scholarly journals A Novel Mutation in Murine Hepatitis Virus nsp5, the Viral 3C-Like Proteinase, Causes Temperature-Sensitive Defects in Viral Growth and Protein Processing

2008 ◽  
Vol 82 (12) ◽  
pp. 5999-6008 ◽  
Author(s):  
Jennifer S. Sparks ◽  
Eric F. Donaldson ◽  
Xiaotao Lu ◽  
Ralph S. Baric ◽  
Mark R. Denison
Keyword(s):  
Hepatitis Virus ◽  
Novel Mutation ◽  
Mutant Virus ◽  
Proteinase Activity ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Nonstructural Proteins ◽  
Murine Hepatitis Virus ◽  
Partial Restoration ◽  
Active Site Cavity

ABSTRACT Sequencing and reversion analysis of murine hepatitis virus (MHV) temperature-sensitive (ts) viruses has identified putative ts mutations in the replicase nonstructural proteins (nsp's) of these coronaviruses. In this study, reverse transcriptase PCR sequencing of the RNA genome of an isolate of the MHV ts virus Alb ts6, referred to as Alb/ts/nsp5/V148A, identified a putative ts mutation in nsp5 (T10651C, Val148Ala), the viral 3C-like proteinase (3CLpro). The introduction of the T10651C mutation into the infectious MHV clone resulted in the recovery of a mutant virus, the nsp5/V148A virus, that demonstrated reduced growth and nsp5 proteinase activity identical to that of Alb/ts/nsp5/V148A at the nonpermissive temperature. Sequence analysis of 40°C revertants of Alb/ts/nsp5/V148A identified primary reversion to Ala148Val in nsp5, as well as two independent second-site mutations resulting in Ser133Asn and His134Tyr substitutions in nsp5. The introduction of the Ser133Asn or His134Tyr substitution into the cloned nsp5/V148A mutant virus background resulted in the recovery of viruses with increased growth fitness and the partial restoration of nsp5 activity at the nonpermissive temperature. Modeling of the nsp5 structure of Alb/ts/nsp5/V148A predicted that the Val148Ala mutation alters residue 148 interactions with residues of the substrate binding S1 subsite of the nsp5 active-site cavity. This study identifies novel residues in nsp5 that may be important for regulating substrate specificity and nsp5 proteinase activity.

scholarly journals Replication of Murine Hepatitis Virus Is Regulated by Papain-Like Proteinase 1 Processing of Nonstructural Proteins 1, 2, and 3

2006 ◽  
Vol 80 (23) ◽  
pp. 11610-11620 ◽  
Author(s):  
Rachel L. Graham ◽  
Mark R. Denison
Keyword(s):  
Deletion Mutant ◽  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Protein Processing ◽  
Mutant Virus ◽  
Viral Titer ◽  
Wild Type ◽  
Nonstructural Proteins ◽  
Murine Hepatitis Virus ◽  
Positive Strand Rna

ABSTRACT 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 >107 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 ∼104 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 102 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.

scholarly journals Processing of Open Reading Frame 1a Replicase Proteins nsp7 to nsp10 in Murine Hepatitis Virus Strain A59 Replication

2007 ◽  
Vol 81 (19) ◽  
pp. 10280-10291 ◽  
Author(s):  
Damon J. Deming ◽  
Rachel L. Graham ◽  
Mark R. Denison ◽  
Ralph S. Baric
Keyword(s):  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Proteolytic Processing ◽  
Mutant Virus ◽  
Open Reading Frame ◽  
Cleavage Sites ◽  
Nonstructural Proteins ◽  
Murine Hepatitis Virus ◽  
Reading Frame ◽  
Genes Encoding

ABSTRACT Coronaviruses express open reading frame 1a (ORF1a) and ORF1b polyproteins from which 16 nonstructural proteins (nsp) are derived. The highly conserved region at the carboxy terminus of ORF1a is processed by the nsp5 proteinase (Mpro) into mature products, including nsp7, nsp8, nsp9, and nsp10, proteins with predicted or identified activities involved in RNA synthesis. Although continuous translation and proteolytic processing of ORF1ab by Mpro is required for replication, it is unknown whether specific cleavage events within the polyprotein are dispensable. We determined the requirement for the nsp7 to nsp10 proteins and their processing during murine hepatitis virus (MHV) replication. Through use of an MHV reverse genetics system, in-frame deletions of the coding sequences for nsp7 to nsp10, or ablation of their flanking Mpro cleavage sites, were made and the effects upon replication were determined. Viable viruses were characterized by analysis of Mpro processing, RNA transcription, and growth fitness. Deletion of any of the regions encoding nsp7 to nsp10 was lethal. Disruption of the cleavage sites was lethal with the exception of that of the nsp9-nsp10 site, which resulted in a mutant virus with attenuated replication. Passage of the attenuated nsp9-nsp10 cleavage mutant increased fitness to near-wild-type kinetics without reversion to a virus capable of processing nsp9-nsp10. We also confirmed the presence of a second cleavage site between nsp7 and nsp8. In order to determine whether a distinct function could be attributed to preprocessed forms of the polyprotein, including nsp7 to nsp10, the genes encoding nsp7 and nsp8 were rearranged. The mutant virus was not viable, suggesting that the uncleaved protein may be essential for replication or proteolytic processing.

scholarly journals A New Cistron in the Murine Hepatitis Virus Replicase Gene

2010 ◽  
Vol 84 (19) ◽  
pp. 10148-10158 ◽  
Author(s):  
Helen L. Stokes ◽  
Surendranath Baliji ◽  
Chang Guo Hui ◽  
Stanley G. Sawicki ◽  
Susan C. Baker ◽  
...  
Keyword(s):  
Gene Locus ◽  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Temperature Sensitive ◽  
Replicase Gene ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Murine Hepatitis Virus ◽  
Infected Cells

ABSTRACT We report an RNA-negative, temperature-sensitive (ts) mutant of Murine hepatitis virus, Bristol ts31 (MHV-Brts31), that defines a new complementation group within the MHV replicase gene locus. MHV-Brts31 has near-normal levels of RNA synthesis at the permissive temperature of 33°C but is unable to synthesize viral RNA when the infection is initiated and maintained at the nonpermissive temperature of 39.5°C. Sequence analysis of MHV-Brts31 RNA indicated that a single G-to-A transition at codon 1307 in open reading frame 1a, which results in a replacement of methionine-475 with isoleucine in nonstructural protein 3 (nsp3), was responsible for the ts phenotype. This conclusion was confirmed using a vaccinia virus-based reverse genetics system to produce a recombinant virus, Bristol tsc31 (MHV-Brtsc31), which has the same RNA-negative ts phenotype and complementation profile as those of MHV-Brts31. The analysis of protein synthesis in virus-infected cells showed that, at the nonpermissive temperature, MHV-Brtsc31 was not able to proteolytically process either p150, the precursor polypeptide of the replicase nonstructural proteins nsp4 to nsp10, or the replicase polyprotein pp1ab to produce nsp12. The processing of replicase polyprotein pp1a in the region of nsp1 to nsp3 was not affected. Transmission electron microscopy showed that, compared to revertant virus, the number of double-membrane vesicles in MHV-Brts31-infected cells is reduced at the nonpermissive temperature. These results identify a new cistron in the MHV replicase gene locus and show that nsp3 has an essential role in the assembly of a functional MHV replication-transcription complex.

scholarly journals Topology and Membrane Anchoring of the Coronavirus Replication Complex: Not All Hydrophobic Domains of nsp3 and nsp6 Are Membrane Spanning

2008 ◽  
Vol 82 (24) ◽  
pp. 12392-12405 ◽  
Author(s):  
Monique Oostra ◽  
Marne C. Hagemeijer ◽  
Michiel van Gent ◽  
Cornelis P. J. Bekker ◽  
Eddie G. te Lintelo ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Cultured Cells ◽  
Hepatitis Virus ◽  
Membrane Vesicles ◽  
Transmembrane Domains ◽  
Nonstructural Proteins ◽  
Murine Hepatitis Virus ◽  
Replication Complex ◽  
Membrane Spanning ◽  
Membrane Anchoring

ABSTRACT Coronaviruses express two very large replicase polyproteins, the 16 autoproteolytic cleavage products of which collectively form the membrane-anchored replication complexes. How these structures are assembled is still largely unknown, but it is likely that the membrane-spanning members of these nonstructural proteins (nsps) are responsible for the induction of the double-membrane vesicles and for anchoring the replication complexes to these membranes. For 3 of the 16 coronavirus nsps—nsp3, nsp4, and nsp6—multiple transmembrane domains are predicted. Previously we showed that, consistent with predictions, nsp4 occurs in membranes with both of its termini exposed in the cytoplasm (M. Oostra et al., J. Virol. 81:12323-12336, 2007). Strikingly, however, for both nsp3 and nsp6, predictions based on a multiple alignment of 27 coronavirus genome sequences indicate an uneven number of transmembrane domains. As a consequence, the proteinase domains present in nsp3 and nsp5 would be separated from their target sequences by the lipid bilayer. To look into this incongruity, we studied the membrane disposition of nsp3 and nsp6 of the severe acute respiratory syndrome coronavirus and murine hepatitis virus by analyzing tagged forms of the proteins expressed in cultured cells. Contrary to the predictions, in both viruses, both proteins had their amino terminus, as well as their carboxy terminus, exposed in the cytoplasm. We established that two of the three hydrophobic domains in nsp3 and six of the seven in nsp6 are membrane spanning. Subsequently, we verified that in nsp4, all four hydrophobic domains span the lipid bilayer. The occurrence of conserved non-membrane-spanning hydrophobic domains in nsp3 and nsp6 suggests an important function for these domains in coronavirus replication.

scholarly journals Murine hepatitis virus nsp4 N258T mutants are not temperature-sensitive

Virology ◽  
2013 ◽  
Vol 435 (2) ◽  
pp. 210-213 ◽  
Author(s):  
Dia C. Beachboard ◽  
Xiaotao Lu ◽  
Susan C. Baker ◽  
Mark R. Denison
Keyword(s):  
Hepatitis Virus ◽  
Temperature Sensitive ◽  
Murine Hepatitis Virus

scholarly journals The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus Are Dispensable for Viral Replication

2005 ◽  
Vol 79 (21) ◽  
pp. 13399-13411 ◽  
Author(s):  
Rachel L. Graham ◽  
Amy C. Sims ◽  
Sarah M. Brockway ◽  
Ralph S. Baric ◽  
Mark R. Denison
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Viral Replication ◽  
Cleavage Site ◽  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Nonstructural Proteins ◽  
Murine Hepatitis Virus ◽  
Retroviral Transduction ◽  
Coding Sequence ◽  
And Function

ABSTRACT 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 (MHVΔnsp2 and SARSΔnsp2, respectively). Infectious MHVΔnsp2 and SARSΔnsp2 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 Δnsp2 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 MHVΔnsp2-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 MHVΔnsp2. 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.

scholarly journals Analysis of Murine Hepatitis Virus Strain A59 Temperature-Sensitive Mutant TS-LA6 Suggests that nsp10 Plays a Critical Role in Polyprotein Processing

2007 ◽  
Vol 81 (13) ◽  
pp. 7086-7098 ◽  
Author(s):  
Eric F. Donaldson ◽  
Rachel L. Graham ◽  
Amy C. Sims ◽  
Mark R. Denison ◽  
Ralph S. Baric
Keyword(s):  
Hepatitis Virus ◽  
Infectious Clone ◽  
Critical Role ◽  
Critical Factor ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Murine Hepatitis Virus ◽  
Negative Strand ◽  
New Findings ◽  
Single Amino Acid Change

ABSTRACT Coronaviruses are the largest RNA viruses, and their genomes encode replication machinery capable of efficient replication of both positive- and negative-strand viral RNAs as well as enzymes capable of processing large viral polyproteins into putative replication intermediates and mature proteins. A model described recently by Sawicki et al. (S. G. Sawicki, D. L. Sawicki, D. Younker, Y. Meyer, V. Thiel, H. Stokes, and S. G. Siddell, PLoS Pathog. 1:e39, 2005), based upon complementation studies of known temperature-sensitive (TS) mutants of murine hepatitis virus (MHV) strain A59, proposes that an intermediate comprised of nsp4 to nsp10/11 (∼150 kDa) is involved in negative-strand synthesis. Furthermore, the mature forms of nsp4 to nsp10 are thought to serve as cofactors with other replicase proteins to assemble a larger replication complex specifically formed to transcribe positive-strand RNAs. In this study, we introduced a single-amino-acid change (nsp10:Q65E) associated with the TS-LA6 phenotype into nsp10 of the infectious clone of MHV. Growth kinetic studies demonstrated that this mutation was sufficient to generate the TS phenotype at permissive and nonpermissive temperatures. Our results demonstrate that the TS mutant variant of nsp10 inhibits the main protease, 3CLpro, blocking its function completely at the nonpermissive temperature. These results implicate nsp10 as being a critical factor in the activation of 3CLpro function. We discuss how these findings challenge the current hypothesis that nsp4 to nsp10/11 functions as a single cistron in negative-strand RNA synthesis and analyze recent complementation data in light of these new findings.

scholarly journals Mutations across Murine Hepatitis Virus nsp4 Alter Virus Fitness and Membrane Modifications

2014 ◽  
Vol 89 (4) ◽  
pp. 2080-2089 ◽  
Author(s):  
Dia C. Beachboard ◽  
Jordan M. Anderson-Daniels ◽  
Mark R. Denison
Keyword(s):  
Virus Replication ◽  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Membrane Vesicles ◽  
Nonstructural Proteins ◽  
Murine Hepatitis Virus ◽  
Double Membrane ◽  
Cytoplasmic Membranes ◽  
Positive Sense ◽  
Virus Fitness

ABSTRACTA common feature of infection by positive-sense RNA virus is the modification of host cell cytoplasmic membranes that serve as sites of viral RNA synthesis. Coronaviruses induce double-membrane vesicles (DMVs), but the role of DMVs in replication and virus fitness remains unclear. Coronaviruses encode 16 nonstructural proteins (nsps), three of which, nsp3, nsp4, and nsp6, are necessary and sufficient for DMV formation. It has been shown previously that mutations in murine hepatitis virus (MHV) nsp4 loop 1 that alter nsp4 glycosylation are associated with disrupted DMV formation and result in changes in virus replication and RNA synthesis. However, it is not known whether DMV morphology or another function of nsp4 glycosylation is responsible for effects on virus replication. In this study, we tested whether mutations across nsp4, both alone and in combination with mutations that abolish nsp4 glycosylation, affected DMV formation, replication, and fitness. Residues in nsp4 distinct from glycosylation sites, particularly in the endoplasmic reticulum (ER) luminal loop 1, independently disrupted both the number and morphology of DMVs and exacerbated DMV changes associated with loss of glycosylation. Mutations that altered DMV morphology but not glycosylation did not affect virus fitness while viruses lacking nsp4 glycosylation exhibited a loss in fitness. The results support the hypothesis that DMV morphology and numbers are not key determinants of virus fitness. The results also suggest that nsp4 glycosylation serves roles in replication in addition to the organization and stability of MHV-induced double-membrane vesicles.IMPORTANCEAll positive-sense RNA viruses modify host cytoplasmic membranes for viral replication complex formation. Thus, defining the mechanisms of virus-induced membrane modifications is essential for both understanding virus replication and development of novel approaches to virus inhibition. Coronavirus-induced membrane changes include double-membrane vesicles (DMVs) and convoluted membranes. Three viral nonstructural proteins (nsps), nsp3, nsp4, and nsp6, are known to be required for DMV formation. It is unknown how these proteins induce membrane modification or which regions of the proteins are involved in DMV formation and stability. In this study, we show that mutations across nsp4 delay virus replication and disrupt DMV formation and that loss of nsp4 glycosylation is associated with a substantial fitness cost. These results support a critical role for nsp4 in DMV formation and virus fitness.

The Viral Spike Protein Is Not Involved in the Polarized Sorting of Coronaviruses in Epithelial Cells

1998 ◽  
Vol 72 (1) ◽  
pp. 497-503 ◽  
Author(s):  
J. W. A. Rossen ◽  
R. de Beer ◽  
G.-J. Godeke ◽  
M. J. B. Raamsman ◽  
M. C. Horzinek ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Secretory Pathway ◽  
Hepatitis Virus ◽  
Spike Protein ◽  
Transmissible Gastroenteritis Virus ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Murine Hepatitis Virus ◽  
Basolateral Membranes ◽  
Transmissible Gastroenteritis

ABSTRACT Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to the apical domain or to the basolateral plasma membrane domain. In this study, we investigated the role of the coronavirus spike protein, because of its prominent position in the virion the prime sorting candidate, in the directionality of virus release. Three independent approaches were taken. (i) The inhibition of N glycosylation by tunicamycin resulted in the synthesis of spikeless virions. The absence of spikes, however, did not influence the polarity in the release of virions. Thus, murine hepatitis virus strain A59 (MHV-A59) was still secreted from the basolateral membranes of mTAL and LMR cells and from the apical sides of MDCKMHVRcells, whereas transmissible gastroenteritis virus (TGEV) was still released from the apical surfaces of LMR cells. (ii) Spikeless virions were also studied by using the MHV-A59 temperature-sensitive mutant Albany 18. When these virions were produced in infected LMR and MDCKMHVR cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. (iii) We recently demonstrated that coronavirus-like particles resembling normal virions were assembled and released when the envelope proteins M and E were coexpressed in cells (H. Vennema, G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek, D.-J. E. Opstelten, and P. J. M. Rottier, EMBO J. 15:2020–2028, 1996). The spikeless particles produced in mTAL cells by using recombinant Semliki Forest viruses to express these two genes of MHV-A59 were specifically released from basolateral membranes, i.e., with the same polarity as that of wild-type MHV-A59. Our results thus consistently demonstrate that the spike protein is not involved in the directional sorting of coronaviruses in epithelial cells. In addition, our observations with tunicamycin show that contrary to the results with some secretory proteins, the N-linked oligosaccharides present on the viral M proteins of coronaviruses such as TGEV also play no role in viral sorting. The implications of these conclusions are discussed.

Sign in / Sign up

Export Citation Format

Share Document