Functional Roles of Human Immunodeficiency Virus Type 1 Integrase During Reverse Transcription

Samson Chow, UC-Los Angeles
Biomedical and Clinical Sciences

After cell entry, the RNA genome of retroviruses is converted to a cDNA copy by reverse transcriptase (RT).This viral cDNA, incorporated into a nucleoprotein complex termed the preintegration complex, enters the nucleus and is integrated into the host cell chromosome. Retroviral integrase (IN) catalyzes the integration of viral DNA. Both reverse transcription and integration are essential steps in the viral life cycle. The function of IN is not restricted to integration. For human immunodeficiency virus type 1 (HIV-1) certain mutations of IN, such as substituting the Cys residue at position 130 of IN to Ser (NL-C130S), can cause a specific defect in reverse transcription, but the underlying mechanism for this defect is poorly understood. In vitro, IN and RT can physically interact, and we have determined previously by co-immunoprecipitation that HIV-1 RT interacts with the C-terminal domain of IN. We hypothesize that the IN-RT interaction is functional and critical for reverse transcription. To confirm that the lack of the reverse transcription is caused by the IN mutation, wild type (WT) IN was packaged into the NL-C130S mutant virions by the in-trans method. Incorporation of IN restored reverse transcription to 25% of the WT virus. To understand the mechanism by which IN stimulates reverse transcription, we employed cell-free reverse transcription assays to examine the effects of IN on the RNA- and DNA-dependent polymerase activity of RT. Our preliminary results showed thatIN can stimulate both the initiation and elongation mode of reverse transcription. In the presence of 10 molar excess IN, initiation and elongation products increased nearly 3-and 2-fold, respectively. In addition to initiation and elongation, IN may help stimulate the early steps of reverse transcription by promoting annealing of the tRNA primer to the template, stabilizing the RT-templateprimer ternary complex, or destabilizing secondary structures of the RNA primer and template, all of which could lead to an increase in initiation and extension products. We are currently investigating such possibilities with aims to identify the critical step that IN is required during early events of reverse transcription. The experiments will shed light on the interaction between two key retroviral enzymes and the effect of such an interaction on the essential step of reverse transcription. Characterizing the RT-IN interaction and determining its biological significance may reveal new functional roles for IN as well as potential targets for devising new approaches to inhibit viral replication.