The Network of Interactions Among Porcine Reproductive and Respiratory Syndrome Virus Non-structural Proteins.
ABSTRACT: The RNA synthesis of porcine reproductive and respiratory syndrome virus (PRRSV), a positive-strand RNA virus, is compartmentalized in virus-induced double-membrane vesicles where viral proteins and some cellular proteins assemble into replication and transcription complexes (RTCs). The viral replicase proteins are the major components of the RTCs but the physical associations among these non-structural proteins (nsps) remain elusive. In this study, we investigated the potential interactions between PRRSV nsps by yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and pull-down assays. Our analyses revealed a complex network of interactions involving most of PRRSV nsps. Among them, nsp9 and nsp12 were identified as the hubs of the nsp interactome; transmembrane proteins nsp2 and nsp5 both interacted with nsp3, indicating that the three membrane-bound proteins might bind together to form the scaffold to support the association of RTCs with the intracellular membrane. The PRRSV nsp interactions identified in this study may provide valuable clues for future researches on the RTC formation and function.
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:Our knowledge about the structure and function of the nonstructural proteins (nsps) encoded by the arterivirus replicase gene has advanced in recent years. The continued characterization of the nsps of the arterivirus prototype equine arteritis virus has not only corroborated several important functional predictions, but also revealed various novel features of arteriviral replication. For porcine reproductive and respiratory syndrome virus (PRRSV), based on bioinformatics predictions and experimental studies, a processing map for the pp1a and pp1ab replicase polyproteins has been developed. Crystal structures have been resolved for two of the PRRSV nonstructural proteins that possess proteinase activity (nsp1? and nsp4). The functional characterization of the key enzymes for arterivirus RNA synthesis, the nsp9 RNA polymerase and nsp10 helicase, has been initiated. In addition, progress has been made on nsp functions relating to the regulation of subgenomic mRNAs synthesis (nsp1), the induction of replication-associated membrane rearrangements (nsp2 and nsp3), and an intriguing replicative endoribonuclease (nsp11) for which the natural substrate remains to be identified. The role of nsps in viral pathogenesis and host immunity is also being explored, and specific nsps (including nsp1?/?, nsp2, nsp4, nsp7, and nsp11) have been implicated in the modulation of host immune responses to PRRSV infection. The nsp3-8 region was identified as containing major virulence factors, although mechanistic information is scarce. The biological significance of PRRSV nsps in virus-host interactions and the technical advancements in engineering the PRRSV genome by reverse genetics are also reflected in recent developments in the area of vaccines and diagnostic assays.
Project description:Atypical porcine reproductive and respiratory syndrome (PRRS), which is caused by the Chinese highly pathogenic PRRS virus (HP-PRRSV), has resulted in large economic loss to the swine industry since its outbreak in 2006. However, to date, the region(s) within the viral genome that are related to the fatal virulence of HP-PRRSV remain unknown. In the present study, we generated a series of full-length infectious cDNA clones with swapped coding regions between the highly pathogenic RvJXwn and low pathogenic RvHB-1/3.9. Next, the in vitro and in vivo replication and pathogenicity for piglets of the rescued chimeric viruses were systematically analyzed and compared with their backbone viruses. First, we swapped the regions including the 5'UTR+ORF1a, ORF1b, and structural proteins (SPs)-coding region between the two viruses and demonstrated that the nonstructural protein-coding region, ORF1b, is directly related to the fatal virulence and increased replication efficiency of HP-PRRSV both in vitro and in vivo. Furthermore, we substituted the nonstructural protein (Nsp) 9-, Nsp10-, Nsp11- and Nsp12-coding regions separately; or Nsp9- and Nsp10-coding regions together; or Nsp9-, Nsp10- and Nsp11-coding regions simultaneously between the two viruses. Our results indicated that the HP-PRRSV Nsp9- and Nsp10-coding regions together are closely related to the replication efficiency in vitro and in vivo and are related to the increased pathogenicity and fatal virulence for piglets. Our findings suggest that Nsp9 and Nsp10 together contribute to the fatal virulence of HP-PRRSV emerging in China, helping to elucidate the pathogenesis of this virus.
Project description:The nonstructural protein 9 (Nsp9) of porcine reproductive and respiratory syndrome virus (PRRSV) is a RNA-dependent RNA polymerase (RdRp) that plays a vital role in viral replication. This study first demonstrated that the Nsp9 of genotype 2 PRRSV interacted with cellular retinoblastoma protein (pRb), and Nsp9 co-localized with pRb in the cytoplasm of PRRSV-infected MARC-145 cells and pulmonary alveolar macrophages (PAMs). Next, the overexpression of truncated pRb was shown to inhibit the PRRSV replication and silencing the pRb gene could facilitate the PRRSV replication in MARC-145 cells. Finally, the pRb level was confirmed to be down-regulated in PRRSV-infected MARC-145 cells, and Nsp9 was shown to promote the pRb degradation by proteasome pathway. These findings indicate that the interaction of Nsp 9 with pRb benefits the replication of genotype 2 PRRSV in vitro, helping to understand the roles of Nsp9 in the replication and pathogenesis of PRRSV.
Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) is a member within the family Arteriviridae of the order Nidovirales. Replication of this positive-stranded RNA virus within the host cell involves expression of viral replicase proteins encoded by two ORFs, namely ORF1a and ORF1b. In particular, translation of ORF1b depends on a -1-ribosomal frameshift strategy. Thus, nonstructural protein 9 (nsp9), the first protein within ORF1b that specifies the function of the viral RNA-dependent RNA polymerase, is expressed as the C-terminal extension of nsp8, a small nsp that is encoded by ORF1a. However, it has remained unclear whether the mature form of nsp9 in virus-infected cells still retains nsp8, addressing which is clearly critical to understand the biological function of nsp9. By taking advantage of specific antibodies to both nsp8 and nsp9, we report the following findings. (1) In infected cells, PRRSV nsp9 was identified as a major product with a size between 72 and 95 kDa (72-95 KDa form), which exhibited the similar mobility on the gel to the in vitro expressed nsp8-9ORF1b, but not the ORF1b-coded portion (nsp9ORF1b). (2) The antibodies to nsp8, but not to nsp7 or nsp10, could detect a major product that had the similar mobility to the 72-95 KDa form of nsp9. Moreover, nsp9 could be co-immunoprecipitated by antibodies to nsp8, and vice versa. (3) Neither nsp4 nor nsp2 PLP2 was able to cleave nsp8-nsp9 in vitro. Together, our studies provide experimental evidence to suggest that nsp8 is an N-terminal extension of nsp9. Our findings here paves way for further charactering the biological function of PRRSV nsp9.
Project description:Equine arteritis virus (EAV) replicase consists of two polyproteins (pp1a and pp1ab) that are encoded by open reading frames (ORFs) 1a and 1b of the viral genome. These two replicase polyproteins are posttranslationally processed by three ORF 1a-encoded proteinases to yield at least 13 nonstructural proteins (nsp1 to nsp12, including nsp7? and 7?). These nsps are expressed in EAV-infected cells, but the equine immune response they induce has not been studied. Therefore, the primary purpose of this study was to evaluate the humoral immune response of horses to each of the nsps following EAV infection. Individual nsp coding regions were cloned and expressed in both mammalian and bacterial expression systems. Each recombinant protein was used in an immunoprecipitation assay with equine serum samples from horses (n = 3) that were experimentally infected with three different EAV strains (VB, KY77, and KY84), from stallions (n = 4) that were persistently infected with EAV, and from horses (n = 4) that were vaccinated with the modified live-virus (MLV) vaccine strain. Subsequently, protein-antibody complexes were subjected to Western immunoblotting analysis with individual nsp-specific rabbit antisera, mouse anti-His antibody, or anti-FLAG tag antibody. Nsp2, nsp4, nsp5, and nsp12 were immunoprecipitated by most of the sera from experimentally or persistently infected horses, while sera from vaccinated horses did not react with nsp5 and reacted weakly with nsp4. However, serum samples from vaccinated horses were able to immunoprecipitate nsp2 and nsp12 proteins consistently. Information from this study will assist ongoing efforts to develop improved methods for the serologic diagnosis of EAV infection in horses.
Project description:Coronavirus (CoV) replication and transcription are carried out in close proximity to restructured endoplasmic reticulum (ER) membranes in replication/transcription complexes (RTC). Many of the CoV nonstructural proteins (nsps) are required for RTC function; however, not all of their functions are known. nsp15 contains an endoribonuclease domain that is conserved in the CoV family. While the enzymatic activity and crystal structure of nsp15 are well defined, its role in replication remains elusive. nsp15 localizes to sites of RNA replication, but whether it acts independently or requires additional interactions for its function remains unknown. To begin to address these questions, we created an in situ tagged form of nsp15 using the prototypic CoV, mouse hepatitis virus (MHV). In MHV, nsp15 contains the genomic RNA packaging signal (P/S), a 95-bp RNA stem-loop structure that is not required for viral replication or nsp15 function. Utilizing this knowledge, we constructed an internal hemagglutinin (HA) tag that replaced the P/S. We found that nsp15-HA was localized to discrete perinuclear puncta and strongly colocalized with nsp8 and nsp12, both well-defined members of the RTC, but not the membrane (M) protein, involved in virus assembly. Finally, we found that nsp15 interacted with RTC-associated proteins nsp8 and nsp12 during infection, and this interaction was RNA independent. From this, we conclude that nsp15 localizes and interacts with CoV proteins in the RTC, suggesting it plays a direct or indirect role in virus replication. Furthermore, the use of in situ epitope tags could be used to determine novel nsp-nsp interactions in coronaviruses. IMPORTANCE:Despite structural and biochemical data demonstrating that the coronavirus nsp15 protein contains an endoribonuclease domain, its precise function during coronavirus infection remains unknown. In this work, we created a novel in situ tagged form of nsp15 to study interactions and localization during infection. This in situ tag was tolerated by MHV and did not affect viral replication. Utilizing this tag, we established that nsp15 localized to sites of replication but not sites of assembly throughout infection. Furthermore, we found that nsp15 interacted with the putative viral primase nsp8 and polymerase nsp12 during CoV infection. The strong association of nsp15 with replication complexes and interactions with replicative CoV enzymes suggest nsp15 is involved in CoV replication. These data and tools developed in this study help elucidate the function of nsp15 during infection and may be used to uncover other novel viral protein interactions.
Project description:BACKGROUND:Autophagy is an essential process in eukaryotic cells in which autophagosomes form to deliver cellular organelles and long-lived proteins to lysosomes for degradation. Many studies have recently identified the regulatory mechanisms involved in the interaction between viral infection and autophagy. METHODS:LC3 turnover and the proteins in the endoplasmic reticulum (ER) stress pathway were investigated using western blot analysis. The formation and degradation of autophagosomes were detected using immunofluorescence staining. RESULTS:Autophagy was activated by porcine reproductive and respiratory syndrome virus (PRRSV) NSP3, NSP5 and NSP9, which are two transmembrane proteins and an RNA-dependent RNA polymerase, respectively. The formation of autophagosomes was induced by NSP3 and NSP5 and developed from the ER; the fusion of these autophagosomes with lysosomes was limited. Although NSP3 and NSP5 are ER transmembrane proteins, these proteins did not activate the ER stress signaling pathways. In addition, the cytoplasmic domain of NSP3 plays a pivotal role in activating autophagy. CONCLUSIONS:The data presented in this study reveal an important relationship between PRRSV NSPs and autophagy and provide new insights that improve our understanding of the involvement of PRRSV NSPs in the autophagy process.
Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) has had a devastating impact on the pig industry in China, and monitoring its genetic diversity is important for epidemiological surveillance and understanding its evolution. Here, we determine the complete genome sequences of two PRRSV strains, GXYL1403 and GXNN1839. Comparative, phylogenetic, and recombination detection program analyses show that the two isolates are recombinant strains with large-fragment amino acid deletions in nsp2. GXYL1403 possesses a unique deletion region of 124 amino acids in nsp2, and GXNN1839 contains a deletion of 131 amino acids in nsp2 as compared with VR2332. Further analysis of the full-length sequence suggests that GXYL1403 is a natural recombinant between sublineages 8.1 (CH-1a like) and 8.3 (JXA1-like). The recombination site of GXYL1403 is located in nsp9-nsp12 (8961nt-11181nt). GXNN1839 is a natural recombinant between the lineage 5 (VR-2332-like) and lineage 1 (NADC30-like) strains. The recombination events occurred in nsp9 (7872nt-8162nt) and in ORF2 (12587nt-13282nt) in the genome of GXNN1839. These results provide new evidence that PRRSV strains circulating in the environment have undergone recombination among the different lineages or sublineages of field strains, and these add to our understanding of RNA combination events that occur in PRRSV.
Project description:As one of many nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV), nonstructural protein 12 (Nsp12) has received relatively little attention, and its role in virus replication, if any, is essentially unknown. By the application of reverse genetic manipulation of an infectious PRRSV clone, the current study is the first to demonstrate that Nsp12 is a key component of PRRSV replication. In addition, the biochemical properties of Nsp12 were evaluated, revealing that Nsp12 forms dimers when exposed to oxidative conditions. Furthermore, we systemically analyzed the function of Nsp12 in PRRSV RNA synthesis using a strand-specific PCR method. To our surprise, Nsp12 was not found to be involved in minus-strand genomic RNA (-gRNA) synthesis; importantly, our results indicate that Nsp12 is involved in the synthesis of both plus- and minus-strand subgenomic mRNAs (+sgmRNA and -sgmRNA). Finally, we found that the combination of cysteine 35 and cysteine 79 in Nsp12 is required for sgmRNA synthesis. To our knowledge, we are the first to report the biological role of Nsp12 in the PRRSV lifecycle, and we conclude that Nsp12 is involved in the synthesis of both?+?sgRNA and -sgRNA.