Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory in tract of the mouse

Alexander Bukreyev, Stephen S. Whitehead, Brian R. Murphy, Peter L. Collins

Research output: Contribution to journalArticle

179 Citations (Scopus)

Abstract

The small hydrophobic protein SH of human respiratory syncytial virus (RSV) is a short transmembrane surface protein of unknown function. A full- length cDNA of RSV strain A2 (subgroup A) antigenomic RNA was modified such that the entire SH gene, including the transcription signals and the complete mRNA-encoding sequence, was deleted and replaced by a synthetic intergenic region. This reduced the length of the antigenome by 398 nucleotides and ablated expression of 1 of the 10 RSV mRNAs. Recombinant virus containing this engineered deletion was recovered, and the absence of the SH gene was confirmed by reverse transcription in conjunction with PCR. Northern blot analysis of intracellular RNAs and gel electrophoresis of labeled intracellular proteins confirmed the lack of expression of the SH mRNA and protein. The absence of the SH gene did not noticeably affect RNA replication, but two effects on transcription were noted. First, synthesis of the G, F, and M2 mRNAs was increased, presumably due to their being one position closer to the promoter in the gene order. Second, transcription of genes downstream of the engineered site exhibited a steeper gradient of polarity. On monolayers of HEp-2 cells, the SH-minus virus produced syncytia which were at least equivalent in size to those of the wild type and produced plaques which were 70% larger. Furthermore, the SH-minus virus grew somewhat better (up to 12.6-fold) than wild-type recombinant RSV in certain cell lines. While the function of the SH protein remains to be determined, it seems to be completely dispensable for growth in tissue culture and fusion function. When inoculated intranasally into mice, the SH-minus virus resembled the wild-type recombinant virus in its efficiency of replication in the lungs, whereas it replicated 10-fold less efficiently in the upper respiratory tract. In mice, the SH-minus and wild-type recombinant viruses were similarly immunogenic and effective in inducing resistance to virus challenge.

Original languageEnglish (US)
Pages (from-to)8973-8982
Number of pages10
JournalJournal of Virology
Volume71
Issue number12
StatePublished - Dec 1997
Externally publishedYes

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Respiratory Syncytial Viruses
respiratory system
Respiratory System
cell culture
Cell Culture Techniques
Viruses
viruses
mice
Genes
genes
Messenger RNA
RNA
transcription (genetics)
Proteins
Human respiratory syncytial virus
Intergenic DNA
Gene Order
proteins
Giant Cells
varespladib methyl

ASJC Scopus subject areas

  • Immunology

Cite this

Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory in tract of the mouse. / Bukreyev, Alexander; Whitehead, Stephen S.; Murphy, Brian R.; Collins, Peter L.

In: Journal of Virology, Vol. 71, No. 12, 12.1997, p. 8973-8982.

Research output: Contribution to journalArticle

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abstract = "The small hydrophobic protein SH of human respiratory syncytial virus (RSV) is a short transmembrane surface protein of unknown function. A full- length cDNA of RSV strain A2 (subgroup A) antigenomic RNA was modified such that the entire SH gene, including the transcription signals and the complete mRNA-encoding sequence, was deleted and replaced by a synthetic intergenic region. This reduced the length of the antigenome by 398 nucleotides and ablated expression of 1 of the 10 RSV mRNAs. Recombinant virus containing this engineered deletion was recovered, and the absence of the SH gene was confirmed by reverse transcription in conjunction with PCR. Northern blot analysis of intracellular RNAs and gel electrophoresis of labeled intracellular proteins confirmed the lack of expression of the SH mRNA and protein. The absence of the SH gene did not noticeably affect RNA replication, but two effects on transcription were noted. First, synthesis of the G, F, and M2 mRNAs was increased, presumably due to their being one position closer to the promoter in the gene order. Second, transcription of genes downstream of the engineered site exhibited a steeper gradient of polarity. On monolayers of HEp-2 cells, the SH-minus virus produced syncytia which were at least equivalent in size to those of the wild type and produced plaques which were 70{\%} larger. Furthermore, the SH-minus virus grew somewhat better (up to 12.6-fold) than wild-type recombinant RSV in certain cell lines. While the function of the SH protein remains to be determined, it seems to be completely dispensable for growth in tissue culture and fusion function. When inoculated intranasally into mice, the SH-minus virus resembled the wild-type recombinant virus in its efficiency of replication in the lungs, whereas it replicated 10-fold less efficiently in the upper respiratory tract. In mice, the SH-minus and wild-type recombinant viruses were similarly immunogenic and effective in inducing resistance to virus challenge.",
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