PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
 
J Virol. 1994 December; 68(12): 7684–7688.
PMCID: PMC237228

Evaluation of the genetic stability of the temperature-sensitive PB2 gene mutation of the influenza A/Ann Arbor/6/60 cold-adapted vaccine virus.

Abstract

A single-gene reassortant bearing the PB2 gene of the A/Ann Arbor/6/60 cold-adapted virus in the background of the A/Korea/82 (H3N2) wild-type virus is a temperature-sensitive (ts) virus with an in vitro shutoff temperature of 38 degrees C. A single mutation at amino acid (aa) at 265 (Asp-Ser) of the PB2 protein is responsible for the ts phenotype. This ts single-gene PB2 reassortant virus was serially passaged at elevated temperatures in Madin-Darby canine kidney cells to generate ts+ phenotypic revertant viruses. Four ts+ phenotypically revertant viruses were derived independently, and each possessed a shutoff temperature for replication in vitro of > 40 degrees C. Each of the four phenotypically revertant viruses replicated efficiently in the upper and lower respiratory tracts of mice and hamsters, unlike the PB2 single-gene reassortant virus, confirming that the ts phenotype was responsible for the attenuation of this virus in rodents. Mating the ts+ revertants with wild-type virus yielded ts progeny in high frequency, indicating that the loss of ts phenotype was due to a suppressor mutation which was mapped to the PA gene in each of the four independently derived ts phenotypic revertants. Nucleotide sequence analysis confirmed the absence of new mutations on the PB2 gene and the presence of predicted amino acid changes in the PA proteins of the revertant viruses. These studies suggest that single amino acid changes at aa 245 (Glu-Lys) or 347 (Asp-Asn) of the PA protein can completely suppress the ts and attenuation phenotypes specified by the Asp-Ser mutation at aa 265 of the PB2 protein of the A/Ann Arbor/6/60 cold-adapted virus.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.0M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Braam J, Ulmanen I, Krug RM. Molecular model of a eucaryotic transcription complex: functions and movements of influenza P proteins during capped RNA-primed transcription. Cell. 1983 Sep;34(2):609–618. [PubMed]
  • Cox NJ, Kitame F, Kendal AP, Maassab HF, Naeve C. Identification of sequence changes in the cold-adapted, live attenuated influenza vaccine strain, A/Ann Arbor/6/60 (H2N2). Virology. 1988 Dec;167(2):554–567. [PubMed]
  • Ghendon Y, Markushin S, Lisovskaya K, Penn CR, Mahy BW. Extragenic suppression of a ts phenotype during recombination between ts mutants of two fowl plague virus strains with a ts mutation in gene 1. J Gen Virol. 1982 Oct;62(Pt 2):239–248. [PubMed]
  • Lawson CM, Subbarao EK, Murphy BR. Nucleotide sequence changes in the polymerase basic protein 2 gene of temperature-sensitive mutants of influenza A virus. Virology. 1992 Nov;191(1):506–510. [PubMed]
  • Mücke K, Scholtissek C. Extragenic and intragenic suppression of a transport mutation in the hemagglutinin gene of an influenza A virus as revealed by backcross and sequence determination. Virology. 1987 May;158(1):112–117. [PubMed]
  • Murphy BR, Markoff LJ, Hosier NT, Massicot JG, Chanock RM. Production and level of genetic stability of an influenza A virus temperature-sensitive mutant containing two genes with ts mutations. Infect Immun. 1982 Jul;37(1):235–242. [PMC free article] [PubMed]
  • Murphy BR, Tierney EL, Spring SB, Chanock RM. Temperature-sensitive mutants of influenza A virus. XI. Transfer of ts lesions in the Hong Kong/68-ts-1 [a] virus to the influenza A/Udorn/72 wild type. J Infect Dis. 1976 Dec;134(6):577–584. [PubMed]
  • Murphy BR, Tolpin MD, Massicot JG, Kim HY, Parrott RH, Chanock RM. Escape of a highly defective influenza A virus mutant from its temperature sensitive phenotype by extragenic suppression and other types of mutation. Ann N Y Acad Sci. 1980;354:172–182. [PubMed]
  • Scholtissek C, Spring SB. Extragenic suppression of temperature-sensitive mutations in RNA segment 8 by replacement of different rna segments with those of other influenza A virus prototype strains. Virology. 1982 Apr 15;118(1):28–34. [PubMed]
  • Shimizu K, Mullinix MG, Chanock RM, Murphy BR. Temperature-sensitive mutants of influenza A/Udorn/72 (H3N2) virus. I. Isolation of temperature-sensitive mutants some of which exhibit host-dependent temperature sensitivity. Virology. 1982 Feb;117(1):38–44. [PubMed]
  • Snyder MH, Betts RF, DeBorde D, Tierney EL, Clements ML, Herrington D, Sears SD, Dolin R, Maassab HF, Murphy BR. Four viral genes independently contribute to attenuation of live influenza A/Ann Arbor/6/60 (H2N2) cold-adapted reassortant virus vaccines. J Virol. 1988 Feb;62(2):488–495. [PMC free article] [PubMed]
  • Snyder MH, Clements ML, De Borde D, Maassab HF, Murphy BR. Attenuation of wild-type human influenza A virus by acquisition of the PA polymerase and matrix protein genes of influenza A/Ann Arbor/6/60 cold-adapted donor virus. J Clin Microbiol. 1985 Nov;22(5):719–725. [PMC free article] [PubMed]
  • Spring SB, Maassab HF, Kendal AP, Murphy BR, Chanock RM. Cold adapted variants of influenza A. II. Comparison of the genetic and biological properties of ts mutants and recombinants of the cold adapted A/AA/6/60 strain. Arch Virol. 1977;55(3):233–246. [PubMed]
  • Subbarao EK, Kawaoka Y, Murphy BR. Rescue of an influenza A virus wild-type PB2 gene and a mutant derivative bearing a site-specific temperature-sensitive and attenuating mutation. J Virol. 1993 Dec;67(12):7223–7228. [PMC free article] [PubMed]
  • Subbarao EK, Perkins M, Treanor JJ, Murphy BR. The attenuation phenotype conferred by the M gene of the influenza A/Ann Arbor/6/60 cold-adapted virus (H2N2) on the A/Korea/82 (H3N2) reassortant virus results from a gene constellation effect. Virus Res. 1992 Sep 1;25(1-2):37–50. [PubMed]
  • Tian SF, Buckler-White AJ, London WT, Reck LJ, Chanock RM, Murphy BR. Nucleoprotein and membrane protein genes are associated with restriction of replication of influenza A/Mallard/NY/78 virus and its reassortants in squirrel monkey respiratory tract. J Virol. 1985 Mar;53(3):771–775. [PMC free article] [PubMed]
  • Tolpin MD, Massicot JG, Mullinix MG, Kim HW, Parrott RH, Chanock RM, Murphy BR. Genetic factors associated with loss of the temperature-sensitive phenotype of the influenza A/Alaska/77-ts-1A2 recombinant during growth in vivo. Virology. 1981 Jul 30;112(2):505–517. [PubMed]
  • Treanor JJ, Buja R, Murphy BR. Intragenic suppression of a deletion mutation of the nonstructural gene of an influenza A virus. J Virol. 1991 Aug;65(8):4204–4210. [PMC free article] [PubMed]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)