The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension

Uniquely among RNA viruses, replication of the ~30-kb SARS-coronavirus genome is believed to involve two RNA-dependent RNA polymerase (RdRp) activities. The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase. Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps]. To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity. We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity. Moreover, this 'improved' recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far. The latter activity is, however, ~20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations. Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.     

Alternative proteolytic processing of the arterivirus replicase ORF1a polyprotein: evidence that NSP2 acts as a cofactor for the NSP4 serine protease.

The C-terminal half of the replicase ORF1a polyprotein of the arterivirus equine arteritis virus is processed by a chymotrypsinlike serine protease (SP) (E. J. Snijder et al., J. Biol. Chem. 271:4864-4871, 1996) located in nonstructural protein 4 (nsp4). Three probable SP cleavage sites had previously been identified in the ORF1a protein. Their proteolysis explained the main processing products generated from the C-terminal part of the ORF1a protein in infected cells (E. J. Snijder et al., J. Virol. 68:5755-5764, 1994). By using sequence comparison, ORF1a expression systems, and site-directed mutagenesis, we have now identified two additional SP cleavage sites: Glu-1430 / Gly and Glu-1452 / Ser. This means that the ORF1a protein can be cleaved into eight processing end products: nsp1 to nsp8. By microsequence analysis of the nsp5 and nsp7 N termini, we have now formally confirmed the specificity of the SP for Glu / (Gly/Ser) substrates. Importantly, our studies revealed that the C-terminal half of the ORF1a protein (nsp3-8) can be processed by the SP following two alternative pathways, which appear to be mutually exclusive. In the majority of the nsp3-8 precursors the SP cleaves the nsp4/5 site, yielding nsp3-4 and nsp5-8. Subsequently, the latter product is cleaved at the nsp7/8 site only, whereas the newly identified nsp5/6 and nsp6/7 sites appear to be inaccessible to the protease. In the alternative proteolytic cascade, which is used at a low but significant level in infected cells, it is the nsp4/5 site which remains uncleaved, while the nsp5/6 and nsp6/7 sites are processed to yield a set of previously unnoticed processing products. Coexpression studies revealed that nsp3-8 has to interact with cleaved nsp2 to allow processing of the nsp4/5 junction, the first step of the major processing pathway. When the nsp2 cofactor is absent, the nsp4/5 site cannot be processed and nsp3-8 is processed following the alternative, minor pathway.     

Structure and genetic analysis of the arterivirus nonstructural protein 7alpha

Arterivirus replicase polyproteins are cleaved into at least 13 mature nonstructural proteins (nsps), and in particular the nsp5-to-nsp8 region is subject to a complex processing cascade. The function of the largest subunit from this region, nsp7, which is further cleaved into nsp7α and nsp7β, is unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the solution structure of nsp7α of equine arteritis virus, revealing an interesting unique fold for this protein but thereby providing little clue to its possible functions. Nevertheless, structure-based reverse genetics studies established the importance of nsp7/nsp7α for viral RNA synthesis, thus providing a basis for future studies.     

Mutagenesis analysis of the nsp4 main proteinase reveals determinants of arterivirus replicase polyprotein autoprocessing

Nonstructural protein 4 (nsp4; 204 amino acids) is the chymotrypsin-like serine main proteinase of the arterivirus Equine arteritis virus (order Nidovirales), which controls the maturation of the replicase complex. nsp4 includes a unique C-terminal domain (CTD) connected to the catalytic two-beta-barrel structure by the poorly conserved residues 155 and 156. This dipeptide might be part of a hinge region (HR) that facilitates interdomain movements and thereby regulates (in time and space) autoprocessing of replicase polyproteins pp1a and pp1ab at eight sites that are conserved in arteriviruses. To test this hypothesis, we characterized nsp4 proteinase mutants carrying either point mutations in the putative HR domain or a large deletion in the CTD. When tested in a reverse genetics system, three groups of mutants were recognized (wild-type-like, debilitated, and dead), which was in line with the expected impact of mutations on HR flexibility. When tested in a transient expression system, the effects of the mutations on the production and turnover of replicase proteins varied widely. They were cleavage product specific and revealed a pronounced modulating effect of moieties derived from the nsp1-3 region of pp1a. Mutations that were lethal affected the efficiency of polyprotein autoprocessing most strongly. These mutants may be impaired in the accumulation of nsp5-7 and/or suffer from delayed or otherwise perturbed processing at the nsp5/6 and nsp6/7 junctions. On average, the production of nsp7-8 seems to be the most resistant to debilitating nsp4 mutations. Our results further prove that the CTD is essential for a vital nsp4 property other than catalysis.     

NMR Structure of the SARS-CoV Nonstructural Protein 7 in Solution at pH 6.5

The NMR structure of the severe acute respiratory syndrome coronavirus nonstructural protein (nsp) 7 in aqueous solution at pH 6.5 was determined and compared with the results of previous structure determinations of nsp7 in solution at pH 7.5 and in the crystals of a hexadecameric nsp7/nsp8 complex obtained from a solution at pH 7.5. All three structures contain four helices as the only regular secondary structures, but there are differences in the lengths and sequence locations of the four helices, as well as between the tertiary folds. The present study includes data on conformational equilibria and intramolecular rate processes in nsp7 in solution at pH 6.5, which provide further insights into the polymorphisms implicated by a comparison of the three presently available nsp7 structures.     

Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex

The 5'-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5'-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2'-O positions, catalyzed by nsp14 N7-MTase and nsp16 2'-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2'-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs.     

Characterization of the SARS-CoV-2 ExoN (nsp14ExoN–nsp10) complex: implications for its role in viral genome stability and inhibitor identification

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14–nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14–nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14–nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3′-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14–nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12–nsp7–nsp8 (nsp12–7–8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14–nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14–nsp10, including the known SARS-CoV-2 major protease (M^pro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.     

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.     

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.     

Comparative analysis of 22 coronavirus HKU1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus HKU1

We sequenced and compared the complete genomes of 22 strains of coronavirus HKU1 (CoV HKU1) obtained from nasopharyngeal aspirates of patients with respiratory tract infections over a 2-year period. Phylogenetic analysis of 24 putative proteins and polypeptides showed that the 22 CoV HKU1 strains fell into three clusters (genotype A, 13 strains; genotype B, 3 strains and genotype C, 6 strains). However, different phylogenetic relationships among the three clusters were observed in different regions of their genomes. From nsp4 to nsp6, the genotype A strains were clustered with the genotype B strains. For nsp7 and nsp8 and from nsp10 to nsp16, the genotype A strains were clustered with the genotype C strains. From hemagglutinin esterase (HE) to nucleocapsid (N), the genotype B strains were clustered closely with the genotype C strains. Bootscan analysis showed possible recombination between genotypes B and C from nucleotide positions 11,500 to 13,000, corresponding to the nsp6-nsp7 junction, giving rise to genotype A, and between genotypes A and B from nucleotide positions 21,500 to 22,500, corresponding to the nsp16-HE junction, giving rise to genotype C. Multiple alignments further narrowed the sites of crossover to a 143-bp region between nucleotide positions 11,750 and 11,892 and a 29-bp region between nucleotide positions 21,502 and 21,530. Genome analysis also revealed various numbers of tandem copies of a perfect 30-base acidic tandem repeat (ATR) which encodes NDDEDVVTGD and various numbers and sequences of imperfect repeats in the N terminus of nsp3 inside the acidic domain upstream of papain-like protease 1 among the 22 genomes. All 10 CoV HKU1 strains with incomplete imperfect repeats (1.4 and 4.4) belonged to genotype A. The present study represents the first evidence for natural recombination in coronavirus associated with human infection. Analysis of a single gene is not sufficient for the genotyping of CoV HKU1 strains but requires amplification and sequencing of at least two gene loci, one from nsp10 to nsp16 (e.g., pol or helicase) and another from HE to N (e.g., spike or N). Further studies will delineate whether the ATR is useful for the molecular typing of CoV HKU1.     

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).     

Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.     

Supermolecular structure of photosystem II and location of the PsbS protein

This paper addresses the question of whether the PsbS protein of photosystem two (PS II) is located within the LHC II PS II supercomplex for which a three-dimensional structure has been obtained by cryoelectron microscopy and single particle analysis. The PsbS protein has recently been implicated as the site for non-photochemical quenching. Based both on immunoblotting analyses and structural considerations of an improved model of the spinach LHC II PS II supercomplex, we conclude that the PsbS protein is not located within the supercomplex. Analyses of other fractions resulting from the solubilization of PS Il-enriched membranes derived from spinach suggest that the PsbS protein is located in the LHC II-rich regions that interconnect the supercomplex within the membrane.