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.

Further characterization and determination of the single amino acid change in thetsI138reovirus thermosensitive mutant

2012 ◽  
Vol 58 (5) ◽  
pp. 589-595
Author(s):  
Guy Lemay ◽  
Martin Bisaillon
Keyword(s):  
Amino Acid ◽  
Amino Acid Change ◽  
Genetic Alterations ◽  
Biological Properties ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Gene Encoding ◽  
Viral Particles ◽  
Single Amino Acid Change

Many temperature-sensitive mutants have been isolated in early studies of mammalian reovirus. However, the biological properties and nature of the genetic alterations remain incompletely explored for most of these mutants. The mutation harbored by the tsI138 mutant was already assigned to the L3 gene encoding the λ1 protein. In the present study, this mutant was further studied as a possible tool to establish the role of the putative λ1 enzymatic activities in viral multiplication. It was observed that synthesis of viral proteins is only marginally reduced, while it was difficult to recover viral particles at the nonpermissive temperature. A single nucleotide substitution resulting in an amino acid change was found; the position of this amino acid is consistent with a probable defect in assembly of the inner capsid at the nonpermissive temperature.

scholarly journals Temperature-sensitive recombinant subtilisin protease variants that efficiently degrade molecular biology enzymes

2020 ◽  
Vol 367 (19) ◽  
Author(s):  
Vanessa C Thompson ◽  
Bailey E McGuire ◽  
Mia S Frier ◽  
Max S G Legg ◽  
Tyler W Dyer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Biology ◽  
Temperature Stability ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Nickel Affinity Chromatography ◽  
Fold Reduction ◽  
Single Amino Acid Change ◽  
Secreted Proteases ◽  
Encoding Gene

ABSTRACT We used error-prone PCR to generate mutations in a subtilisin protease-encoding gene, and screened for recombinants that expressed temperature-sensitive (TS) variants. From the dozens of mutations that we detected in the recombinant genes we found that those mutations that affected aspartate-75 had the most profound effect on temperature stability. We thus focused our analysis on two variants of subtilisin C, the more heat-sensitive variant 24 (V24), with amino acid changes D75G, L234M and Q274P; and variant 25 (V25), with a single amino acid change, D75A. For V24 a two log-fold reduction in activity occurs in under 10 min at 50°C. For V25, a two log-fold reduction occurs at 60°C, a temperature that reduces the activity of the wild type enzyme by about 30%. The V24 variant fully inactivates enzymes commonly used in molecular biology research and in molecular diagnostics, and is stabilized against autolysis with propylene glycol concentrations of 10% or greater. The subtilisin variants are produced by a strain of Bacillus subtilis that lacks expression of its native secreted proteases, and the variants can be isolated from the supernatants using nickel affinity chromatography.

scholarly journals Expression of Hemagglutinin Esterase Protein from Recombinant Mouse Hepatitis Virus Enhances Neurovirulence

2005 ◽  
Vol 79 (24) ◽  
pp. 15064-15073 ◽  
Author(s):  
Lubna Kazi ◽  
Arjen Lissenberg ◽  
Richard Watson ◽  
Raoul J. de Groot ◽  
Susan R. Weiss
Keyword(s):  
Receptor Binding ◽  
Demyelinating Disease ◽  
Hepatitis Virus ◽  
Critical Role ◽  
Spike Glycoprotein ◽  
Murine Hepatitis Virus ◽  
Recombinant Viruses ◽  
Viral Spread ◽  
The Central Nervous System

ABSTRACT Murine hepatitis virus (MHV) infection provides a model system for the study of hepatitis, acute encephalitis, and chronic demyelinating disease. The spike glycoprotein, S, which mediates receptor binding and membrane fusion, plays a critical role in MHV pathogenesis. However, viral proteins other than S also contribute to pathogenicity. The JHM strain of MHV is highly neurovirulent and expresses a second spike glycoprotein, the hemagglutinin esterase (HE), which is not produced by MHV-A59, a hepatotropic but only mildly neurovirulent strain. To investigate a possible role for HE in MHV-induced neurovirulence, isogenic recombinant MHV-A59 viruses were generated that produced either (i) the wild-type protein, (ii) an enzymatically inactive HE protein, or (iii) no HE at all (A. Lissenberg, M. M. Vrolijk, A. L. W. van Vliet, M. A. Langereis, J. D. F. de Groot-Mijnes, P. J. M. Rottier, and R. J. de Groot, J. Virol. 79:15054-15063, 2005 [accompanying paper]). A second, mirror set of recombinant viruses was constructed in which, in addition, the MHV-A59 S gene had been replaced with that from MHV-JHM. The expression of HE in combination with A59 S did not affect the tropism, pathogenicity, or spread of the virus in vivo. However, in combination with JHM S, the expression of HE, regardless of whether it retained esterase activity or not, resulted in increased viral spread within the central nervous system and in increased neurovirulence. Our findings suggest that the properties of S receptor utilization and/or fusogenicity mainly determine organ and host cell tropism but that HE enhances the efficiency of infection and promotes viral dissemination, at least in some tissues, presumably by serving as a second receptor-binding protein.

scholarly journals A Single Amino Acid Change in nsP1 Attenuates Neurovirulence of the Sindbis-Group Alphavirus S.A.AR86

2000 ◽  
Vol 74 (9) ◽  
pp. 4207-4213 ◽  
Author(s):  
Mark T. Heise ◽  
Dennis A. Simpson ◽  
Robert E. Johnston
Keyword(s):  
Weight Loss ◽  
Hind Limb ◽  
Nonstructural Protein ◽  
Infectious Clone ◽  
Severe Disease ◽  
Clinical Signs ◽  
Single Amino Acid ◽  
Wild Type ◽  
Single Amino Acid Change ◽  
The Brain

ABSTRACT S.A.AR86, a member of the Sindbis group of alphaviruses, is neurovirulent in adult mice and has a unique threonine at position 538 of nsP1; nonneurovirulent members of this group of alphaviruses encode isoleucine. Isoleucine was introduced at position 538 in the wild-type S.A.AR86 infectious clone, ps55, and virus derived from this mutant clone, ps51, was significantly attenuated for neurovirulence compared to that derived from ps55. Intracranial (i.c.) s55 infection resulted in severe disease, including hind limb paresis, conjunctivitis, weight loss, and death in 89% of animals. In contrast, s51 caused fewer clinical signs and no mortality. Nevertheless, comparison of the virus derived from the mutant (ps51) and wild-type (ps55) S.A.AR86 molecular clones demonstrated that s51 grew as well as or better than the wild-type s55 virus in tissue culture and that viral titers in the brain following i.c. infection with s51 were equivalent to those of wild-type s55 virus. Analysis of viral replication within the brain by in situ hybridization revealed that both viruses established infection in similar regions of the brain at early times postinfection (12 to 72 h). However, at late times postinfection, the wild-type s55 virus had spread throughout large areas of the brain, while the s51 mutant exhibited a restricted pattern of replication. This suggests that s51 is either defective in spreading throughout the brain at late times postinfection or is cleared more rapidly than s55. Further evidence for the contribution of nsP1 Thr 538 to S.A.AR86 neurovirulence was provided by experiments in which a threonine residue was introduced at nsP1 position 538 of Sindbis virus strain TR339, which is nonneurovirulent in weanling mice. The resulting virus, 39ns1, demonstrated significantly increased neurovirulence and morbidity, including weight loss and hind limb paresis. These results demonstrate a role for alphavirus nonstructural protein genes in adult mouse neurovirulence.

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 Murine Hepatitis Virus Nonstructural Protein 4 Regulates Virus-Induced Membrane Modifications and Replication Complex Function

2009 ◽  
Vol 84 (1) ◽  
pp. 280-290 ◽  
Author(s):  
Mark J. Gadlage ◽  
Jennifer S. Sparks ◽  
Dia C. Beachboard ◽  
Reagan G. Cox ◽  
Joshua D. Doyle ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Complex Function ◽  
Microscopic Analysis ◽  
Murine Hepatitis Virus ◽  
Electron Microscopic ◽  
Virus Growth ◽  
Positive Strand Rna

ABSTRACT Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.

scholarly journals Crystal Structure of Nonstructural Protein 10 from the Severe Acute Respiratory Syndrome Coronavirus Reveals a Novel Fold with Two Zinc-Binding Motifs

2006 ◽  
Vol 80 (16) ◽  
pp. 7894-7901 ◽  
Author(s):  
Jeremiah S. Joseph ◽  
Kumar Singh Saikatendu ◽  
Vanitha Subramanian ◽  
Benjamin W. Neuman ◽  
Alexei Brooun ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Severe Acute Respiratory Syndrome ◽  
Structural Integrity ◽  
Rna Binding ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Sequence Specificity ◽  
Nucleic Acid Binding ◽  
Murine Hepatitis Virus

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The first open reading frame encodes replicase polyproteins 1a and 1ab, which are cleaved to generate 16 “nonstructural” proteins, nsp1 to nsp16, involved in viral replication and/or RNA processing. Among these, nsp10 plays a critical role in minus-strand RNA synthesis in a related coronavirus, murine hepatitis virus. Here, we report the crystal structure of SARS-CoV nsp10 at a resolution of 1.8 Å as determined by single-wavelength anomalous dispersion using phases derived from hexatantalum dodecabromide. nsp10 is a single domain protein consisting of a pair of antiparallel N-terminal helices stacked against an irregular β-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn2+ ion in a unique conformation. The second Zn finger, with four cysteines, is a distant member of the “gag-knuckle fold group” of Zn2+-binding domains and appears to maintain the structural integrity of the C-terminal tail. A distinct clustering of basic residues on the protein surface suggests a nucleic acid-binding function. Gel shift assays indicate that in isolation, nsp10 binds single- and double-stranded RNA and DNA with high-micromolar affinity and without obvious sequence specificity. It is possible that nsp10 functions within a larger RNA-binding protein complex. However, its exact role within the replicase complex is still not clear.

scholarly journals Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes.

1989 ◽  
Vol 9 (7) ◽  
pp. 2989-2999 ◽  
Author(s):  
H M Traglia ◽  
N S Atkinson ◽  
A K Hopper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Open Reading Frame ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Reading Frame ◽  
Genomic Deletions ◽  
Single Amino Acid Change ◽  
Carboxy Terminal

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

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