Dataset Information

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion.

ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) blocks host mRNA nuclear export to the cytoplasm, and nonstructural protein 1 beta (nsp1?) of PRRSV has been identified as the protein that disintegrates the nuclear pore complex. In the present study, the molecular basis for the inhibition of host mRNA nuclear export was investigated. Nucleoporin 62 (Nup62) was found to bind to nsp1?, and the region representing the C-terminal residues 328 to 522 of Nup62 was determined to be the binding domain for nsp1?. The nsp1? L126A mutant in the SAP domain did not bind to Nup62, and in L126A-expressing cells, host mRNA nuclear export occurred normally. The vL126A mutant PRRSV generated by reverse genetics replicated at a lower rate, and the titer was lower than for wild-type virus. In nsp1?-overexpressing cells or small interfering RNA (siRNA)-mediated Nup62 knockdown cells, viral protein synthesis increased. Notably, the production of type I interferons (IFN-?/?), IFN-stimulated genes (PKR, OAS, Mx1, and ISG15 genes), IFN-induced proteins with tetratricopeptide repeats (IFITs) 1 and 2, and IFN regulatory factor 3 decreased in these cells. As a consequence, the growth of vL126A mutant PRRSV was rescued to the level of wild-type PRRSV. These findings are attributed to nuclear pore complex (NPC) disintegration by nsp1?, resulting in increased viral protein production and decreased host protein production, including antiviral proteins in the cytoplasm. Our study reveals a new strategy of PRRSV for immune evasion and enhanced replication during infection.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS and is known to effectively suppress host innate immunity. The PRRSV nsp1? protein blocks host mRNA nuclear export, which has been shown to be one of the viral mechanisms for inhibition of antiviral protein production. nsp1? binds to the cellular protein nucleoporin 62 (Nup62), and as a consequence, the nuclear pore complex (NPC) is disintegrated and the nucleocytoplasmic trafficking of host mRNAs and host proteins is blocked. We show the dual benefits of Nup62 and nsp1? binding for PRRSV replication: the inhibition of host antiviral protein expression and the exclusive use of host translation machinery by the virus. Our study unveils a novel strategy of PRRSV for immune evasion and enhanced replication during infection.

SUBMITTER: Ke H

PROVIDER: S-EPMC6600190 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion.

Ke Hanzhong H Han Mingyuan M Kim Jineui J Gustin Kurt E KE Yoo Dongwan D

Journal of virology 20190628 14

Porcine reproductive and respiratory syndrome virus (PRRSV) blocks host mRNA nuclear export to the cytoplasm, and nonstructural protein 1 beta (nsp1β) of PRRSV has been identified as the protein that disintegrates the nuclear pore complex. In the present study, the molecular basis for the inhibition of host mRNA nuclear export was investigated. Nucleoporin 62 (Nup62) was found to bind to nsp1β, and the region representing the C-terminal residues 328 to 522 of Nup62 was determined to be the bindi ...[more]

PMID: 31043527

Similar Datasets

Project description:Porcine reproductive and respiratory syndrome (PRRS) is of great concern to the swine industry due to pandemic outbreaks of the disease, current ineffective vaccinations, and a lack of efficient antiviral strategies. In our previous study, a PRRSV Nsp9-specific nanobody, Nb6, was successfully isolated, and the intracellularly expressed Nb6 could dramatically inhibit PRRSV replication in MARC-145 cells. However, despite its small size, the application of Nb6 protein in infected cells is greatly limited, as the protein itself cannot enter the cells physically. In this study, a trans-activating transduction (TAT) peptide was fused with Nb6 to promote protein entry into cells. TAT-Nb6 was expressed as an inclusion body in Escherichia coli, and indirect enzyme-linked immunosorbent assays and pulldown assays showed that E. coli-expressed TAT-Nb6 maintained the binding ability to E. coli-expressed or PRRSV-encoded Nsp9. We demonstrated that TAT delivered Nb6 into MARC-145 cells and porcine alveolar macrophages (PAMs) in a dose- and time-dependent manner, and TAT-Nb6 efficiently inhibited the replication of several PRRSV genotype 2 strains as well as a genotype 1 strain. Using a yeast two-hybrid assay, Nb6 recognition sites were identified in the C-terminal part of Nsp9 and spanned two discontinuous regions (Nsp9aa454-551 and Nsp9aa599-646). Taken together, these results suggest that TAT-Nb6 can be developed as an antiviral drug for the inhibition of PRRSV replication and controlling PRRS disease.IMPORTANCE The pandemic outbreak of PRRS, which is caused by PRRSV, has greatly affected the swine industry. We still lack an efficient vaccine, and it is an immense challenge to control its infection. An intracellularly expressed Nsp9-specific nanobody, Nb6, has been shown to be able to inhibit PRRSV replication in MARC-145 cells. However, its application is limited, because Nb6 cannot physically enter cells. Here, we demonstrated that the cell-penetrating peptide TAT could deliver Nb6 into cultured cells. In addition, TAT-Nb6 fusion protein could suppress the replication of various PRRSV strains in MARC-145 cells and PAMs. These findings may provide a new approach for drug development to control PRRS.

Project description:Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly identified phlebovirus associated with severe hemorrhagic fever in humans. Studies have shown that SFTSV nucleoprotein (N) induces BECN1-dependent autophagy to promote viral assembly and release. However, the function of other SFTSV proteins in regulating autophagy has not been reported. In this study, we identify SFTSV NSs, a nonstructural protein that forms viroplasm-like structures in the cytoplasm of infected cells as the virus component mediating SFTSV-induced autophagy. We found that SFTSV NSs-induced autophagy was inclusion body independent, and most phenuivirus NSs had autophagy-inducing effects. Unlike N protein-induced autophagy, SFTSV NSs was key in regulating autophagy by interacting with the host's vimentin in an inclusion body-independent manner. NSs interacted with vimentin and induced vimentin degradation through the K48-linked ubiquitin-proteasome pathway. This negatively regulating Beclin1-vimentin complex formed and promoted autophagy. Furthermore, we identified the NSs-binding domain of vimentin and found that overexpression of wild-type vimentin antagonized the induced effect of NSs on autophagy and inhibited viral replication, suggesting that vimentin is a potential antiviral target. The present study shows a novel mechanism through which SFTSV nonstructural protein activates autophagy, which provides new insights into the role of NSs in SFTSV infection and pathogenesis. IMPORTANCE Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerging tick-borne pathogen that causes multifunctional organ failure and even death in humans. As a housekeeping mechanism for cells to maintain steady state, autophagy plays a dual role in viral infection and the host's immune response. However, the relationship between SFTSV infection and autophagy has not been described in detail yet. Here, we demonstrated that SFTSV infection induced complete autophagic flux and facilitated viral proliferation. We also identified a key mechanism underlying NSs-induced autophagy, in which NSs interacted with vimentin to inhibit the formation of the Beclin1-vimentin complex and induced vimentin degradation through K48-linked ubiquitination modification. These findings may help us understand the new functions and mechanisms of NSs and may aid in the identification of new antiviral targets.

Project description:Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids that is in most FMDVs examined to date, and it plays important roles in virus replication, virulence, and host range. To better understand the role of 3A during FMDV infection, we used coimmunoprecipitation followed by mass spectrometry to identify host proteins that interact with 3A in FMDV-infected cells. Here, we report that cellular vimentin is a host binding partner for 3A. The 3A-vimentin interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pull down, and immunofluorescence assays. Alanine-scanning mutagenesis indicated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin. Using reverse genetics, we demonstrate that mutations in 3A that disrupt the interaction between 3A and vimentin are also critical for virus growth. Overexpression of vimentin significantly suppressed the replication of FMDV, whereas knockdown of vimentin significantly enhanced FMDV replication. However, chemical disruption of the vimentin network by acrylamide resulted in a significant decrease in viral yield, suggesting that an intact vimentin network is needed for FMDV replication. These results indicate that vimentin interacts with FMDV 3A and negatively regulates FMDV replication and that the vimentin-3A interaction is essential for FMDV replication. This study provides information that should be helpful for understanding the molecular mechanism of FMDV replication.IMPORTANCE Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, host range, and virulence. To further understand the role of 3A during FMDV infection, identification of host cell factors that interact with FMDV 3A is needed. Here, we found that vimentin is a direct binding partner of FMDV 3A, and manipulation of vimentin has a negative effect on virus replication. We also demonstrated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin and that the 3A-vimentin interaction is critical for viral replication since the full-length cDNA clone harboring mutations in 3A, which were disrupt 3A-vimentin reactivity, could not produce viable virus progeny. This study provides information that not only provides us a better understanding of the mechanism of FMDV replication but also helps in the development of novel antiviral strategies in the future.

Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. Severe interstitial pneumonia is the typical pathological characteristic of PRRSV-infected swine. Accumulating evidence has suggested that hypoxia-inducible factor 1α (HIF-1α) plays vital roles in the development of inflammation and the viral life cycle. However, the role and the underlying mechanism of HIF-1α in PRRSV infection remain elusive. Here, we found that PRRSV infection elevated HIF-1α expression. Furthermore, overexpression of HIF-1α increased PRRSV replication, whereas knockdown of HIF-1α inhibited PRRSV infection. Our further mechanistic analysis revealed that PRRSV-encoded nonstructural protein 1β (nsp1β) promoted HIF-1α transcription via its N-terminal nuclease activity and degraded the polyubiquitin chain of HIF-1α via its C-terminal deubiquitylation (DUB) enzyme activity, collectively stabilizing HIF-1α. Meanwhile, nsp1β interacted with both HIF-1α and von Hippel-Lindau tumor suppressor (pVHL) to form a ternary complex, which may have hindered pVHL-mediated ubiquitination degradation of HIF-1α by impairing the interaction between HIF-1α and pVHL. Interestingly, pVHL also stabilized nsp1β via K63-linked ubiquitination, forming a positive feedback loop to stabilize HIF-1α. Taken together, these results indicate that PRRSV infection stabilizes HIF-1α to facilitate viral proliferation and that viral nsp1β plays a vital role in enhancing the expression and stabilization of HIF-1α. The regulation of HIF-1α may have great therapeutic potential for the development of novel drugs against PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has devastated the swine industry worldwide for over 30 years and shows no signs of slowing down. In this study, we found that PRRSV infection elevated hypoxia-inducible factor 1α (HIF-1α) expression. In addition, overexpressed HIF-1α contributed to PRRSV replication, whereas knockdown of HIF-1α reduced PRRSV growth. The PRRSV-encoded nonstructural protein 1β (nsp1β) exerted a stabilizing effect on HIF-1α through its nuclease protease and papain-like cysteine protease enzymatic domains. PRRSV nsp1β also interacted with von Hippel-Lindau tumor suppressor (pVHL) and HIF-1α, whereby nsp1β impaired the interaction between HIF-1α and pVHL. This work deepens our understanding of the molecular mechanisms involved in PRRSV infection and provides new insights for the development of HIF-1α-based anti-PRRSV therapies.

Dataset Information

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion.

Publications

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets