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Genetic analysis of influenza virus NS1 gene: a temperature-sensitive mutant shows defective formation of virus particles

Academic Article
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Overview

authors

  • Garaigorta De Dios, Urtzi
  • Falcon, A. M.
  • Ortin, J.

publication date

  • December 2005

journal

  • Journal of Virology  Journal

abstract

  • To perform a genetic analysis of the influenza A virus NS1 gene, a library of NS1 mutants was generated by PCR-mediated mutagenesis. A collection of mutant ribonucleic proteins containing the nonstructural genes was generated from the library that were rescued for an infectious virus mutant library by a novel RNP competition virus rescue procedure. Several temperature-sensitive (ts) mutant viruses were obtained by screening of the mutant library, and the sequences of their NS1 genes were determined. Most of the mutations identified led to amino acid exchanges and concentrated in the N-terminal region of the protein, but some of them occurred in the C-terminal region. Mutant 11C contained three mutations that led to amino acid exchanges, V18A, R44K, and S195P, all of which were required for the ts phenotype, and was characterized further. Several steps in the infection were slightly altered: (i) M1, M2, NS1, and neuraminidase (NA) accumulations were reduced and (ii) NS1 protein was retained in the nucleus in a temperature-independent manner, but these modifications could not justify the strong virus titer reduction at restrictive temperature. The most dramatic phenotype was the almost complete absence of virus particles in the culture medium, in spite of normal accumulation and nucleocytoplasmic export of virus RNPs. The function affected in the 11C mutant was required late in the infection, as documented by shift-up and shift-down experiments. The defect in virion production was not due to reduced NA expression, as virus yield could not be rescued by exogenous neuraminidase treatment. All together, the analysis of 11C mutant phenotype may indicate a role for NS1 protein in a late event in virus morphogenesis.

subject areas

  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Cercopithecus aethiops
  • Defective Viruses
  • Influenza A virus
  • Molecular Sequence Data
  • Mutation
  • RNA, Viral
  • Temperature
  • Viral Matrix Proteins
  • Viral Nonstructural Proteins
  • Virus Replication
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Identity

PubMed Central ID

  • PMC1316024

International Standard Serial Number (ISSN)

  • 0022-538X

Digital Object Identifier (DOI)

  • 10.1128/jvi.79.24.15246-15257.2005

PubMed ID

  • 16306596
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Additional Document Info

start page

  • 15246

end page

  • 15257

volume

  • 79

issue

  • 24

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