ACTIVITIES AND SUBSTRATE-SPECIFICITY OF THE EVOLUTIONARILY CONSERVED CENTRAL DOMAIN OF RETROVIRAL INTEGRASE

The retroviral integrase (IN) is a virus-encoded enzyme that is essent ial for insertion of viral DNA into the host chromosome. In order to m ap and define the properties of a minimal functional domain for this u nique viral enzyme, a series of N- and C-terminal deletions of both Ro us sarcoma virus (RSV) and human immunodeficiency virus (HIV) INs were constructed. The RSV IN deletion mutants were first tested for their ability to remove two nucleotides from the end of a substrate represen ting the terminus of viral DNA in order to assess the contribution of N and C regions towards this reaction, referred to as processing. The results suggest that C-terminal amino acids of the intact RSV protein are required to maintain specificity of the processing reaction. Thoug h deficient for processing, the RSV deletion mutants exhibited a secon dary endonucleolytic activity that was indistinguishable from that of wild-type IN, demonstrating that all retained some enzymatic activity. RSV, and a larger set of HIV-1, IN deletion mutants were then tested for their ability to perform an intramolecular, concerted cleavage-lig ation reaction using an oligodeoxynucleotide substrate that mimics the intermediate viral-host DNA junction found prior to the final step of covalent closure. The composite results from such analyses define a m inimal functional central region of similar to 140 amino acids for eac h enzyme that includes the highly conserved D,D(35)E domain. Results w ith HIV-1 and HIV-2 IN also indicate that the efficiency of concerted cleavage-ligation depends upon the presence of CA/GT base pairs within the viral component of the DNA substrate at the reaction site. Even t he isolated central region of HIV-1 IN exhibited this sequence require ment for optimal activity. We conclude that this evolutionarily conser ved central region of IN not only encodes residues that are required f or the catalytic activity of the enzyme but also harbors some or all o f the determinants responsible for recognition of the CA/GT dinucleoti des that are present at the ends of all retroviral DNAs. (C) 1995 Acad emic Press, Inc.