Coupled integration of human immunodeficiency virus type 1 cDNA ends by purified integrase in vitro: stimulation by the viral nucleocapsid protein.

Integration of retroviral cDNA involves coupled joining of the two ends of the viral genome at precisely spaced positions in the host cell DNA. Correct coupled joining is essential for viral replication, as shown, for example, by the finding that viral mutants defective in coupled joining are defective in integration and replication. To date, reactions with purified human immunodeficiency virus type 1 (HIV-1) integrase protein in vitro have supported mainly uncoupled joining of single cDNA ends.

Chromosome structure and human immunodeficiency virus type 1 cDNA integration: centromeric alphoid repeats are a disfavored target.

Integration of retroviral cDNA into host chromosomal DNA is an essential and distinctive step in viral replication. Despite considerable study, the host determinants of sites for integration have not been fully clarified. To investigate integration site selection in vivo, we used two approaches.

Human immunodeficiency virus type 1 nucleocapsid protein specifically stimulates Mg2+-dependent DNA integration in vitro.

The integrase (IN) protein of the human immunodeficiency virus mediates integration of the viral DNA into the cellular genome. In vitro, this reaction can be mimicked by using purified recombinant IN and model DNA substrates. IN mediates two reactions: an endonucleolytic cleavage at each 3' end of the proviral DNA (terminal cleavage) and the joining of the linear viral DNA to 5' phosphates in the target DNA (strand transfer).

Impairment of Moloney murine leukemia virus integration in a cell line underexpressing DNA topoisomerase II.

The possible intervention of nuclear proteins as cofactors of integrase-catalyzed integration of retroviral DNA into the host cell genome is not fully understood. Among various nuclear proteins, DNA topoisomerase II appears to be a plausible candidate. This hypothesis is supported by a series of evidence, including the fact that integration is markedly affected by the topology of the target DNA and mainly occurs in transcribed regions in which topoisomerase II is preferentially located.