|dc.description.abstract||The human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus whose genome encodes 9 genes coding for 15 different proteins. Many of these proteins are required to facilitate the hallmark process of retroviral replication, reverse transcription. Reverse transcription is a complex, multistep process, during which the virus converts single stranded RNA genome into a double stranded DNA, which can then integrate into the host cell genome. Many studies have indicated that reverse transcription is a tightly-regulated process, with multiple factors contributing to this regulation, both viral and cellular. The research herein examines how different factors affect this process. RNA factors have long been believed to play a role in regulating reverse transcription, although the precise mechanisms by which they may regulate the process has never been fully characterised. Chapter two describes for the first time an RNA element called the repressor of reverse transcription (RRT) that appears to downregulates initiation of reverse transcription immediately following viral entry into the cell. Loss of this repression results in severe replication defects in cells. Results suggest that this loss of replication is due to defective formation of the viral pre-integration complex following completion of reverse transcription, resulting in degradation of the viral cDNA following entry into the nucleus. This study potentially explains previous observations that there is a lag between viral entry into the cell and initiation of reverse transcription, as well as showing that deregulation of reverse transcription is detrimental to viral replication.
Multiple studies have shown that protein interactions with RNA elements are required for efficient reverse transcription to occur. Chapter three investigates the role of the viral Tat protein in regulating reverse transcription. Multiple studies have suggested a role for Tat in reverse transcription, but there are conflicting views on what that role may be. In this study an in vitro assay was established to mimic initiation of reverse transcription in a cell-free environment. The results showed that at low concentrations, Tat upregulates initiation of reverse transcription, but at high concentrations it switches to suppression of the same process. The alternate effects of 8 the Tat protein at high and low concentrations could explain the discrepancy observed between various studies. Further analysis showed that Tat does not interact with the RNA template to mediate the effect but instead interacts with the RT protein directly, possibly stabilising the heterodimeric form of the protein to allow more efficient initiation to occur. Upregulation required a number of Tat domains, including the basic domain, which was surprising since previous studies indicated that this domain was not required for Tat to mediate reverse transcription.
The basic domain of Tat is required to mediate several Tat functions. It contains an arginine-rich motif (ARM) that contains the RNA binding domain, as well as an extra-cellular receptor interaction domain, and the nuclear localisation signal that is required for Tat accumulation in the nucleus. Chapter three showed that the basic domain is also required for reverse transcription to occur, a finding that was expanded upon in chapter four. A transdominant mutant of Tat, Nullbasic, was made, and the effects on viral replication analysed. The results showed that expression of Nullbasic significantly suppressed viral production from cells, caused by decreased Env and Gag expression. Nullbasic was found to inhibit Rev mediated export of viral mRNA, resulting in the observed reduction in proteins expressed from unspliced or singlyspliced mRNA transcripts. Furthermore, virus produced from cells expressing Nullbasic was poorly infectious. These viruses were found to be highly defective for reverse transcription, indicating that Nullbasic is impairing efficient initiation of reverse transcription in the virion. The multiple inhibitory effects of Nullbasic on viral replication indicated that it was a possible candidate for treatment of viral infection.
To test this principle, Nullbasic was expressed in a reporter cell line and then challenged with high dose HIV-1. Interestingly, Nullbasic caused a greater than 800- fold decrease in viral replication, indicating that it may potentially be useful as a therapeutic in the future, however there are significant obstacles to be overcome before this becomes a reality.
Overall, this study addresses the role of multiple factors in regulating reverse transcription, while highlighting multiple regions that could provide potential therapeutic applications.||