Completion of Avian Retroviral DNA Replication Intermediates Inhibited by Antisense RNA

Abstract

High level expression of RNA complementary to either neo r or src sequences located near the 3′ end of recombinant retroviral vectors derived from Rous sarcoma virus inhibited viral replication. Stable integration of proviral DNA was not detected in the presence of antisense RNA. We investigated the mechanism of this inhibition by determining the structure of unintegrated viral DNA (vDNA) intermediates accumulating in the presence of the anti-sense RNA. The major vDNA intermediate detected was a full-length duplex linear molecule with complementary single-stranded long terminal repeats (LTRs). These vDNA linears could be joined directly by T4 DNA ligase to form junctions which contained a single normal LTR, These results can be explained by arrest of linear vDNA formation before strand displacement results in completion of the LTRs. Isolation of these sticky-ended intermediates as linear rather than nicked circular molecules suggests that these complementary vDNA LTR segments were not hydrogen bonded in the infected cell and further implies that completion of LTR synthesis is an ordered, controlled process.