The minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus.
A new transcription unit was generated in the 3' noncoding region of the vesicular stomatitis virus (VSV) glycoprotein gene by introducing the smallest conserved sequence found at each VSV gene junction. This sequence was introduced into a DNA copy of the VSV genome from which infectious VSV can be derived. It contained an 11-nucleotide putative transcription stop/polyadenylation signal for the glycoprotein mRNA, an intergenic dinucleotide, and a 10-nucleotide putative transcription start sequence preceding a downstream foreign gene encoding the bacterial enzyme chloramphenicol acetyltransferase. Infectious recombinant VSV was recovered from this construct and was found to express high levels of functional chloramphenicol acetyltransferase mRNA and protein. The recombinant virus grew to wild-type titers of 5 x 10(9)/ml, and expression of the foreign gene was completely stable for at least 15 passages involving 10(6)-fold expansion at each passage. These results define functionally the transcription stop/polyadenylation and start sequences for VSV and also illustrate the utility of VSV as a stable vector that should have wide application in cell biology and vaccine development.
Schnell
M J
MJ
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA.
Buonocore
L
L
Whitt
M A
MA
Rose
J K
JK
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D017124:Conserved Sequence; D015967:Gene Expression Regulation, Viral; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012692:Serial Passage; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics; D010948:Viral Plaque Assay
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D015500:Chloramphenicol O-Acetyltransferase