Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1993 Dec;67(12):7223–7228. doi: 10.1128/jvi.67.12.7223-7228.1993

Rescue of an influenza A virus wild-type PB2 gene and a mutant derivative bearing a site-specific temperature-sensitive and attenuating mutation.

E K Subbarao 1, Y Kawaoka 1, B R Murphy 1
PMCID: PMC238184  PMID: 8230444

Abstract

Live attenuated influenza A virus vaccines are currently produced by the transfer of attenuating genes from a donor virus to new epidemic variants of influenza A virus, with the selection of reassortant viruses that possess the protective antigens (i.e., the two surface glycoproteins) of the epidemic virus and the attenuating genes from the donor virus. The previously studied attenuated donor viruses were produced by conventional methods such as passage of virus at low temperature or chemical mutagenesis. The present paper describes a new strategy for the generation of a donor virus bearing an attenuating, non-surface-glycoprotein gene. This strategy involves the introduction of attenuating mutations into the cDNA copy of the PB2 polymerase gene by site-directed mutagenesis, transfection of in vitro RNA transcripts of PB2 cDNA, and recovery of the transfected PB2 gene into an infectious virus. An avian-human influenza A virus PB2 single-gene reassortant virus (with an avian influenza A virus PB2 gene) that replicates efficiently in avian tissue but poorly in mammalian cells was used as a helper virus to rescue a transfected synthetic RNA derived from a human influenza A virus PB2 gene. The desired human influenza A virus mutant PB2 transfectant was favored in this situation because the avian influenza A virus PB2 gene restricts viral replication in mammalian cells in culture, the system used for rescue, thereby providing strong selection for the virus bearing the human influenza A virus PB2 gene. We validated the feasibility of this approach by rescuing the PB2 gene of the wild-type influenza A/Ann Arbor/6/60 virus and a mutant derivative that had a single amino acid substitution introduced at position 265 by site-directed mutagenesis. Previously, this amino acid substitution had been shown to specify both a temperature-sensitive (ts) and an attenuation (att) phenotype. The rescued mutant 265 PB2 transfectant virus exhibited the ts and att phenotypes, which confirms that these phenotypes were specified by this single amino acid substitution. The transfectant virus was immunogenic and protected hamsters from subsequent challenge with wild-type virus. The cDNA copy of this influenza A/Ann Arbor/6/60 virus mutant 265 PB2 gene will be used as a substrate for the introduction of additional attenuating mutations by site-directed mutagenesis.

Full text

PDF
7223

Images in this article

7225Image
on p.7225

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boulikas T., Hancock R. A highly sensitive technique for staining DNA and RNA in polyacrylamide gels using silver. J Biochem Biophys Methods. 1981 Oct;5(4):219–228. doi: 10.1016/0165-022x(81)90046-4. [DOI] [PubMed] [Google Scholar]
  2. Castrucci M. R., Bilsel P., Kawaoka Y. Attenuation of influenza A virus by insertion of a foreign epitope into the neuraminidase. J Virol. 1992 Aug;66(8):4647–4653. doi: 10.1128/jvi.66.8.4647-4653.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clements M. L., Subbarao E. K., Fries L. F., Karron R. A., London W. T., Murphy B. R. Use of single-gene reassortant viruses to study the role of avian influenza A virus genes in attenuation of wild-type human influenza A virus for squirrel monkeys and adult human volunteers. J Clin Microbiol. 1992 Mar;30(3):655–662. doi: 10.1128/jcm.30.3.655-662.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cox N. J., Kitame F., Kendal A. P., Maassab H. F., 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] [Google Scholar]
  5. Enami M., Luytjes W., Krystal M., Palese P. Introduction of site-specific mutations into the genome of influenza virus. Proc Natl Acad Sci U S A. 1990 May;87(10):3802–3805. doi: 10.1073/pnas.87.10.3802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Enami M., Palese P. High-efficiency formation of influenza virus transfectants. J Virol. 1991 May;65(5):2711–2713. doi: 10.1128/jvi.65.5.2711-2713.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lawson C. M., Subbarao E. K., Murphy B. R. 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. doi: 10.1016/0042-6822(92)90221-a. [DOI] [PubMed] [Google Scholar]
  8. Li S. Q., Schulman J. L., Moran T., Bona C., Palese P. Influenza A virus transfectants with chimeric hemagglutinins containing epitopes from different subtypes. J Virol. 1992 Jan;66(1):399–404. doi: 10.1128/jvi.66.1.399-404.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Luytjes W., Krystal M., Enami M., Parvin J. D., Palese P. Amplification, expression, and packaging of foreign gene by influenza virus. Cell. 1989 Dec 22;59(6):1107–1113. doi: 10.1016/0092-8674(89)90766-6. [DOI] [PubMed] [Google Scholar]
  10. Murphy B. R., Hinshaw V. S., Sly D. L., London W. T., Hosier N. T., Wood F. T., Webster R. G., Chanock R. M. Virulence of avian influenza A viruses for squirrel monkeys. Infect Immun. 1982 Sep;37(3):1119–1126. doi: 10.1128/iai.37.3.1119-1126.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Muster T., Subbarao E. K., Enami M., Murphy B. R., Palese P. An influenza A virus containing influenza B virus 5' and 3' noncoding regions on the neuraminidase gene is attenuated in mice. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5177–5181. doi: 10.1073/pnas.88.12.5177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Parvin J. D., Palese P., Honda A., Ishihama A., Krystal M. Promoter analysis of influenza virus RNA polymerase. J Virol. 1989 Dec;63(12):5142–5152. doi: 10.1128/jvi.63.12.5142-5152.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Snyder M. H., Betts R. F., DeBorde D., Tierney E. L., Clements M. L., Herrington D., Sears S. D., Dolin R., Maassab H. F., Murphy B. R. 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. doi: 10.1128/jvi.62.2.488-495.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Snyder M. H., Buckler-White A. J., London W. T., Tierney E. L., Murphy B. R. The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza A/Pintail/79 reassortant viruses for monkeys. J Virol. 1987 Sep;61(9):2857–2863. doi: 10.1128/jvi.61.9.2857-2863.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Subbarao E. K., London W., Murphy B. R. A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. J Virol. 1993 Apr;67(4):1761–1764. doi: 10.1128/jvi.67.4.1761-1764.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Tolpin M. D., Massicot J. G., Mullinix M. G., Kim H. W., Parrott R. H., Chanock R. M., Murphy B. R. 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. doi: 10.1016/0042-6822(81)90298-1. [DOI] [PubMed] [Google Scholar]

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

RESOURCES