Dataset Information

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.

ABSTRACT: Type I interferons (IFN-?/?) 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-?B activities when stimulated with a dsRNA analogue and TNF-?, 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.

SUBMITTER: Sun Y

PROVIDER: S-EPMC5172586 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

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.

Sun Yan Y Ke Hanzhong H Han Mingyuan M Chen Ning N Fang Weihuan W Yoo Dongwan D

PloS one 20161220 12

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 prot ...[more]

PMID: 27997564

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Project description:Interferons (IFNs) play a crucial role in host antiviral response by activating the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway to induce the expression of myriad genes. STAT2 is a key player in the IFN-activated JAK/STAT signaling. Porcine reproductive and respiratory syndrome virus (PRRSV) is an important viral pathogen, causing huge losses to the swine industry. PRRSV infection elicits a meager protective immune response in pigs. The objective of this study was to investigate the effect of PRRSV on STAT2 signaling. Here, we demonstrated that PRRSV downregulated STAT2 to inhibit IFN-activated signaling. PRRSV strains of both PRRSV-1 and PRRSV-2 species reduced the STAT2 protein level, whereas the STAT2 transcript level had minimal change. PRRSV reduced the STAT2 level in a dose-dependent manner and shortened STAT2 half-life significantly from approximately 30 to 5?h. PRRSV-induced STAT2 degradation could be restored by treatment with the proteasome inhibitor MG132 and lactacystin. In addition, PRRSV nonstructural protein 11 (nsp11) was identified to interact with and reduce STAT2. The N-terminal domain (NTD) of nsp11 was responsible for STAT2 degradation and interacted with STAT2 NTD and the coiled-coil domain. Mutagenesis analysis showed that the amino acid residue K59 of nsp11 was indispensable for inducing STAT2 reduction. Mutant PRRSV with the K59A mutation generated by reverse genetics almost lost the ability to reduce STAT2. Together, these results demonstrate that PRRSV nsp11 antagonizes IFN signaling via mediating STAT2 degradation and provide further insights into the PRRSV interference of the innate immunity.IMPORTANCE PRRSV infection elicits a meager protective immune response in pigs. One of the possible reasons is that PRRSV antagonizes interferon induction and its downstream signaling. Interferons are key components in the innate immunity and play crucial roles against viral infection and in the activation of adaptive immune response via JAK/STAT signaling. STAT2 is indispensable in the JAK/STAT signaling since it is also involved in activation of antiviral activity in the absence of STAT1. Here, we discovered that PRRSV nsp11 downregulates STAT2. Interestingly, the N-terminal domain of nsp11 is responsible for inducing STAT2 degradation and directly interacts with STAT2 N-terminal domain. We also identified a crucial amino acid residue K59 in nsp11 since a mutation of it led to loss of the ability to downregulate STAT2. A mutant PRRSV with mutation of K59 had minimal effect on STAT2 reduction. Our data provide further insights into PRRSV interference with interferon signaling.

Project description:Tembusu virus (TMUV) is a newly emerged avian flavivirus that has caused severe egg-drop syndrome and fatal encephalitis in domestic ducks. It has spread widely throughout the main duck-producing areas in Asia, resulting in substantial economic losses to the duck industry. Previous studies have reported that TMUV has evolved several strategies to counteract the duck's innate immune responses to successfully establish infection in its host cells. However, the mechanisms underlying this phenomenon have not been elucidated. Here, we discovered that TMUV-encoded NS2B is a negative regulator of poly(I:C)-induced duck interferon-β (IFN-β) expression. Mechanistically, TMUV NS2B was found to interact specifically with the mitochondrial antiviral-signaling protein (duMAVS). Consequently, duMAVS was degraded through the K48-linked ubiquitination and proteasomal pathway, leading to the interruption of the RIG-I-like receptor (RLR) signaling. Further analyses also identified K321, K354, K398, and K411 as crucial residues for NS2B-mediated ubiquitination and degradation of duMAVS. Additionally, we demonstrated that NS2B functions by recruiting the E3 ubiquitin ligase duck membrane-associated RING-CH-type finger 5 (duMARCH5) to modify duMAVS via polyubiquitination, blocking the duMAVS-mediated innate immune response and promoting TMUV replication. Taken together, our findings revealed a novel mechanism by which TMUV evades the duck's antiviral innate immune responses. IMPORTANCE Tembusu virus (TMUV), an emerging pathogenic flavivirus, has spread to most duck farming areas in Asia since 2010, causing significant economic losses to the duck industry. Recently, TMUV has expanded its host range and may pose a potential threat to mammals, including humans. Understanding the interaction between TMUV and its host is essential for the development of effective vaccines and therapeutics. Here, we show that NS2B encoded by TMUV inhibits IFN production by interacting with duck MAVS (duMAVS) to mediate ubiquitination and proteasomal degradation. Further studies suggest that the E3 ubiquitin ligase duck membrane-associated RING-CH-type finger 5 (duMARCH5) is recruited by NS2B to mediate proteasomal degradation of duMAVS. As a result, the innate immune response triggered by the RIG-I-like receptor (RLR) is disrupted, facilitating viral replication. Overall, our results reveal a novel mechanism by which TMUV evades host innate immunity and provide new therapeutic strategies to prevent TMUV infection.

Dataset Information

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.

Publications

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.

Similar Datasets

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