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Genetic analysis of HIV-1 integrase protein.
Taddeo B; Shin CG; Haseltine WA; Farnet CM; Dana-Farber Cancer
December 30, 1995
Natl Conf Hum Retroviruses Relat Infect (1st). 1993 Dec 12-16;:126.

Analysis of amino acid sequences of a number of retroviral integrases have demonstrated the presence of conserved motifs that may be important for integrase activity. The most highly conserved region is the middle part of protein, known as the D,D(35)E region, that is believed to contain the catalytic site of integrase. In order to analyze effects of integrase point mutations on viral protein synthesis, virion morphogenesis, and viral replication, point mutations in selected amino acids in the central region of HIV-1 integrase were introduced. Nine integrase point mutants of the infectious molecular clone pHXBc2 were constructed. We observed: 1) mutation of two amino acids that are invariant among retroviral integrases, E152 and D116, as well as mutation of the highly conserved amino acid S147, blocked viral replication in two CD4+ human T cell lines; 2) mutations of four other highly conserved amino acids in the region had no effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with delayed replication phenotypes; 3) defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants. These data suggest that mutant integrase proteins can interfere with a variety of steps in the early and late stages of viral replication cycle. Integrase mutations must be carefully assessed for their effects on all aspects of viral replication before a particular phenotype can be assigned to a specific defect in integrase function. We are now establishing stable virus- producing cell lines for all of the replication-defective mutants in order to analyze their effects on the rate of viral DNA synthesis, viral DNA end processing, nuclear entry and formation of circular viral DNA to pinpoint the basis of the altered phenotypes.

Cell Line CD4-Positive T-Lymphocytes/VIROLOGY *DNA Nucleotidyltransferases/*GENETICS Human HIV-1/*ENZYMOLOGY/PHYSIOLOGY Phenotype Point Mutation Virus Replication ABSTRACT