Cellular factors and the Regulation of HIV-1 Reverse Transcription

Reverse transcription is an essential step of HIV-1 replication during which the viral RNA/DNA polymerase, reverse transcriptase (RT), facilitates the conversion of the positive strand viral genome to double stranded DNA. Following entry into the host cell, the HIV-1 core that contains the viral genomic RNA and viral enzymes reverse transcriptase (RT) and integrase (IN), re-organises to form the reverse transcription complex (RTC). The RTC requires RT and IN to be active and they are believed to recruit cellular factors to facilitate DNA synthesis. More than 50 host proteins have been implicated as important for HIV reverse transcription, however whether any of these proteins are components of the RTC or affect reverse transcription indirectly remains unclear. Previous research has demonstrated that the addition of mammalian cell lysates to HIV-1 particles enhances the efficiency reverse transcription in vitro, supporting the hypothesis that host cell factors are required. The research herein identifies the host protein(s) that contribute to the previously described reverse transcription stimulatory activity and investigates their association with the viral RTC during cell infection. The addition of mammalian cell lysates to partially purified HIV-1 virions in endogenous reverse transcription (ERT) assays has previously been shown to stimulate the production of late reverse transcription products in vitro. In Chapter 2, human T-cell lysates were purified by conventional chromatography and proteins that may contribute to the previously described reverse transcription stimulatory activity were identified by mass spectrometry. Twenty five host proteins were consistently detected in highly purified active fractions including eukaryotic translation factors eEF1A and eEF1G. The aforementioned host proteins are highly conserved and essential subunits of the eukaryotic translation elongation factor 1 (eEF1) complex that recruits aminoacyl-tRNAs onto the ribosome during protein synthesis. In Chapter 3, eEF1A and eEF1G are demonstrated to be co-factors of in vitro HIV-1 reverse transcription as their immunodepletion ablated the ability of active lysate fractions to stimulate reverse transcription. Furthermore, down regulation of the cellular levels of eEF1G in target cells is shown to cause a significant decrease in efficiency of reverse transcription, suggesting that this protein and/or additional subunits of the eEF1 complex are important for HIV-1 reverse transcription during cell infection. Interactions between the eEF1 complex and HIV-1 RTC are investigated in Chapter 4. eEF1A and eEF1G are shown to interact with HIV-1 RT and IN following endogenous reverse transcription, as well as associate with purified RTCs and co-localise with RT during cell infection. These findings provide the first evidence that subunits of the eEF1 complex are components of the HIV-1 RTC and supports the hypothesis that one or more components of the eEF1 complex are required for efficient HIV-1 reverse transcription. Taken together, the results of this study demonstrate for the first time that subunits of the human eEF1 complex are required to stimulate the late stages of HIV-1 reverse transcription and directly associate with the viral RTC during infection. The identification of novel co-factors of reverse transcription provide new insights into HIV-1 replication and represents a new target for the development of anti-HIV therapy