Moloney murine leukemia virus integrase protein augments viral DNA synthesis in infected cells

Mutations in the IN domain of retroviral DNA may affect multiple steps of t he virus life cycle, suggesting that the IN protein may have other function s in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient vi ral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this repo rt, we characterized the structure and function of the Moloney murine leuke mia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector cont aining green fluorescent protein as a sensitive reporter for virus infectio n, we found that mutations in either the catalytic triad (D184A) or the HHC C motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations t hat deleted the entire IN (Delta IN) or 34 C-terminal amino acid residues ( Delta 34) were more severely defective, with infectivity levels consistentl y reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteol ytic processing of the Gag and Pol precursor proteins. Using semiquantitati ve PCR to analyze viral cDNA synthesis in infected cells, we found the Delt a 34 and Delta IN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthe sis defect was rescued by complementing the Delta 34 and Delta IN mutants i n trans with either wild-type IN or the D184A mutant IN, provided as a Gag- IN fusion protein. However, the DNA synthesis defect of Delta IN mutant vir ions could not be complemented with the Delta 34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is requir ed for efficient viral DNA synthesis and that this function may be conserve d among other retroviruses.