| Human immunodeficiency virus type-1 integrase (IN) catalyzes the essential step of integrating double-stranded viral DNA into the host cell chromosome. Besides integration, certain mutations of IN cause defects at multiple steps during viral replication, but the mechanism for the pleiotropic effects are not well understood. We previously found that HIV-1 IN plays an important role during the reverse transcription step of the viral life cycle, possibly through physical interactions with reverse transcriptase (RT).; HIV-1 IN mutant viruses that bear a Cys-to-Ser substitution at position 130 of IN are defective in viral replication. Extensive biochemical studies showed that the entry and postintegration steps of the viral life cycle were normal and viruses with a C130S mutation retained particle-associated reverse transcriptase activity. However, early reverse-transcribed DNA products were absent in the lysate of cells infected with the C130S mutant virus, indicating that the mutation abolished the ability of the virus to initiate intracellular reverse transcription. Coimmunoprecipitation studies using purified IN and RT proteins revealed that the C-terminal domain of IN interacts with RT. Notably the C130S mutation disrupted the interaction, whereas other IN mutants with no effect on reverse transcription retained their ability to interact with RT.; In an attempt to understand the mechanism(s) whereby IN affects this process, we employed a cell-free reverse transcription assay designed to recapitulate endogenous reverse transcription. To this end, reverse transcription reactions were carried out using HIV-1 RNA sequences as the template, natural tRNA 3Lys as the primer, recombinant RT, and varying amounts of IN to monitor the synthesis of early reverse transcription products. Our results showed that IN specifically stimulated the early events of reverse transcription in vitro. Furthermore, IN continued to stimulate RNA- and DNA-dependent polymerization of full length viral cDNA products in the presence of a RT trap. Taken together, our results suggest that IN acts at early steps of reverse transcription by augmenting the processive activity of RT, possibly through functional interactions with RT. Reverse transcription is a critical step in viral replication, and therefore understanding IN role at this step may reveal potential targets for devising new approaches to inhibit viral replication. |
