple roles in regulating the nuclear transcription factor kappa B (NF-κB) activity and a variety of cytokines transcription involved in immune responses. Here, we reported that the nonstructural protein 4 (Nsp4) of PRRSV cleaved IKKβ at the E378 site to inhibit the activation of NF-κB signaling pathway. Additionally, we clearly showed that cleavage of IKKβ by PRRSV Nsp4 depends on the 3 C-like serine protease activity of Nsp4 because the catalytically inactivate mutants of Nsp4 lost the function to cleave IKKβ. Furthermore, we found that hydrophobic patch at the KD-ULD junction of IKKβ could be disrupted by PRRSV Nsp4 via the cleavage of the E378 site, resulting in disruption of NF-κB activity. Of note, the two cleavage fragments of IKKβ lose their function to phosphorylate IκBα and activate NF-κB signaling pathway. Our findings provide a clue to better understand the pathogenic mechanism of PRRSV involved in PRRSV evasion of host antiviral innate immune responses.

PRRSV nonstructural protein 11 degrades swine ISG15 by its endoribonuclease activity to antagonize antiviral immune response

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped positive-stranded RNA virus which causes serious economic losses to pig industry worldwide. Type I IFN induces expression of interferon-stimulated genes 15 (ISG15) to inhibit virus replication. To survive in the host, PRRSV has evolved to antagonize the antiviral response of ISGylation. Previous studies have reported that nonstructural protein 2 of PRRSV inhibits the ISGylation and antiviral function of ISG15 depending on its ovarian tumor (OTU) domain/papain-like protease domain (PLP2). However, whether there are other PRRSV proteins inhibiting ISGylation of cellular proteins is less well understood. In this study, we first found that PRRSV Nsp11 decreased ISGylation of cellular proteins. Meanwhile, the expression level of ISG15 was significantly inhibited by Nsp11. Further mechanistic studies demonstrated that the transcription of ISG15 was reduced by endoribonuclease activity of Nsp11. Finally, we found that the Nsp11-induced degradation of ISG15 was partially relied on autophagy-lysosome system. Taken together, PRRSV Nsp11 antagonizes the antiviral response of ISG15 by its endoribonuclease activity to promote PRRSV replication. Our results reveal a novel mechanism that PRRSV inhibits ISGylation of cellular proteins and impairs host innate immune response.

Porcine reproductive and respiratory syndrome virus nsp4 positively regulates cellular cholesterol to inhibit type I interferon production

Cellular cholesterol plays an important role in the life cycles of enveloped viruses. Previous studies by our group and other groups have demonstrated that the depletion of cellular cholesterol by methyl-β-cyclodextrin (MβCD) reduces the proliferation of porcine reproductive and respiratory syndrome virus (PRRSV), a porcine Arterivirus that has been devastating the swine industry worldwide for over two decades. However, how PRRSV infection regulates cholesterol synthesis is not fully understood. In this study, we showed that PRRSV infection upregulated the activity of protein phosphatase 2 (PP2A), which subsequently activated 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme in the cholesterol synthesis pathway, to increase the levels of cellular cholesterol. By screening the PRRSV-encoded proteins, we showed that nsp4 dominated the upregulation of cellular cholesterol, independently of the 3C-like protease activity of nsp4. A mutation analysis showed that domain I (amino acids 1–80) of PRRSV nsp4 interacted with PR65 alpha (PR65α), the structural subunit, and PP2Ac, the catalytic subunit, of PP2A. Importantly, domain I of nsp4 inhibited Sendai virus-induced interferon β production, and this inhibitory effect was eliminated by Lovastatin, an HMGCR inhibitor, indicating that the upregulation of cellular cholesterol by nsp4 is a strategy used by PRRSV to suppress the antiviral innate immunity of its host. Collectively, we here demonstrated the mechanism by which PRRSV regulates cellular cholesterol synthesis and reported a novel strategy by which PRRSV evades its host's antiviral innate immune response.

Induction of UPR Promotes Interferon Response to Inhibit PRRSV Replication via PKR and NF-κB Pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) was previously shown to induce a certain level of cellular stress during viral replication. Unfolded protein response (UPR) is a cellular stress response responsible for coping with stress and cellular survival. However, the pathway leading to the induction of UPR that may influence PRRSV replication is still unknown. Here, we found that PRRSV infection induced UPR prior to interferon response. Induction of UPR significantly enhanced the expression of interferon and interferon-related genes, thus leading to the suppression of PRRSV infection. Next, we explored the underlying mechanisms of UPR-induced antiviral response. We found that induction of UPR promoted the expression of protein kinase R (PKR), and PKR was highly correlated with the reduction of PRRSV replication. Furthermore, tunicamycin stimulation and PKR overexpression activated NF-κB and interferon response at the early stage of PRRSV infection, thus reinforcing the expression of type I interferons and proinflammatory cytokines and leading to inhibition of PRRSV. In addition, PRRSV nsp4 was shown to reduce the expression of PKR. These findings might have implications for our understandings of the host’s immune mechanism against PRRSV and a new strategy of PRRSV to evade the host antiviral immunity.

Porcine reproductive and respiratory syndrome virus Nsp4 cleaves ZAP to antagonize its antiviral activity

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens impacting the global swine industry. PRRSV has been recognized to modulate the host immune response through a number of mechanisms. In our previous study, we found that over-expression of ZAP, a zinc finger antiviral protein of host, could suppress PRRSV replication, but how PRRSV escape the restriction of ZAP under natural conditions was still unknown. In this study, We found PRRSV infection significantly down-regulate the endogenous ZAP protein expression in Marc-145 cells. And PRRSV nonstructural protein 4 (Nsp4), a 3C-like serine proteinase, was screened to be responsible for ZAP reduction. Nsp4 could cleave ZAP, depending on its protease activity. The anti-PRRSV activity of ZAP was antagonized by Nsp4 in Marc-145 cells. In addition, we identified a unique amino acid, serine 180 of Nsp4 was required for efficient degradation of ZAP, and the mutation at residue 180 could decrease the ability of recombinant PRRSV to degrade ZAP. Those findings reveal a manner of PRRSV Nsp4 antagonizing the antiviral activity of ZAP, and shed light on a new strategy evolved by PRRSV to escape the host defense.

Porcine Reproductive and Respiratory Syndrome Virus Antagonizes PCSK9's Antiviral Effect via Nsp11 Endoribonuclease Activity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry worldwide. Our previous study had indicated that proprotein convertase subtilisin/kexin type 9 (PCSK9) was a responsive gene in porcine alveolar macrophages (PAMs) upon PRRSV infection. However, whether PCSK9 impacts the PRRSV replication and how the PRRSV modulates host PCSK9 remains elusive. Here, we demonstrated that PCSK9 protein suppressed the replication of both type-1 and type-2 PRRSV species. More specifically, the C-terminal domain of PCSK9 was responsible for the antiviral activity. Besides, we showed that PCSK9 inhibited PRRSV replication by targeting the virus receptor CD163 for degradation through the lysosome. In turn, PRRSV could down-regulate the expression of PCSK9 in both PAMs and MARC-145 cells. By screening the nonstructural proteins (nsps) of PRRSV, we showed that nsp11 could antagonize PCSK9’s antiviral activity. Furthermore, mutagenic analyses of PRRSV nsp11 revealed that the endoribonuclease activity of nsp11 was critical for antagonizing the antiviral effect of PCSK9. Collectively, our data provide further insights into the interaction between PRRSV and the cell host and offer a new potential target for the antiviral therapy of PRRSV.

Porcine Reproductive and Respiratory Syndrome Virus nsp11 Antagonizes Type I Interferon Signaling by Targeting IRF9.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus from the Nidovirales order that causes reproductive failure and respiratory disease in pigs and poses a constant threat to the global pig industry. The PRRSV-encoded nonstructural protein 11 (nsp11) is a nidovirus-specific endoribonuclease (NendoU) that is conserved throughout the Arteriviridae and Coronaviridae families. Previously, our research and that of others demonstrated that PRRSV nsp11 inhibits type I interferon (IFN) production through NendoU activity-dependent mechanisms. Here, we found that PRRSV nsp11 also inhibited IFN-stimulated response element (ISRE) promoter activity and subsequent transcription of IFN-stimulated genes (ISGs). Detailed analysis showed that nsp11 targeted interferon regulatory factor 9 (IRF9), but not transducer and activator of transcription 1 (STAT1) or STAT2, key molecules in the type I IFN signaling pathway. Furthermore, the nsp11-IRF9 interaction impaired the formation and nuclear translocation of the transcription factor complex IFN-stimulated gene factor 3 (ISGF3) in both nsp11-overexpressed and PRRSV-infected cells. Importantly, nsp11 mutations (H129A, H144A, and K173A) that ablate NendoU activity or its cell cytotoxicity also interacted with IRF9 and retained the ability to block IFN signaling, indicating that the nsp11-IRF9 interaction is independent of NendoU activity or cell cytotoxicity of nsp11. Taking the results together, our study demonstrated that PRRSV nsp11 antagonizes type I IFN signaling by targeting IRF9 via a NendoU activity-independent mechanism, and this report describes a novel strategy evolved by PRRSV to counteract host innate antiviral responses, revealing a potential new function for PRRSV nsp11 in type I IFN signaling.IMPORTANCE The nidovirus-specific endoribonuclease (NendoU) encoded by PRRSV nonstructural protein 11 (nsp11) is a unique NendoU of nidoviruses that infect vertebrates; thus, it is an attractive target for the development of antinidovirus drugs. Previous studies have revealed that the NendoU of nidoviruses, including porcine reproductive and respiratory syndrome virus (PRRSV) and human coronavirus 229E (HCoV-229E), acts as a type I interferon (IFN) antagonist. Here, for the first time, we demonstrated that overexpression of PRRSV nsp11 also inhibits IFN signaling by targeting the C-terminal interferon regulatory factor (IRF) association domain of IRF9. This interaction impaired the ability of IRF9 to form the transcription factor complex IFN-stimulated gene factor 3 (ISGF3) and to act as a signaling protein of IFN signaling. Collectively, our data identify IRF9 as a natural target of PRRSV NendoU and reveal a novel mechanism evolved by an arterivirus to counteract innate immune signaling.

Arterivirus nsp4 Antagonizes Interferon Beta Production by Proteolytically Cleaving NEMO at Multiple Sites.

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose major threats for the horse- and swine-breeding industries worldwide. A previous study suggested that PRRSV nsp4, a 3C-like protease, antagonizes interferon beta (IFN-β) production by cleaving the NF-κB essential modulator (NEMO) at a single site, glutamate 349 (E349). Here, we demonstrated that EAV nsp4 also inhibited virus-induced IFN-β production by targeting NEMO for proteolytic cleavage and that the scission occurred at four sites: E166, E171, glutamine 205 (Q205), and E349. Additionally, we found that, besides the previously reported cleavage site E349 in NEMO, scission by PRRSV nsp4 took place at two additional sites, E166 and E171. These results imply that while cleaving NEMO is a common strategy utilized by EAV and PRRSV nsp4 to antagonize IFN induction, EAV nsp4 adopts a more complex substrate recognition mechanism to target NEMO. By analyzing the abilities of the eight different NEMO fragments resulting from EAV or PRRSV nsp4 scission to induce IFN-β production, we serendipitously found that a NEMO fragment (residues 1 to 349) could activate IFN-β transcription more robustly than full-length NEMO, whereas all other NEMO cleavage products were abrogated for the IFN-β-inducing capacity. Thus, NEMO cleavage at E349 alone may not be sufficient to completely inactivate the IFN response via this signaling adaptor. Altogether, our findings suggest that EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is critical for disarming the innate immune response for viral survival.IMPORTANCE The arterivirus nsp4-encoded 3C-like protease (3CLpro) plays an important role in virus replication and immune evasion, making it an attractive target for antiviral therapeutics. Previous work suggested that PRRSV nsp4 suppresses type I IFN production by cleaving NEMO at a single site. In contrast, the present study demonstrates that both EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is essential for disruption of type I IFN production. Moreover, we reveal that EAV nsp4 also cleaves NEMO at glutamine 205 (Q205), which is not targeted by PRRSV nsp4. Notably, targeting a glutamine in NEMO for cleavage has been observed only with picornavirus 3C proteases (3Cpro) and coronavirus 3CLpro In aggregate, our work expands knowledge of the innate immune evasion mechanisms associated with NEMO cleavage by arterivirus nsp4 and describes a novel substrate recognition characteristic of EAV nsp4.

The Superimposed Deubiquitination Effect of OTULIN and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Nsp11 Promotes Multiplication of PRRSV.

Linear ubiquitination plays an important role in the regulation of the immune response by regulating nuclear factor κB (NF-κB). The linear ubiquitination-specific deubiquitinase ovarian tumor domain deubiquitinase with linear linkage specificity (OTULIN) can control the immune signaling transduction pathway by restricting the Met1-linked ubiquitination process. In our study, the porcine OTLLIN gene was cloned and deubiquitin functions were detected in a porcine reproductive and respiratory syndrome virus (PRRSV)-infected-cell model. PRRSV infection promotes the expression of the OTULIN gene; in turn, overexpression of OTULIN contributes to PRRSV proliferation. There is negative regulation of innate immunity with OTULIN during viral infection. The cooperative effects of swine OTULIN and PRRSV Nsp11 potentiate the ability to reduce levels of cellular protein ubiquitin associated with innate immunity. Importantly, PRRSV Nsp11 recruits OTULIN through a nonenzymatic combination to enhance its ability to remove linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I interferons (IFNs). Our report presents a new model of virus utilization of the ubiquitin-protease system in vivo from the perspective of the viral proteins that interact with cell deubiquitination enzymes, providing new ideas for prevention and control of PRRSV.IMPORTANCE Deubiquitination effects of swine OTULIN were identified. The interaction between porcine OTULIN and PRRSV Nsp11 is dependent on the OTU domain. PRRSV Nsp11 recruits OTULIN through a nonenzymatic combination to promote removal of linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I IFNs.

Structure and Function of PRRSV nsp11 Endoribonuclease

Endoribonuclease (NendoU) is conserved and plays essential roles in viral life cycle among all nidoviruses. Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 11 (nsp11) is shown to have NendoU activity and its multifunctions have been demonstrated, such as processing RNA, suppressing innate immunity system of infected hosts and regulating the progression of S phase cells. We solve the crystal structure of PRRSV nsp11 mutant (K170A), and the overall structure is greatly different from severe acute respiratory syndrome coronaviruses (SARS-CoV) nsp15, the counterpart of nsp11 in coronaviruses. Compared with SARS-CoV nsp15 which has three domains, PRRSV nsp11 only have two domains: the N-terminal domain (NTD) and C-terminal domain (CTD). Smallangle X-ray scattering data shows that the PRRSV nsp11 has several kinds of oligomeric conformations in solution, this may be due to the missing domain, which mediates the hexamerization of SARS-CoV nsp15. The active center of NendoU is located in the CTD, where the conserved catalytic residues form a positively charged groove and bind the negatively charged RNA. The catalytic regions have several conformations, which are quite diverse. The NTD shares similar structural element with ubiquitin binding protein, but nsp11 cannot bind to ubiquitin according to our data. The flexible catalytic region of nidoviruses endoribonuclease confers an excellent target for drug design. These structural and functional information of PRRSV nsp11 would lay a foundation for better understanding of the molecular mechanism and antiviral drug development.

Identification of a novel linear B-cell epitope in nonstructural protein 11 of porcine reproductive and respiratory syndrome virus that are conserved in both genotypes.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens, that hinder the development of global pork industry. Its nonstructural protein 11 (nsp11), with the nidoviral uridylate-specific endoribonuclease (NendoU) domain, is essential for PRRSV genome replication and it also contributes to host innate immunity suppression. However, the immunogenicity and immune structure of PRRSV nsp11 have not been well investigated yet. In this study, a monoclonal antibody (mAb), designated 3F9, that against nsp11 was generated. Subsequently, a series of partially overlapped fragments, covered the nsp1140-223aa, were expressed to test the reactivity with mAb 3F9, and the 111DCREY115 was found to be the core unit of the B-cell epitope recognized by mAb 3F9. Further investigation indicated that both genotype 1 and genotype 2 PRRSV can be recognized by mAb 3F9, due to the 111DCREY115 is conserved in both genotype virus. Meanwhile, this epitope, localized at the surface of nsp11 in 3D structure, is confirmed to be able to induce humoral immune response in PRRSV infected pigs. These findings do not only provide an mAb tool to further investigate the function of nsp11, they also indicate the diagnostic potential for this epitope.

Critical role of cytochrome c1 and its cleavage in porcine reproductive and respiratory syndrome virus nonstructural protein 4-induced cell apoptosis via interaction with nsp4

Porcine reproductive and respiratory syndrome virus (PRRSV) actively induces cell apoptosis both in vitro and in vivo, which can contribute critically to viral pathogenesis. Previous studies have shown that the PRRSV nonstructural protein 4 (nsp4) is an important mediator of this process, but the underlying molecular details remain poorly understood. In this study, we found that the PRRSV nsp4 interacted with the mitochondrial inner membrane protein cytochrome c1 (cyto.c1) and induced its proteolytic cleavage. Interestingly, the cleaved N-terminal fragment of cyto.c1 was found to exert apoptotic activity, which could cause mitochondrial fragmentation, resulting in apoptotic cell death. And RNA interference (RNAi) silencing experiments further confirmed the crucial role which cyto.c1 played in nsp4- and PRRSV-induced cell apoptosis. Thus, our data provide an important piece of mechanistic clues for PRRSV-induced cell apoptosis and also elucidate a novel mechanism for the 3C-like proteases in this finding.

Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus Suppresses Both MAVS and RIG-I Expression as One of the Mechanisms to Antagonize Type I Interferon Production

Type I interferons (IFN-α/β) play a key role in antiviral defense, and porcine reproductive and respiratory syndrome virus (PRRSV) is known to down-regulate the IFN response in virus-infected cells and pigs. In this study, we showed that the overexpression of nsp11 of PRRSV induced a strong suppression of IFN production. Nsp11 suppressed both IRF3 and NF-κB activities when stimulated with a dsRNA analogue and TNF-α, respectively. This suppression was RLR dependent, since the transcripts and proteins of MAVS and RIG-I, two critical factors in RLR-mediated pathway, were both found to be reduced in the presence of overexpressed nsp11. Since nsp11 is an endoribonuclease (EndoU), the structure function relationship was examined using a series of nsp11 EndoU mutant plasmids. The mutants that impaired the EndoU activity failed to suppress IFN and led to the normal expression of MAVS. Seven single amino acid substitutions (4 in subdomain A and 3 in subdomain B) plus one insertion (frame-shift in nsp11) were then introduced into PRRSV infectious cDNA clones to generate nsp11 mutant viruses. Unfortunately, all EndoU knock-out nsp11 mutant viruses appeared replication-defective and no progenies were produced. Three mutations in EndoU subdomain A expressed the N and nsp2/3 proteins but their infectivity diminished after 2 passages. Taken together, our data show that PRRSV nsp11 endoribonuclease activity is critical for both viral replication and IFN antagonism. More importantly, the endoribonuclease activity of nsp11 demonstrates the substrate specificity towards MAVS and RIG-I (transcripts and proteins) over p65 and IRF3 in the context of gene transfection and overexpression. This is likely a mechanism of nsp11 suppression of type I IFN production.

Nonstructural protein 11 (nsp11) of porcine reproductive and respiratory syndrome virus (PRRSV) promotes PRRSV infection in MARC-145 cells.

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) induces one of most important devastating disease of swine worldwide, and the current methods poorly control it. Previous studies have indicated that the nonstructural protein 11 (nsp11) of PRRSV may be an important protein for the immune escape of PRRSV.ResultsHere, we firstly explored the effect of over-expression of nsp11 on PRRSV infection and found that over-expression of nsp11 enhanced the PRRSV titers while the small interfering RNA (siRNAs) specifically targeting nsp11 could reduce the PRRSV titers in MARC-145 cells.ConclusionIn conclusion, PRRSV nsp11 promotes PRRSV infection in MARC-145 cells and siRNAs targeting nsp11 may be a potential therapeutic strategy to control PRRSV in future.

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Induces Apoptosis Dependent on Its 3C-Like Serine Protease Activity

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease in pigs caused by PRRS virus (PRRSV). Although PRRSV infection-induced cell apoptosis has been established, the related viral protein is still unknown. Here, we reported that PRRSV nonstructural protein 4 (nsp4) was a critical apoptosis inducer. Nsp4 could activate caspase-3, -8, and -9. Using truncated constructs without different domains in nsp4, we demonstrated that the full-length of nsp4 structure was required for its apoptosis-inducing activity. Furthermore, using site-directed mutagenesis to inactivate the 3C-like serine protease activity of nsp4, we showed that nsp4-induced apoptosis was dependent on its serine protease activity. The ability of nsp4 to induce apoptosis was significantly impaired by His39, Asp64, and Ser118 mutations, suggesting that His39, Asp64, and Ser118 were essential for nsp4 to trigger apoptosis. In conclusion, our present work showed that PRRSV nsp4 could induce apoptosis in host cells and might be partially responsible for the apoptosis induced by PRRSV infection. PRRSV 3C-like protease-mediated apoptosis represents the first report in the genus Arterivirus, family Arteriviridae.