Identification of Nonstructural Protein 8 as the N-Terminus of the RNA-Dependent RNA Polymerase of Porcine Reproductive and Respiratory Syndrome Virus

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 ORF1 a and ORF1 b. In particular, translation of ORF1 b depends on a-1-ribosomal frameshift strategy. Thus, nonstructural protein 9 (nsp9), the first protein within ORF1 b 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 ORF1 a. 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 k Da (72–95 KDa form), which exhibited the similar mobility on the gel to the in vitro expressed nsp8–9 ORF1 b, but not the ORF1 b-coded portion (nsp9 ORF1 b). (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.     

Protein products of the open reading frames encoding nonstructural proteins of human astrovirus serotype 8

Human astroviruses have a positive-strand RNA genome, which contains three open reading frames (ORF1a, ORF1b, and ORF2). The genomic RNA is translated into two nonstructural polyproteins, nsp1a and nsp1ab, that contain sequences derived from ORF1a and from both ORF1a and ORF1b, respectively. Proteins nsp1a and nsp1ab are thought to be proteolytically processed to yield the viral proteins implicated in the replication of the virus genome; however, the intermediate and final products of this processing have been poorly characterized. To identify the cleavage products of the nonstructural polyproteins of a human astrovirus serotype 8 strain, antisera to selected recombinant proteins were produced and were used to analyze the viral proteins synthesized in astrovirus-infected Caco-2 cells and in cells transfected with recombinant plasmids expressing the ORF1a and ORF1b polyproteins. Pulse-chase experiments identified proteins of approximately 145, 88, 85, and 75 kDa as cleavage intermediates during the polyprotein processing. In addition, these experiments and kinetic analysis of the synthesis of the viral proteins identified polypeptides of 57, 20, and 19 kDa, as well as two products of around 27 kDa, as final cleavage products, with the 57-kDa polypeptide most probably being the virus RNA polymerase and the two approximately 27-kDa products being the viral protease. Based on the differential reactivities of the astrovirus proteins with the various antisera used, the individual polypeptides detected were mapped to the virus ORF1a and ORF1b regions.     

Nsp9 and Nsp10 Contribute to the Fatal Virulence of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Emerging in China

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.     

Murine coronaviruses encoding nsp2 at different genomic loci have altered replication, protein expression, and localization

Partial or complete deletion of several coronavirus nonstructural proteins (nsps), including open reading frame 1a (ORF1a)-encoded nsp2, results in viable mutant proteins with specific replication defects. It is not known whether expression of nsps from alternate locations in the genome can complement replication defects. In this report, we show that the murine hepatitis virus nsp2 sequence was tolerated in ORF1b with an in-frame insertion between nsp13 and nsp14 and in place of ORF4. Alternate encoding or duplication of the nsp2 gene sequence resulted in differences in nsp2 expression, processing, and localization, was neutral or detrimental to replication, and did not complement an ORF1a Δnsp2 replication defect. The results suggest that wild-type genomic organization and expression of nsps are required for optimal replication.     

Coronavirus Nsp10, a Critical Co-factor for Activation of Multiple Replicative Enzymes

The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1–16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3′-5′ exoribonuclease and 2′-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2′-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses.     

The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication

The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVDeltansp2 and SARSDeltansp2, respectively). Infectious MHVDeltansp2 and SARSDeltansp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Deltansp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVDeltansp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVDeltansp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function.     

The nsp9 replicase protein of SARS-coronavirus, structure and functional insights

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single beta-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).     

与PRRSV nsp11互作的宿主细胞蛋白鉴定及生物信息学分析

【目的】研究猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)nsp11与宿主细胞蛋白之间的相互作用,对于揭示nsp11在病毒复制过程中发挥的功能至关重要。【方法】在病毒感染细胞的基础上,利用nsp11的单克隆抗体,采用免疫沉淀结合串联质谱的方法,筛选与PRRSV nsp11相互作用的宿主细胞蛋白,并对所筛选出的宿主细胞蛋白进行了GO注释、COG注释和KEGG代谢通路注释;选取筛选出的宿主细胞蛋白IRAK1,利用免疫共沉淀技术和激光共聚焦技术鉴定其与nsp11之间的相互作用。【结果】与空白对照组相比,病毒感染组中出现3条差异带;经质谱分析共筛选得到了201个与nsp11相互作用的宿主细胞蛋白,分别与蛋白质代谢、细胞信号通路转导以及病原致病性等密切相关;在生物信息学分析的基础上,实验验证了nsp11确与宿主细胞蛋白IRAK1进行相互作用。【结论】鉴定出与PRRSV nsp11相互作用的宿主细胞蛋白,生物信息学分析显示它们在病毒的复制和致病过程中发挥重要作用。研究结果为探究nsp11的生物学功能指明了方向,也为研究宿主细胞蛋白与病毒蛋白间的相互作用及其调控病毒复制和致病性的分子机制奠定了基础。     

Replication of murine hepatitis virus is regulated by papain-like proteinase 1 processing of nonstructural proteins 1, 2, and 3

Coronaviruses are positive-strand RNA viruses that translate their genome RNA into polyproteins that are co- and posttranslationally processed into intermediate and mature replicase nonstructural proteins (nsps). In murine hepatitis virus (MHV), nsps 1, 2, and 3 are processed by two papain-like proteinase activities within nsp3 (PLP1 and PLP2) to yield nsp1, an nsp2-3 intermediate, and mature nsp2 and nsp3. To determine the role in replication of processing between nsp2 and nsp3 at cleavage site 2 (CS2) and PLP1 proteinase activity, mutations were engineered into the MHV genome at CS2, at CS1 and CS2, and at the PLP1 catalytic site, alone and in combination. Mutant viruses with abolished cleavage at CS2 were delayed in growth and RNA synthesis but grew to wild-type titers of >10(7) PFU/ml. Mutant viruses with deletion of both CS1 and CS2 exhibited both a delay in growth and a decrease in peak viral titer to approximately 10(4) PFU/ml. Inactivation of PLP1 catalytic residues resulted in a mutant virus that did not process at either CS1 or CS2 and was severely debilitated in growth, achieving only 10(2) PFU/ml. However, when both CS1 and CS2 were deleted in the presence of inactivated PLP1, the growth of the resulting mutant virus was partially compensated, comparable to that of the CS1 and CS2 deletion mutant. These results demonstrate that interactions of PLP1 with CS1 and CS2 are critical for protein processing and suggest that the interactions play specific roles in regulation of the functions of nsp1, 2, and 3 in viral RNA synthesis.     

An RNA stem-loop within the bovine coronavirus nsp1 coding region is a cis-acting element in defective interfering RNA replication

Higher-order cis-acting RNA replication structures have been identified in the 3'- and 5'-terminal untranslated regions (UTRs) of a bovine coronavirus (BCoV) defective interfering (DI) RNA. The UTRs are identical to those in the viral genome, since the 2.2-kb DI RNA is composed of only the two ends of the genome fused between an internal site within the 738-nucleotide (nt) 5'-most coding region (the nsp1, or p28, coding region) and a site just 4 nt upstream of the 3'-most open reading frame (ORF) (the N gene). The joined ends of the viral genome in the DI RNA create a single continuous 1,635-nt ORF, 288 nt of which come from the 738-nt nsp1 coding region. Here, we have analyzed features of the 5'-terminal 288-nt portion of the nsp1 coding region within the continuous ORF that are required for DI RNA replication. We observed that (i) the 5'-terminal 186 nt of the nsp1 coding region are necessary and sufficient for DI RNA replication, (ii) two Mfold-predicted stem-loops within the 186-nt sequence, named SLV (nt 239 to 310) and SLVI (nt 311 to 340), are supported by RNase structure probing and by nucleotide covariation among closely related group 2 coronaviruses, and (iii) SLVI is a required higher-order structure for DI RNA replication based on mutation analyses. The function of SLV has not been evaluated. We conclude that SLVI within the BCoV nsp1 coding region is a higher-order cis-replication element for DI RNA and postulate that it functions similarly in the viral genome.     

Structure of arterivirus nsp4. The smallest chymotrypsin-like proteinase with an alpha/beta C-terminal extension and alternate conformations of the oxyanion hole

Arteriviruses are enveloped, positive-stranded RNA viruses and include pathogens of major economic concern to the swine- and horse-breeding industries. The arterivirus replicase gene encodes two large precursor polyproteins that are processed by the viral main proteinase nonstructural protein 4 (nsp4). The three-dimensional structure of the 21-kDa nsp4 from the arterivirus prototype equine arteritis virus has been determined to 2.0 A resolution. Nsp4 adopts the smallest known chymotrypsin-like fold with a canonical catalytic triad of Ser-120, His-39, and Asp-65, as well as a novel alpha/beta C-terminal extension domain that may play a role in mediating protein-protein interactions. In different copies of nsp4 in the asymmetric unit, the oxyanion hole adopts either a collapsed inactive conformation or the standard active conformation, which may be a novel way of regulating proteolytic activity.     

Crystal structure of SARS-CoV-2 nsp10 bound to nsp14-ExoN domain reveals an exoribonuclease with both structural and functional integrity

The emergence of SARS-CoV-2 infection has posed unprecedented threat to global public health. The virus-encoded non-structural protein 14 (nsp14) is a bi-functional enzyme consisting of an exoribonuclease (ExoN) domain and a methyltransferase (MTase) domain and plays a pivotal role in viral replication. Here, we report the structure of SARS-CoV-2 nsp14-ExoN domain bound to its co-factor nsp10 and show that, compared to the SARS-CoV nsp10/nsp14-full-length complex, SARS-CoV-2 nsp14-ExoN retains an integral exoribonuclease fold and preserves an active configuration in the catalytic center. Analysis of the nsp10/nsp14-ExoN interface reveals a footprint in nsp10 extensively overlapping with that observed in the nsp10/nsp16 structure. A marked difference in the co-factor when engaging nsp14 and nsp16 lies in helix-α1′, which is further experimentally ascertained to be involved in nsp14-binding but not in nsp16-engagement. Finally, we also show that nsp10/nsp14-ExoN is enzymatically active despite the absence of nsp14-MTase domain. These data demonstrate that SARS-CoV-2 nsp10/nsp14-ExoN functions as an exoribonuclease with both structural and functional integrity.