Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC7354446 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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

Zhang Yujiao Y Gao Fei F Li Liwei L Zhao Kuan K Jiang Shan S Jiang Yifeng Y Yu Lingxue L Zhou Yanjun Y Liu Changlong C Tong Guangzhi G

Viruses 20200617 6

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 a ...[more]

PMID: 32560445

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Project description:Monocyte chemotactic protein-induced protein 1 (MCPIP1) is an inflammatory regulator in immune response and has broad antiviral effects by targeting viral RNA. Porcine reproductive and respiratory syndrome virus (PRRSV), a major viral pathogen in pigs, causes immune suppression leading to coinfection of swine pathogens, but the mechanisms are not fully clarified. In this study, MCPIP1 expression was found to be significantly upregulated in lungs of PRRSV-infected piglets, as well as in Marc-145 and porcine pulmonary alveolar macrophage (PAM) cells upon PRRSV stimulation. MCPIP1 overexpression significantly inhibited PRRSV replication, while MCPIP1 knockdown increased the virus titer. Various mutations in RNase functional domains of MCPIP1 impaired the inhibitory activity against PRRSV, while those in deubiquitinase domains failed to do so. MCPIP1 expression started to decrease from 60 h after PRRSV infection in PAMs. Meanwhile, infection with higher dose of PRRSV further downregulated MCPIP1, indicating the antagonizing effects from PRRSV against MCPIP1. Moreover, it was confirmed that MCPIP1 expression was downregulated in 3D4 cells with either interleukin-17 (IL-17) or nsp11 overexpression, while IL-17 inhibitor abolished the decrease of MCPIP1 caused by nsp11, indicating nsp11 employs IL-17 induction to inhibit MCPIP1. Furthermore, the PRRSV nsp11 mutant with a deficiency in IL-17 induction showed the recovered expression of MCPIP1 in infected cells, inspiring a strategy for virus attenuation. This is the first report about the role of MCPIP1 against PRRSV and the function of PRRSV nsp11 against innate immunity to facilitate virus replication via IL-17. The study not only illuminates PRRSV infection machinery but also enlightens alternative antiviral strategies, such as vaccine candidates. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses the innate immunity and leads to coinfection of swine pathogens. Monocyte chemotactic protein-induced protein 1 (MCPIP1) is a broad-spectrum host antiviral protein. Therefore, to further clarify the mechanism of PRRSV against innate immunity, we explored the relationship between MCPIP1 and PRRSV infection. The results showed that MCPIP1 inhibited PRRSV infection in the early stage of virus infection. Importantly, PRRSV nsp11 subsequently employed IL-17 induction to suppress MCPIP1 expression and antagonized anti-PRRSV effects. Furthermore, PRRSV with mutation of nsp11 S74A failed to induce MCPIP1 reduction. These findings confirmed the function of MCPIP1 against PRRSV and revealed that PRRSV nsp11 plays an important role in virus against innate immunity. This study enlightens a new strategy to develop safer attenuated vaccines against PRRSV by nsp11 mutation.

Project description:UnlabelledPorcine reproductive and respiratory syndrome virus (PRRSV) RNA endoribonuclease nsp11 belongs to the XendoU superfamily and plays a crucial role in arterivirus replication. Here, we report the first crystal structure of the arterivirus nsp11 protein from PRRSV, which exhibits a unique structure and assembles into an asymmetric dimer whose structure is completely different from the hexameric structure of coronavirus nsp15. However, the structures of the PRRSV nsp11 and coronavirus nsp15 catalytic domains were perfectly superimposed, especially in the "active site loop" (His129 to His144) and "supporting loop" (Val162 to Thr179) regions. Importantly, our biochemical data demonstrated that PRRSV nsp11 exists mainly as a dimer in solution. Mutations of the major dimerization site determinants (Ser74 and Phe76) in the dimerization interface destabilized the dimer in solution and severely diminished endoribonuclease activity, indicating that the dimer is the biologically functional unit. In the dimeric structure, the active site loop and supporting loop are packed against one another and stabilized by monomer-monomer interactions. These findings may help elucidate the mechanism underlying arterivirus replication and may represent great potential for the development of antiviral drugs.ImportancePorcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirales PRRSV is a major agent of respiratory diseases in pigs, causing tremendous economic losses to the swine industry worldwide. The PRRSV nsp11 endoribonuclease plays a vital role in arterivirus replication, but its precise roles and mechanisms of action are poorly understood. Here, we report the first dimeric structure of the arterivirus nsp11 from PRRSV at 2.75-Å resolution. Structural and biochemical experiments demonstrated that nsp11 exists mainly as a dimer in solution and that nsp11 may be fully active as a dimer. Mutagenesis and structural analysis revealed NendoU active site residues, which are conserved throughout the order Nidovirales(families Arteriviridae and Coronaviridae) and the major determinants of dimerization (Ser74 and Phe76) in Arteriviridae Importantly, these findings may provide a new structural basis for antiviral drug development.

Project description: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.

Project description:To fulfill virus replication and persistent infection in hosts, viruses have to find ways to compromise innate immunity, including timely impedance on antiviral RNases and inflammatory responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a major swine pathogen causing immune suppression. MALT1 is a central immune regulator in both innate and adaptive immunity. In this study, MALT1 was confirmed to be induced rapidly upon PRRSV infection and mediate the degradation of two anti-PRRSV RNases, MCPIP1 and N4BP1, relying on its proteolytic activity, consequently facilitating PRRSV replication. Multiple PRRSV nsps, including nsp11, nsp7β, and nsp4, contributed to MALT1 elicitation. Interestingly, the elevated expression of MALT1 began to decrease once intracellular viral expression reached a high enough level. Higher infection dose brought earlier MALT1 inflection. Further, PRRSV nsp6 mediated significant MALT1 degradation via ubiquitination-proteasome pathway. Downregulation of MALT1 suppressed NF-κB signals, leading to the decrease in proinflammatory cytokine expression. In conclusion, MALT1 expression was manipulated by PRRSV in an elaborate manner to antagonize precisely the antiviral effects of host RNases without excessive and continuous activation of inflammatory responses. These findings throw light on the machinery of PRRSV to build homeostasis in infected immune system for viral settlement. IMPORTANCE PRRSV is a major swine pathogen, suppresses innate immunity, and causes persistent infection and coinfection with other pathogens. As a central immune mediator, MALT1 plays essential roles in regulating immunity and inflammation. Here, PRRSV was confirmed to manipulate MALT1 expression in an accurate way to moderate the antiviral immunity. Briefly, multiple PRRSV nsps induced MALT1 protease to antagonize anti-PRRSV RNases N4BP1 and MCPIP1 upon infection, thereby facilitating viral replication. In contrast, PRRSV nsp6 downregulated MALT1 expression via ubiquitination-proteasome pathway to suppress the inflammatory responses upon infection aggravation, contributing to immune defense alleviation and virus survival. These findings revealed the precise expression control on MALT1 by PRRSV for antagonizing antiviral RNases, along with recovering immune homeostasis. For the first time, this study enlightens a new mechanism of PRRSV adapting antiviral innate immunity by modulating MALT1 expression.

Project description:Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic signaling mediator of many cytokines, including interleukin-6 (IL-6) and IL-10. STAT3 is known to play critical roles in cell growth, proliferation, differentiation, immunity and inflammatory responses. The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on the STAT3 signaling since PRRSV induces a weak protective immune response in host animals. We report here that PRRSV infection of MARC-145 cells and primary porcine pulmonary alveolar macrophages led to significant reduction of STAT3 protein level. Several strains of both PRRSV type 1 and type 2 led to a similar reduction of STAT3 protein level but had a minimal effect on its transcripts. The PRRSV-mediated STAT3 reduction was in a dose-dependent manner as the STAT3 level decreased, along with incremental amounts of PRRSV inocula. Further study showed that nonstructural protein 5 (nsp5) of PRRSV induced the STAT3 degradation by increasing its polyubiquitination level and shortening its half-life from 24 h to ∼3.5 h. The C-terminal domain of nsp5 was shown to be required for the STAT3 degradation. Moreover, the STAT3 signaling in the cells transfected with nsp5 plasmid was significantly inhibited. These results indicate that PRRSV antagonizes the STAT3 signaling by accelerating STAT3 degradation via the ubiquitin-proteasomal pathway. This study provides insight into the PRRSV interference with the JAK/STAT3 signaling, leading to perturbation of the host innate and adaptive immune responses. The typical features of immune responses in PRRSV-infected pigs are delayed onset and low levels of virus neutralizing antibodies, as well as weak cell-mediated immunity. Lymphocyte development and differentiation rely on cytokines, many of which signal through the JAK/STAT signaling pathway to exert their biological effects. Here, we discovered that PRRSV antagonizes the JAK/STAT3 signaling by inducing degradation of STAT3, a master transcription activator involved in multiple cellular processes and the host immune responses. The nsp5 protein of PRRSV is responsible for the accelerated STAT3 degradation. The PRRSV-mediated antagonizing STAT3 could lead to suppression of a broad spectrum of cytokines and growth factors to allow virus replication and spread in host animals. This may be one of the reasons for the PRRSV interference with the innate immunity and its poor elicitation of protective immunity. This finding provides insight into PRRSV pathogenesis and its interference with the host immune responses.

Dataset Information

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

Publications

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

Similar Datasets

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