• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Dec 5, 1995; 92(25): 11563–11567.
PMCID: PMC40442

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Abstract

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

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 (1.5M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Hall CB. Prospects for a respiratory syncytial virus vaccine. Science. 1994 Sep 2;265(5177):1393–1394. [PubMed]
  • Murphy BR, Hall SL, Kulkarni AB, Crowe JE, Jr, Collins PL, Connors M, Karron RA, Chanock RM. An update on approaches to the development of respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) vaccines. Virus Res. 1994 Apr;32(1):13–36. [PubMed]
  • Pattnaik AK, Ball LA, LeGrone AW, Wertz GW. Infectious defective interfering particles of VSV from transcripts of a cDNA clone. Cell. 1992 Jun 12;69(6):1011–1020. [PubMed]
  • Calain P, Roux L. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol. 1993 Aug;67(8):4822–4830. [PMC free article] [PubMed]
  • Conzelmann KK, Schnell M. Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins. J Virol. 1994 Feb;68(2):713–719. [PMC free article] [PubMed]
  • Yu Q, Hardy RW, Wertz GW. Functional cDNA clones of the human respiratory syncytial (RS) virus N, P, and L proteins support replication of RS virus genomic RNA analogs and define minimal trans-acting requirements for RNA replication. J Virol. 1995 Apr;69(4):2412–2419. [PMC free article] [PubMed]
  • Grosfeld H, Hill MG, Collins PL. RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA. J Virol. 1995 Sep;69(9):5677–5686. [PMC free article] [PubMed]
  • Schnell MJ, Mebatsion T, Conzelmann KK. Infectious rabies viruses from cloned cDNA. EMBO J. 1994 Sep 15;13(18):4195–4203. [PMC free article] [PubMed]
  • Lawson ND, Stillman EA, Whitt MA, Rose JK. Recombinant vesicular stomatitis viruses from DNA. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4477–4481. [PMC free article] [PubMed]
  • Collins PL, Hill MG, Johnson PR. The two open reading frames of the 22K mRNA of human respiratory syncytial virus: sequence comparison of antigenic subgroups A and B and expression in vitro. J Gen Virol. 1990 Dec;71(Pt 12):3015–3020. [PubMed]
  • Wyatt LS, Moss B, Rozenblatt S. Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells. Virology. 1995 Jun 20;210(1):202–205. [PubMed]
  • Murphy BR, Sotnikov AV, Lawrence LA, Banks SM, Prince GA. Enhanced pulmonary histopathology is observed in cotton rats immunized with formalin-inactivated respiratory syncytial virus (RSV) or purified F glycoprotein and challenged with RSV 3-6 months after immunization. Vaccine. 1990 Oct;8(5):497–502. [PubMed]
  • Johnson PR, Collins PL. The A and B subgroups of human respiratory syncytial virus: comparison of intergenic and gene-overlap sequences. J Gen Virol. 1988 Nov;69(Pt 11):2901–2906. [PubMed]
  • Zoller MJ, Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. [PubMed]
  • Connors M, Crowe JE, Jr, Firestone CY, Murphy BR, Collins PL. A cold-passaged, attenuated strain of human respiratory syncytial virus contains mutations in the F and L genes. Virology. 1995 Apr 20;208(2):478–484. [PubMed]
  • Sidhu MS, Chan J, Kaelin K, Spielhofer P, Radecke F, Schneider H, Masurekar M, Dowling PC, Billeter MA, Udem SA. Rescue of synthetic measles virus minireplicons: measles genomic termini direct efficient expression and propagation of a reporter gene. Virology. 1995 Apr 20;208(2):800–807. [PubMed]
  • Crowe JE, Jr, Bui PT, Davis AR, Chanock RM, Murphy BR. A further attenuated derivative of a cold-passaged temperature-sensitive mutant of human respiratory syncytial virus retains immunogenicity and protective efficacy against wild-type challenge in seronegative chimpanzees. Vaccine. 1994 Jul;12(9):783–790. [PubMed]
  • Randhawa JS, Chambers P, Pringle CR, Easton AJ. Nucleotide sequences of the genes encoding the putative attachment glycoprotein (G) of mouse and tissue culture-passaged strains of pneumonia virus of mice. Virology. 1995 Feb 20;207(1):240–245. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

  • Production of infectious human respiratory syncytial virus from cloned cDNA conf...
    Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.
    Proceedings of the National Academy of Sciences of the United States of America. Dec 5, 1995; 92(25)11563
    PMC

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...