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.     

Temperature-sensitive mutants and revertants in the coronavirus nonstructural protein 5 protease (3CLpro) define residues involved in long-distance communication and regulation of protease activity

Positive-strand RNA virus genomes are translated into polyproteins that are processed by viral proteases to yield functional intermediate and mature proteins. Coronaviruses (CoVs) carry genes that encode an nsp5 protease (also known as 3CLpro or Mpro) responsible for 11 maturation cleavages. The nsp5 structure contains two chymotrypsin-like domains (D1 and D2) and a unique domain (D3), and forms functional dimers. However, little is known of interactions or communication across the structure of the protease during nsp5 activity. Using reverse genetic mutagenesis of the CoV murine hepatitis virus (MHV) nsp5, we identified a new temperature-sensitive (ts) mutation in D2 of nsp5 (Ser133Ala) and confirmed a ts residue in D3 (Phe219Leu). Both D2-tsS133A and D3-tsF219L were impaired for viral replication and nsp5-mediated polyprotein processing at the nonpermissive temperature. Passage of tsS133A and tsF219L at the nonpermissive temperature resulted in emergence of multiple second-site suppressor mutations, singly and in combinations. Among the second-site mutations, a D2 His134Tyr change suppressed the ts phenotype of D2-tsS133A and D3-tsF219L, as well as the previously reported D2-tsV148A. Analysis of multiple CoV nsp5 structures, and alignment of nonredundant nsp5 primary sequences, demonstrated that ts and suppressor residues are not conserved across CoVs and are physically distant (>10 Å) from each other, from catalytic and substrate-binding residues, and from the nsp5 dimer interface. These findings demonstrate that long-distance communication pathways between multiple residues and domains of nsp5 play a significant role in nsp5 activity and viral replication, suggesting possible novel targets for non-active site inhibitors of nsp5.     

Genetic analysis of Murine hepatitis virus nsp4 in virus replication

Coronavirus replicase polyproteins are translated from the genomic positive-strand RNA and are proteolytically processed by three viral proteases to yield 16 mature nonstructural proteins (nsp1 to nsp16). nsp4 contains four predicted transmembrane-spanning regions (TM1, -2, -3, and -4), demonstrates characteristics of an integral membrane protein, and is thought to be essential for the formation and function of viral replication complexes on cellular membranes. To determine the requirement of nsp4 for murine hepatitis virus (MHV) infection in culture, engineered deletions and mutations in TMs and intervening soluble regions were analyzed for effects on virus recovery, growth, RNA synthesis, protein expression, and intracellular membrane modifications. In-frame partial or complete deletions of nsp4; deletions of TM1, -2, and -3; and alanine substitutions of multiple conserved, clustered, charged residues in nsp4 resulted in viruses that were nonrecoverable, viruses highly impaired in growth and RNA synthesis, and viruses that were nearly wild type in replication. The results indicate that nsp4 is required for MHV replication and that while putative TM1, -2, and -3 and specific charged residues may be essential for productive virus infection, putative TM4 and the carboxy-terminal amino acids K(398) through T(492) of nsp4 are dispensable. Together, the experiments identify important residues and regions for studies of nsp4 topology, function, and interactions.     

Chimeric Exchange of Coronavirus nsp5 Proteases (3CLpro) Identifies Common and Divergent Regulatory Determinants of Protease Activity

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp''s 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp''s at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.     

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.     

Poliovirus 5'-terminal cloverleaf RNA is required in cis for VPg uridylylation and the initiation of negative-strand RNA synthesis

Chimeric poliovirus RNAs, possessing the 5' nontranslated region (NTR) of hepatitis C virus in place of the 5' NTR of poliovirus, were used to examine the role of the poliovirus 5' NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3' NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5'-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5'-terminal cloverleaf and 3' NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.     

A second nonstructural protein functions in the regulation of alphavirus negative-strand RNA synthesis.

Previous studies (D.L. Sawicki, D. B. Barkhimer, S. G. Sawicki, C. M. Rice, and S. Schlesinger, Virology 174:43-52, 1990) identified a temperature-sensitive (ts) defect in Sindbis virus nonstructural protein 4 (nsP4) that reactivated negative-strand synthesis after its normal cessation at the end of the early phase of replication. We now report identification of two different ts alterations in nsP2 of Ala-517 to Thr in ts17 or Asn-700 to Lys in ts133 that also reactivated negative-strand synthesis. These same mutations caused severely reduced protease processing by nsP2 and recognition of the internal promoter for subgenomic mRNA synthesis and were responsible for the conditional lethality and RNA negativity of these mutants. Reactivation of negative-strand synthesis by mutations in nsP2 resembled that in nsP4: it was a reversible property of stable replication complexes and did not require continuation of viral protein synthesis. Recombinant viruses expressing both mutant nsP2 and nsP4 reactivated negative-strand synthesis more efficiently than did either mutant protein alone, consistent with the hypothesis that both nsP2 and nsP4 participate in template recognition. We propose that these alterations cause nsP2 and nsP4 to switch from their normal preference to recognize negative strands as templates to recognize positive strands and thereby mimic the initial formation of a replication complex.     

5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis

A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.     

Replication of poliovirus RNA with complete internal ribosome entry site deletions

cis-acting RNA sequences and structures in the 5' and 3' nontranslated regions of poliovirus RNA interact with host translation machinery and viral replication proteins to coordinately regulate the sequential translation and replication of poliovirus RNA. The poliovirus internal ribosome entry site (IRES) in the 5' nontranslated region (NTR) has been implicated as a cis-active RNA required for both viral mRNA translation and viral RNA replication. To evaluate the role of the IRES in poliovirus RNA replication, we exploited the advantages of cell-free translation-replication reactions and preinitiation RNA replication complexes. Genetic complementation with helper mRNAs allowed us to create preinitiation RNA replication complexes containing RNA templates with defined deletions in the viral open reading frame and the IRES. A series of deletions revealed that no RNA elements of either the viral open reading frame or the IRES were required in cis for negative-strand RNA synthesis. The IRES was dispensable for both negative- and positive-strand RNA syntheses. Intriguingly, although small viral RNAs lacking the IRES replicated efficiently, the replication of genome length viral RNAs was stimulated by the presence of the IRES. These results suggest that RNA replication is not directly dependent on a template RNA first functioning as an mRNA. These results further suggest that poliovirus RNA replication is not absolutely dependent on any protein-RNA interactions involving the IRES.     

Localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication

The coronavirus nonstructural proteins (nsp's) derived from the replicase polyproteins collectively constitute the viral replication complexes, which are anchored to double-membrane vesicles. Little is known about the biogenesis of these complexes, the membrane anchoring of which is probably mediated by nsp3, nsp4, and nsp6, as they contain several putative transmembrane domains. As a first step to getting more insight into the formation of the coronavirus replication complex, the membrane topology, processing, and subcellular localization of nsp4 of the mouse hepatitis virus (MHV) and severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were elucidated in this study. Both nsp4 proteins became N glycosylated, while their amino and carboxy termini were localized to the cytoplasm. These observations imply nsp4 to assemble in the membrane as a tetraspanning transmembrane protein with a Nendo/Cendo topology. The amino terminus of SARS-CoV nsp4, but not that of MHV nsp4, was shown to be (partially) processed by signal peptidase. nsp4 localized to the endoplasmic reticulum (ER) when expressed alone but was recruited to the replication complexes in infected cells. nsp4 present in these complexes did not colocalize with markers of the ER or Golgi apparatus, while the susceptibility of its sugars to endoglycosidase H indicated that the protein had also not traveled trough the latter compartment. The important role of the early secretory pathway in formation of the replication complexes was also demonstrated by the inhibition of coronaviral replication when the ER export machinery was blocked by use of the kinase inhibitor H89 or by expression of a mutant, Sar1[H79G].     

SYNCRIP, a member of the heterogeneous nuclear ribonucleoprotein family, is involved in mouse hepatitis virus RNA synthesis

Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5' and 3' untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5'-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5'-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5'-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.     

High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants

Replication fidelity of RNA virus genomes is constrained by the opposing necessities of generating sufficient diversity for adaptation and maintaining genetic stability, but it is unclear how the largest viral RNA genomes have evolved and are maintained under these constraints. A coronavirus (CoV) nonstructural protein, nsp14, contains conserved active-site motifs of cellular exonucleases, including DNA proofreading enzymes, and the severe acute respiratory syndrome CoV (SARS-CoV) nsp14 has 3'-to-5' exoribonuclease (ExoN) activity in vitro. Here, we show that nsp14 ExoN remarkably increases replication fidelity of the CoV murine hepatitis virus (MHV). Replacement of conserved MHV ExoN active-site residues with alanines resulted in viable mutant viruses with growth and RNA synthesis defects that during passage accumulated 15-fold more mutations than wild-type virus without changes in growth fitness. The estimated mutation rate for ExoN mutants was similar to that reported for other RNA viruses, whereas that of wild-type MHV was less than the established rates for RNA viruses in general, suggesting that CoVs with intact ExoN replicate with unusually high fidelity. Our results indicate that nsp14 ExoN plays a critical role in prevention or repair of nucleotide incorporation errors during genome replication. The established mutants are unique tools to test the hypothesis that high replication fidelity is required for the evolution and stability of large RNA genomes.