Temperature-Sensitive Mutants of MHV-A59

Sturman, Lawrence S.; Eastwood, C.; Frana, Mark F.; Duchala, Cynthia S.; Baker, Frederick D.; Ricard, Cynthia S.; Sawicki, Stanley G.; Holmes, Kathryn V.

doi:10.1007/978-1-4684-1280-2_20

Cited by 20 publications

(18 citation statements)

References 12 publications

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“…Previous studies predicted that nsp10 acts as a cofactor in the MHV replication complex (3,28,30,41,44,50), which led us to hypothesize that mutations in nsp10 could impact several specific areas of viral RNA synthesis including but not limited to (i) positive-strand synthesis, resulting in reductions of both subgenomic and genomic RNAs; (ii) negative-strand synthesis, resulting in reductions of minus-strand templates; and/or (iii) polyprotein processing, resulting in reductions of essential proteins or intermediates processed from the nascent polyprotein that are essential for the formation of the replication complex. To further examine the functional role of nsp10 in MHV RNA transcription/replication, we applied a reverse-genetics approach incorporating alanine-scanning mutations into the nsp10 gene of the molecular clone of MHV based upon two strategies.…”

Section: Discussionmentioning

confidence: 99%

Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis

Donaldson¹,

Sims

Graham

et al. 2007

J Virol

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Coronavirus replication requires proteolytic processing of the large polyprotein encoded by ORF1a/ab into putative functional intermediates and eventually ϳ15 mature proteins. The C-terminal ORF1a protein nsp10 colocalizes with viral replication complexes, but its role in transcription/replication is not well defined. To investigate the role of nsp10 in coronavirus transcription/replication, alanine replacements were engineered into a murine hepatitis virus (MHV) infectious clone in place of conserved residues in predicted functional domains or charged amino acid pairs/triplets, and rescued viruses were analyzed for mutant phenotypes. Of the 16 engineered clones, 5 viable viruses were rescued, 3 mutant viruses generated no cytopathic effect but were competent to synthesize viral subgenomic RNAs, and 8 were not viable. All viable mutants showed reductions in growth kinetics and overall viral RNA synthesis, implicating nsp10 as being a cofactor in positive-or negative-strand synthesis. Viable mutant nsp10-E2 was compromised in its ability to process the nascent polyprotein, as processing intermediates were detected in cells infected with this virus that were not detectable in wild-type infections. Mapping the mutations onto the crystal structure of severe acute respiratory syndrome virus nsp10 identified a central core resistant to mutation. Mutations targeting residues in or near either zinc-binding finger generated nonviable phenotypes, demonstrating that both domains are essential to nsp10 function and MHV replication. All mutations resulting in viable phenotypes mapped to loops outside the central core and were characterized by a global decrease in RNA synthesis. These results demonstrate that nsp10 is a critical regulator of coronavirus RNA synthesis and may play an important role in polyprotein processing.Coronaviruses (CoVs) have historically been associated with severe disease in important domestic animals and mild upper respiratory tract infections in humans (human CoV strains HCoV-229E and HCoV-OC43), rarely leading to severe disease (24). However, the rapid identification of a CoV as the etiological agent responsible for severe acute respiratory syndrome (SARS) redefined historic perceptions. SARS-CoV spread globally and infected over 8,000 people, with mortality rates of about 10% (49). More recent studies have identified human CoVs NL63 and HKU-1 as being important lower respiratory tract pathogens (25, 53). With ubiquitous populations of CoVs found in many animals, it is not surprising that several newly identified CoVs that likely emerged from zoonotic reservoirs over the past 20 years have been described. An understanding of the basic biology and pathogenesis of CoV infection is of urgent importance.CoVs are the largest known RNA viruses encoding plussense, 5Ј-capped, and polyadenylated genomes of 27 to 31 kb in length. CoVs are classified as members of the order Nidovirales, family Coronaviridae, genus Coronavirus, and are divided into three groups based on serological and phylogenetic categorizat...

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Section: Discussionmentioning

confidence: 99%

Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis

Donaldson¹,

Sims

Graham

et al. 2007

J Virol

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“…Over the past 30 years, several panels of MHV temperaturesensitive (ts) mutant viruses were established by chemical mu-tagenesis and were proposed to have ts mutations in the replicase nsp's based on viral complementation studies (41). Recently, sequencing of the replicase genes of several of these previously known ts viruses identified mutations predicted to be responsible for the ts phenotypes (39), one of which has experimentally been confirmed to occur in nsp10 using reverse genetic introduction of the identified mutation into the infectious MHV clone (17).…”

mentioning

confidence: 99%

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

Sparks

Donaldson²,

et al. 2008

J Virol

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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.Coronaviruses (CoVs) belong to a family of enveloped, positive-strand RNA viruses that cause important diseases in humans and animals. The identification of a novel human CoV as the etiological agent of severe acute respiratory syndrome (SARS) highlights the potential of this virus family to cause severe and acute disease in the human population (13,18,36). Additionally, the increasing identification of CoVs in bats and other animals (37), combined with the threat of CoV transspecies transmission, underscores the need to better understand the replication strategies of these viruses in order to identify improved targets for attenuation and interference with replication.The CoV murine hepatitis virus (MHV) has been used as a model system to investigate the functions of viral proteins in replication and pathogenesis. The MHV genome is approximately 32 kb in size and contains seven genes, with the first encoding the nonstructural proteins (nsp's) and genes 2 to 7 encoding the structural and accessory proteins of the virus. The life cycle of MHV occurs exclusively in the host cell cytoplasm. Following the entry of MHV into a cell, the RNA genome is translated by host cell ribosomes from open reading frame 1 (ORF1), which consists of approximately 22 kb and encodes nsp1 to nsp16 (Fig. 1A). ORF1 is comprised of two ORFs (ORF1a and ORF1ab) that are connected by a Ϫ1 ribosomal frameshift element (7,9,29,34). Translation of the ORF1a or the ORF1ab fusion polyprotein (pp1a or pp1ab, respectively) results in 49...

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“…Sawicki et al analyzed a known temperature-sensitive(ts) mutant of MHV, Alb ts6, by sequence and reversion analysis. They identified within the nsp4 coding region an AA 9494 T-to-A C 9494 T nucleotide (nt) change resulting in an Asn258Thr (N258T) substitution as the putative ts mutation (Sawicki et al, 2005;Sturman et al, 1987). Clementz et al engineered the N258T substitution in recombinant MHV using a two nt change AAT 9494-9495 -to-ACA 9494-9495 .…”

Section: The Studymentioning

confidence: 99%

Murine hepatitis virus nsp4 N258T mutants are not temperature-sensitive

Beachboard

Baker

et al. 2013

Virology

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Coronavirus replicase nsp4 is critical for virus-induced membrane modifications. An nsp4 mutant (N258T) of murine hepatitis virus (MHV) has been reported to be temperature-sensitive (ts) and to alter membrane targeting. We engineered and recovered all four possible codon variants of N258T in the cloned MHV-A59 background. All mutant viruses demonstrated impaired replication compared to wildtype MHV, but no nsp4 N258T mutant virus was ts, and all variants colocalized with viral protein markers for replication complexes, but not with markers for mitochondria. This study emphasizes that complete genome sequencing may be necessary, even with directed and confirmed reverse genetic mutants.

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Temperature-Sensitive Mutants of MHV-A59

Cited by 20 publications

References 12 publications

Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis

Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis

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

Murine hepatitis virus nsp4 N258T mutants are not temperature-sensitive

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