Genomic stability of murine leukemia viruses containing insertions at the Env-3 ' untranslated region boundary

Retroviruses containing inserts of exogenous sequences frequently eliminate the inserted sequences upon spread in susceptible cells, We have construct ed replication-competent murine leukemia virus (MLV) vectors containing int ernal ribosome entry site (IRES)-transgene cassettes at the env-3' untransl ated region boundary in order to examine the effects of insert sequence and size on the loss of inserts during viral replication, A virus containing a n insertion of 1.6 kb replicated with greatly attenuated kinetics relative to wild-type virus and lost the inserted sequences in a single infection cy cle. In contrast, MLVs containing inserts of 1.15 to 1.30 kb replicated wit h kinetics only slightly attenuated compared to wild-type MLV and exhibited much greater stability, maintaining their genomic integrity over multiple serial infection cycles. Eventually, multiple species of deletion mutants w ere detected simultaneously in later infection cycles; once detected, these variants rapidly dominated the population and thereafter appeared to be ma intained at a relative equilibrium. Sequence analysis of these variants ide ntified preferred sites of recombination in the parental viruses, including both short direct repeats and inverted repeats. One instance of insert del etion through recombination with an endogenous retrovirus was also observed . When specific sequences involved in these recombination events were elimi nated, deletion variants still arose with the same kinetics upon virus pass age and by apparently similar mechanisms, although at different locations i n the vectors. Our results suggest that while lengthened, insert-containing genomes can be maintained over multiple replication cycles, preferential d eletions resulting in loss of the inserted sequences confer a strong select ive advantage.