scholarly journals Role for Conserved Residues of Sindbis Virus Nonstructural Protein 2 Methyltransferase-Like Domain in Regulation of Minus-Strand Synthesis and Development of Cytopathic Infection

2008 ◽  
Vol 82 (15) ◽  
pp. 7284-7297 ◽  
Author(s):  
Mayuri ◽  
Todd W. Geders ◽  
Janet L. Smith ◽  
Richard J. Kuhn
Keyword(s):  
Defense Mechanisms ◽  
Sindbis Virus ◽  
Nonstructural Protein ◽  
Temperature Sensitive ◽  
Minus Strand ◽  
Nonstructural Proteins ◽  
Equine Encephalitis Virus ◽  
Nonstructural Protein 2 ◽  
Transcriptional Inhibition ◽  
Lysine Residues

ABSTRACT The plus-strand RNA genome of Sindbis virus (SINV) encodes four nonstructural proteins (nsP1 to nsP4) that are involved in the replication of the viral RNA. The ∼800-amino-acid nsP2 consists of an N-terminal domain with nucleoside triphosphatase and helicase activities and a C-terminal protease domain. Recently, the structure determined for Venezuelan equine encephalitis virus nsP2 indicated the presence of a previously unrecognized methyltransferase (MTase)-like domain within the C-terminal ∼200 residues and raised a question about its functional importance. To assess the role of this MTase-like region in viral replication, highly conserved arginine and lysine residues were mutated to alanine. The plaque phenotypes of these mutants ranged from large/wild-type to small plaques with selected mutations demonstrating temperature sensitive lethality. The proteolytic polyprotein processing activity of nsP2 was unaffected in most of the mutants. Some of the temperature-sensitive mutants showed reduction in the minus-strand RNA synthesis, a function that has not yet been ascribed to nsP2. Mutation of SINV residue R615 rendered the virus noncytopathic and incapable of inhibiting the host cell translation but with no effects on the transcriptional inhibition. This property differentiated the mutation at R615 from previously described noncytopathic mutations. These results implicate nsP2 in regulation of minus-strand synthesis and suggest that different regions of the nsP2 MTase-like domain differentially modulate host defense mechanisms, independent of its role as the viral protease.

scholarly journals Ribosomal Protein S6 Associates with Alphavirus Nonstructural Protein 2 and Mediates Expression from Alphavirus Messages

2006 ◽  
Vol 80 (15) ◽  
pp. 7729-7739 ◽  
Author(s):  
Stephanie A. Montgomery ◽  
Peter Berglund ◽  
Clayton W. Beard ◽  
Robert E. Johnston
Keyword(s):  
Ribosomal Protein ◽  
Nonstructural Protein ◽  
Cellular Proteins ◽  
Nonstructural Proteins ◽  
Replication Complex ◽  
Equine Encephalitis Virus ◽  
Nonstructural Protein 2 ◽  
Ribosomal Protein S6 ◽  
Cellular Translation ◽  
Rps6 Phosphorylation

ABSTRACT Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.

scholarly journals Molecular Defects Caused by Temperature-Sensitive Mutations in Semliki Forest Virus nsP1

2008 ◽  
Vol 82 (18) ◽  
pp. 9236-9244 ◽  
Author(s):  
Valeria Lulla ◽  
Dorothea L. Sawicki ◽  
Stanley G. Sawicki ◽  
Aleksei Lulla ◽  
Andres Merits ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Minus Strand ◽  
Nonstructural Proteins ◽  
Mutant Proteins ◽  
Recombinant Viruses ◽  
Mrna Capping

ABSTRACT Alphavirus replicase protein nsP1 has multiple functions during viral RNA synthesis. It catalyzes methyltransferase and guanylyltransferase activities needed in viral mRNA capping, attaches the viral replication complex to cytoplasmic membranes, and is required for minus-strand RNA synthesis. Two temperature-sensitive (ts) mutations in Semliki Forest virus (SFV) were previously identified within nsP1: ts10 (E529D) and ts14 (D119N). Recombinant viruses containing these individual mutations reproduced the features of the original ts strains. We now find that the capping-associated enzymatic activities of recombinant nsP1, containing ts10 or ts14 lesions, were not ts. The mutant proteins and polyproteins also were membrane bound, mutant nsP1 interacted normally with the other nonstructural proteins, and there was no major defect in nonstructural polyprotein processing in the mutants, although ts14 surprisingly displayed slightly retarded processing. The two mutant viruses were specifically defective in minus-strand RNA synthesis at the restrictive temperature. Integrating data from SFV and Sindbis virus, we discuss the domain structure of nsP1 and the relative positioning of and interactions between the replicase proteins. nsP1 is suggested to contain a specific subdomain involved in minus-strand synthesis and interaction with the polymerase nsP4 and the protease nsP2.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 2 Interacts with a Host Protein Complex Involved in Mitochondrial Biogenesis and Intracellular Signaling

2009 ◽  
Vol 83 (19) ◽  
pp. 10314-10318 ◽  
Author(s):  
Cromwell T. Cornillez-Ty ◽  
Lujian Liao ◽  
John R. Yates ◽  
Peter Kuhn ◽  
Michael J. Buchmeier
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Intracellular Signaling ◽  
Nonstructural Protein ◽  
Host Protein ◽  
Nonstructural Proteins ◽  
Host Proteins ◽  
Catalytic Activities ◽  
Nonstructural Protein 2 ◽  
Host Signaling ◽  
Prohibitin 1

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) generates 16 nonstructural proteins (nsp's) through proteolytic cleavage of a large precursor protein. Although several nsp's exhibit catalytic activities that are important for viral replication and transcription, other nsp's have less clearly defined roles during an infection. In order to gain a better understanding of their functions, we attempted to identify host proteins that interact with nsp's during SARS-CoV infections. For nsp2, we identified an interaction with two host proteins, prohibitin 1 (PHB1) and PHB2. Our results suggest that nsp2 may be involved in the disruption of intracellular host signaling during SARS-CoV infections.

Temperature sensitive shut-off of alphavirus minus strand RNA synthesis maps to a nonstructural protein, nsP4

Virology ◽  
1990 ◽  
Vol 174 (1) ◽  
pp. 43-52 ◽  
Author(s):  
Dorothea L. Sawicki ◽  
David B. Barkhimer ◽  
Stanley G. Sawicki ◽  
Charles M. Rice ◽  
Sondra Schlesinger
Keyword(s):  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Temperature Sensitive ◽  
Minus Strand

scholarly journals Alphavirus Minus-Strand RNA Synthesis: Identification of a Role for Arg183 of the nsP4 Polymerase

2002 ◽  
Vol 76 (17) ◽  
pp. 8632-8640 ◽  
Author(s):  
Cori L. Fata ◽  
Stanley G. Sawicki ◽  
Dorothea L. Sawicki
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Temperature Sensitive ◽  
Minus Strand ◽  
Mrna Synthesis ◽  
Wild Type ◽  
Lethal Temperature ◽  
Host Restriction ◽  
Mosquito Cells ◽  
Promoter Recognition

ABSTRACT A partially conserved region spanning amino acids 142 to 191 of the Sindbis virus (SIN) nsP4 core polymerase is implicated in host restriction, elongation, and promoter recognition. We extended the analysis of this region by substituting Ser, Ala, or Lys for a highly conserved Arg183 residue immediately preceding its absolutely conserved Ser184-Ala-Val-Pro-Ser188 sequence. In chicken cells, the nsP4 Arg183 mutants had a nonconditionally lethal, temperature-sensitive (ts) growth phenotype caused by a ts defect in minus-strand synthesis whose extent varied with the particular amino acid substituted (Ser>Ala>Lys). Plus-strand synthesis by nsP4 Arg183 mutant polymerases was unaffected when corrected for minus-strand numbers, although 26S mRNA synthesis was enhanced at the elevated temperature compared to wild type. The ts defect was not due to a failure to form or accumulate nsP4 at 40°C. In contrast to their growth in chicken cells, the nsP4 Arg183 mutants replicated equally poorly, if at all, in mosquito cells. We conclude that Arg183 within the Pro180-Asn-Ile-Arg-Ser184 sequence of the SIN nsP4 polymerase contributes to the efficient initiation of minus strands or the formation of its replicase and that a host factor(s) participates in this event.

scholarly journals Random Insertion Mutagenesis of Sindbis Virus Nonstructural Protein 2 and Selection of Variants Incapable of Downregulating Cellular Transcription

2009 ◽  
Vol 83 (18) ◽  
pp. 9031-9044 ◽  
Author(s):  
Ilya Frolov ◽  
Natalia Garmashova ◽  
Svetlana Atasheva ◽  
Elena I. Frolova
Keyword(s):  
Sindbis Virus ◽  
Nonstructural Protein ◽  
Insertion Mutagenesis ◽  
Type I ◽  
Structural Domains ◽  
Nonstructural Protein 2 ◽  
New Information ◽  
Protein Variants ◽  
Cellular Transcription ◽  
In Cells

ABSTRACT Sindbis virus nonstructural protein 2 (SINV nsP2) is an important determinant of virus pathogenesis and downregulation of virus-induced cell response. This protein efficiently inhibits transcription of cellular messenger and ribosomal RNAs and, thus, is capable of inhibiting the activation of genes whose products are involved in development of the antiviral response. Alphavirus nsP2 has a number of predicted functional domains, some of which were confirmed by crystal structure. Our current study demonstrated that none of the putative or known structural domains alone or their combinations was capable of functioning in transcription inhibition. By using random, transposon-mediated mutagenesis, we generated a library of SINV nsP2 variants having short peptide insertions and selected those that lost the ability to inhibit cellular transcription and cause a cytopathic effect. Insertions abrogating the nuclear functions of the protein were found in the three different functional nsP2 domains. Some of the mutated protein variants retained the enzymatic functions required for replication of the viral genome. Such viruses were capable of efficient, productive replication in cells defective in interferon (IFN) signaling but were attenuated and incapable of spreading in cells with an intact type I IFN response. These results revealed new information about the structure of SINV nsP2 and interaction of its domains.

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