Analysis of the interaction of HIV-1 reverse transcriptase and integrase proteins
Hehl, Eric Adam
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HIV-1 Reverse Transcriptase (RT) and Integrase (IN) proteins are proteolytically separated during maturation of the virus. However several retroviruses such as AKR Murine Leukemia Virus (AKR-MuLV), Avian Sarcoma Leukosis Virus (ASLV) and Human T-cell Leukemia Virus-1 (HTLV-1) all show evidence of a physical association between Reverse Transcriptase (RT) and Integrase (IN). In addition, the association of RT and IN may be evolutionarily conserved as seen in the retrotransposon R2Bm.;In an attempt to investigate if this association occurs in HIV-1 as well, we created glutathione-S-transferase-IN (GST-IN) fusion proteins and via GST 'pull down' assays, we were able to show that GST-IN is able to specifically bring down RT from bacterially expressed crude lysates. HIV-1 IN needs to oligomerize in order to be functionally active. Studies using an oligomerization-deficient mutant of IN, V260E, showed that IN oligomerization is not required for interaction with RT. A similar study was performed using a heterodimerization-defective mutant of RT, L234A. The results indicated that in the absence of heterodimerization of RT, the RT-IN interaction was diminished.;In an attempt to localize the interaction domains on each protein, nested deletions of RT and IN were generated. RT has been described as resembling a right hand containing Fingers, Palm, Thumb, Connection and RNase H domains. Deletion analysis has shown that IN binds to two separate subdomains on RT; Fingers-Palm and the carboxy terminal portion of Connection.;Several mutations in IN, when introduced into viruses, have been shown to cause a reduction in viral DNA synthesis. Wu et al. (2000) proposed that this defect may be due to an inability for RT and IN to interact with each other. In an attempt to further investigate this proposal, we wished to identify mutations that confer an RT-IN interaction-negative phenotype. Functional assays were performed to investigate any influence either may exert on the activity of the other protein.;In order to investigate whether IN has any influence on reverse transcription in vitro, RT processivity assays were performed in the absence and presence of IN. No effect of IN on RT processivity was observed. We tested to see whether RT influences IN's 3' end processing and joining activity. Although no effect was observed for 3' cleavage, RT significantly stimulated in vitro joining by IN. (Abstract shortened by UMI.).