Recovery of infectious respiratory syncytial virus expressing an additional, foreign gene

Alexander Bukreyev, Ena Camargo, Peter L. Collins

Research output: Contribution to journalArticle

67 Citations (Scopus)

Abstract

A previous report described the recovery from cDNA of infectious recombinant respiratory syncytial virus (RSV) strain A2 (P. L. Collins, M. G. Hill, E. Camargo, H. Grosfeld, R. M. Chanock, and B. R. Murphy, Proc. Natl. Acad. Sci. USA, 92:11563-11567, 1995). Here, the system was used to construct recombinant RSV containing an additional gene encoding chloramphenicol acetyltransferase (CAT). The CAT coding sequence was flanked by RSV-specific gene-start and gene-end motifs, the transcription signals for the vital RNAdependent RNA polymerase. The RSV-CAT chimeric transcription cassette was inserted into the region between the G and F genes of the complete cDNA- encoded positive-sense RSV antigenome, and infectious CAT-expressing recombinant RSV was recovered. Transcription of the inserted gene into the predicted subgertomic polyadenylated mRNA was demonstrated by Northern (RNA) blot hybridization analysis, and the encoded protein was detected by enzyme assay and by radioimmunoprecipitation. Quantitation of intracellular CAT, SH, G, and F mRNAs showed that the CAT mRNA was efficiently expressed and that the levels of the G and F mRNAs (which represent the genes on either side of the inserted CAT gene) were comparable to those expressed by a wild-type recombinant RSV. Consistent with this finding, the CAT-containing and wild- type viruses were very similar with regard to the levels of synthesis of the major viral proteins. Each of 25 RSV isolates obtained by plaque purification following eight serial passages expressed CAT, showing that the foreign gene was faithfully maintained in functional form. Analysis by reverse transcription and PCR did not reveal evidence of deletion of the foreign sequence. This finding demonstrated that the RSV genome can accept and maintain an increase in length of 762 nucleotides of foreign sequence and can be engineered to encode an additional, 11th mRNA. The presence of the additional gene resulted in a 10% decrease in plaque diameter and was associated with delay in virus growth and 20-fold decrease in virus yield in vitro. Thus, introduction of an additional gene into the RSV genome might represent a method of attenuation. The ability to express foreign genes by recombinant RSV has implications for basic studies as well as for the development of live recombinant vaccines.

Original languageEnglish (US)
Pages (from-to)6634-6641
Number of pages8
JournalJournal of Virology
Volume70
Issue number10
StatePublished - Oct 1996
Externally publishedYes

Fingerprint

Respiratory Syncytial Viruses
Chloramphenicol O-Acetyltransferase
chloramphenicol acetyltransferase
viruses
Genes
genes
Messenger RNA
Viruses
transcription (genetics)
Complementary DNA
Genome
Serial Passage
Synthetic Vaccines
Sequence Deletion
Enzyme Assays
Viral Proteins
DNA-Directed RNA Polymerases
varespladib methyl
Northern Blotting
reverse transcription

ASJC Scopus subject areas

  • Immunology

Cite this

Recovery of infectious respiratory syncytial virus expressing an additional, foreign gene. / Bukreyev, Alexander; Camargo, Ena; Collins, Peter L.

In: Journal of Virology, Vol. 70, No. 10, 10.1996, p. 6634-6641.

Research output: Contribution to journalArticle

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