MUTATION OF POLYADENYLATION SIGNALS GENERATES MURINE RETROVIRUSES THAT PRODUCE FUSED VIRUS-CELL RNA TRANSCRIPTS AT HIGH-FREQUENCY

Retroviruses act as insertional mutagens and can also capture cellular sequences through a mechanism which initially requires the generation of RNA transcripts which fail to cleave and polyadenylate correctly. The correct termination of retroviral transcripts at the 3' LTR R/U5 j unction is primarily dependent on the canonical AAUAAA polyadenylation signal, so we have analyzed the effect of mutating the polyadenylatio n signal sequences on the properties of a selectable murine retroviral vector. Mutation of consensus polyadenylation signal sequences in the 5' and/or 3' proviral LTRs demonstrated that a UA to CC change genera ted larger sized virus-specific RNA, consistent with loss of normal po lyadenylation. Cell clones infected with viruses generated by proviral constructs containing this mutation in the 5' LTR express either norm al-length or elongated viral RNA. Fused transcripts contained the muta nt polyadenylation signal, while sequence analysis was consistent with the hypothesis that premature 5' to 3' primer strand transfer was res ponsible for the high frequency (80%) of wild-type polyadenylation. Ce lls infected by viruses from constructs mutated in both 5' and 3' prov iral LTRs expressed poly(A)(+) viral RNA between 0.3 and 3 kb larger t han normal virus in 100% of infected clones, and sequence analysis of clones derived from either infected rodent or human cells confirmed th at these transcripts contained both viral and adjacent cellular sequen ces. While mutant virus exhibits no increased ability to alter cell ph enotypes, the read-through transcripts contain both unique and repetit ive cell-derived sequences and can easily be recovered using PCR techn iques, suggesting that these viruses may serve as effective tools for rapidly cloning cellular sequences and generating random genomic marke rs for gene mapping. (C) 1998 Academic Press.