FUNCTIONAL DOMAINS OF MOLONEY MURINE LEUKEMIA-VIRUS INTEGRASE DEFINEDBY MUTATION AND COMPLEMENTATION ANALYSIS

Retroviral integrases perform two catalytic steps, 3' processing and s trand transfer, that result in the stable insertion of the retroviral DNA into the host genome. Mutant M-MuLV integrases were constructed to define the functional domains important for 3' processing, strand tra nsfer, and disintegration by in vitro assays. N-terminal mutants had n o detectable 3' processing activity, and only one mutant which lacks t he HHCC domain, N Delta 105, had strand transfer activity. Strand tran sfer mediated by N Delta 105 showed preference for one site in the tar get DNA. Disintegration activity of N-terminal mutants decreased only minimally. In contrast, all C-terminal mutants truncated by more than 28 amino acids had no integration or disintegration activity. Activity on a single-strand disintegration substrate did not require a functio nal HHCC domain but did require most of the C-terminal region. Complem entation analysis found that the HHCC region alone was able to functio n in trans to a promoter containing only the DD(35)E and C-terminal re gions and to enhance integration site selection. Increasing the reduci ng conditions or adding the HHCC domain to N Delta 105 reaction mixtur es restored the wild-type strand transfer activity and range of target sites. The reducing agent affected Cys-209 in the DD(35)E region. The presence of C-209 was required for complementation of N Delta 105 by the HHCC region.