Mechanisms of loss of foreign gene expression in recombinant vesicular stomatitis viruses

We investigated the stability and mechanisms of loss of foreign gene expres sion In two recombinant vesicular stomatitis viruses (VSVs), A recombinant expressing the cellular CD4 protein exhibited remarkable stability of forei gn gene expression. However, after 26 sequential passages, a mutant no long er expressing CD4 was recovered from the virus stock, Sequencing of the CD4 coding region in this mutant revealed a single nucleotide deletion causing a frameshift and termination of protein synthesis. A second VSV recombinan t expressing the measles virus F protein grew poorly and exhibited extreme instability of expression of the F protein. Expression of F protein was los t rapidly through mutations of the upstream transcription termination site from (3')AUAC(5') to (3')AUAU(5'), as well as lengthening of the subsequent U-7 tract that is the template for poly(A) addition to VSV G mRNA. Such mu tations resulted in fusion of the F mRNA to the 3' end of the G mRNA, makin g the F protein translation initiation codon inaccessible. We suggest that the VSV polymerase is error prone during replication of the U7 tract, provi ding a rapid means for complete elimination of expression of proteins that are toxic to the virus life cycle. (C) 2001 Academic Press.