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The crystal structure of porcine reproductive and respiratory syndrome virus nonstructural protein Nsp1beta reveals a novel metal-dependent nuclease.

ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family of Nidovirales, is the causative agent of porcine reproductive and respiratory syndrome, which results in enormous economic losses in the swine industry. As the second protein encoded by the PRRSV genome, nsp1beta cleaves itself from the downstream nsp2 protein via a C-terminal papain-like cysteine protease (PCP) domain. Although nsp1beta is known to be involved in virulence, its precise role in the process of viral infection remains unclear. In this work, we describe the homodimeric crystal structure of PRRSV nsp1beta in its natural, self-processed form. We show that the architecture of its N-terminal domain (NTD) adopts a fold closely resembling that of several known nucleases and has intrinsic nuclease activity that is strongly activated by manganese ions in vitro. Key features, however, distinguish nsp1beta from characterized nucleases, including the C-terminal PCP domain (which is responsible for the self-release of nsp1beta from nsp2), a linker domain (LKD) that connects the NTD and the PCP domain, and a C-terminal extension (CTE) that binds to and is stabilized by the putative substrate binding site of the PCPbeta domain. Combined with the reported nuclear localization of this protein, these results shed light on the self-processing mode and precise biological function of nsp1beta and thus offer a multitarget template for future drug discovery.

SUBMITTER: Xue F

PROVIDER: S-EPMC2903276 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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The crystal structure of porcine reproductive and respiratory syndrome virus nonstructural protein Nsp1beta reveals a novel metal-dependent nuclease.

Xue Fei F Sun Yuna Y Yan Liming L Zhao Cong C Chen Ji J Bartlam Mark M Li Xuemei X Lou Zhiyong Z Rao Zihe Z

Journal of virology 20100421 13

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family of Nidovirales, is the causative agent of porcine reproductive and respiratory syndrome, which results in enormous economic losses in the swine industry. As the second protein encoded by the PRRSV genome, nsp1beta cleaves itself from the downstream nsp2 protein via a C-terminal papain-like cysteine protease (PCP) domain. Although nsp1beta is known to be involved in virulence, its precise role in the ...[more]

PMID: 20410261

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Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus belonging to the family Arteriviridae Synthesis of the viral RNA is directed by replication/transcription complexes (RTC) that are mainly composed of a network of PRRSV nonstructural proteins (nsps) and likely cellular proteins. Here, we mapped the interaction network among PRRSV nsps by using yeast two-hybrid screening in conjunction with coimmunoprecipitation (co-IP) and cotransfection assays. We identified a total of 24 novel interactions and found that the interactions were centered on open reading frame 1b (ORF1b)-encoded nsps that were mainly connected by the transmembrane proteins nsp2, nsp3, and nsp5. Interestingly, the interactions of the core enzymes nsp9 and nsp10 with transmembrane proteins did not occur in a straightforward manner, as they worked in the co-IP assay but were poorly capable of finding each other within intact mammalian cells. Further proof that they can interact within cells required the engineering of N-terminal truncations of both nsp9 and nsp10. However, despite the poor colocalization relationship in cotransfected cells, both nsp9 and nsp10 came together with membrane proteins (e.g., nsp2) at the viral replication and transcription complexes (RTC) in PRRSV-infected cells. Thus, our results indicate the existence of a complex interaction network among PRRSV nsps and raise the possibility that the recruitment of key replicase proteins to membrane-associated nsps may involve some regulatory mechanisms during infection.IMPORTANCE Synthesis of PRRSV RNAs within host cells depends on the efficient and correct assembly of RTC that takes places on modified intracellular membranes. As an important step toward dissecting this poorly understood event, we investigated the interaction network among PRRSV nsps. Our studies established a comprehensive interaction map for PRRSV nsps and revealed important players within the network. The results also highlight the likely existence of a regulated recruitment of the PRRSV core enzymes nsp9 and nsp10 to viral membrane nsps during PRRSV RTC assembly.

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: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:Infection by most viruses triggers apoptosis in host cells, and viruses manipulate this cell response to promote viral replication, virus spread, and cell killing. Porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to induce apoptosis both in vitro and in vivo, while the regulatory roles of PRRSV-encoded products in apoptosis are not fully understood. In the present study, we first showed a biphasic apoptosis regulation by a highly pathogenic PRRSV strain JXwn06. It was indicated that PRRSV infection delays apoptosis at early infection but activates apoptosis at late infection in MARC-145 cells. In PRRSV-infected MARC-145 cells, procaspase-8, -9 and -12 were activated at late infection, demonstrating the involvements of death receptor pathway, mitochondrial pathway and endoplasmic reticulum (ER) stress pathway in inducing apoptosis. PRRSV was also shown to induce a similar apoptosis process in pulmonary alveolar macrophages (PAMs) with an early initiation. Next, the PRRSV-encoded apoptosis inducers were screened, indicating that the nonstructural protein (Nsp) 4 and Nsp10 of PRRSV are pro-apoptotic. In the presence of Nsp4, it was confirmed that procaspase-8, -9 and -12 were cleaved, and Nsp4 facilitates the cleavage of procaspase-9 by activating B-cell lymphoma 2 interacting mediator of cell death (Bim), a pro-apoptotic protein. In addition, Nsp4 was shown to induce the degradation of an anti-apoptotic protein, B-cell lymphoma-extra large (Bcl-xL). Nsp10 was shown to activate procaspase-8 and -9 but procaspase-12 and to upregulate the expression of BH3-only pro-apoptotic protein BH3 interacting-domain death agonist (Bid) and its active form, truncated Bid (tBid). Clearly, the participation of both activated caspase-8 and Bid is required for Nsp10-induced apoptosis, indicating a crosstalk between extrinsic- and mitochondria-dependent pathways. Together, our findings suggest that PRRSV infection regulates apoptosis in a two-phase manner and activates all three apoptotic pathways; the Nsp4 and Nsp10 of PRRSV function as apoptosis inducers with different molecular basis.

Dataset Information

The crystal structure of porcine reproductive and respiratory syndrome virus nonstructural protein Nsp1beta reveals a novel metal-dependent nuclease.

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The crystal structure of porcine reproductive and respiratory syndrome virus nonstructural protein Nsp1beta reveals a novel metal-dependent nuclease.

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