Proteomic Analysis of HIV-1 Viral Cores and the Identification of HIV-1 Replication Modulators

During the course of HIV viral assembly and budding, an abundance of cellular proteins are incorporated into virus particles. The importance of these cellular proteins has become of great interest in recent years, with the discovery of multiple restriction factors, along with the fact that some cellular factors can modulate the early stages of HIV-1 replication. Many cellular proteins have been described previously via traditional biochemical methods and will be discussed in this dissertation. However, the functions of many other cellular factors incorporated into viruses have not been elucidated, such as whether or not these factors can affect the reverse transcription complex (RTC) and its eventual nuclear import. A mechanism that remains to be completely understood and identifying the cellular factors involved could assist with fully understanding how the reverse transcription complex is formed and imported into the nucleus.;In this dissertation, through the use of proteomics, we will describe how pure viral cores were isolated and their proteomes identified, in particular within viruses derived from monocytes, macrophages and T cells. Taking this a step further, to help shed some light on how cellular factors can affect the RTCs within an infected cell, we have identified two potential modulators of the HIV-1 life cycle, the first of which, MCM5 -- mini-chromosome maintenance complex component 5, was identified by observing cellular factors that were not randomly incorporated amongst differing cell types. Further analysis revealed that MCM5 binds to Gag, and that when MCM5 levels are manipulated in the producer cell reverse transcription is impaired, potentially through alteration of the RTC infrastructure. The second factor, inter-alpha-trypsin inhibitor isoform 4 (ITIH4) was identified within the viral cores of a unique cell clone that is non-permissive to HIV-1 infection, except in the presence of Vpr. This suggested that this might be the elusive cellular factor that Vpr is counteracting. Further analysis demonstrated that Vpr does indeed reduce ITIH4 expression and is inducible by interferon-alpha. Mechanistic analysis showed that ITIH4 works by dramatically decreasing HIV-1 protein expression. Preliminary evidence suggests that HIV transcription is targeted, potentially via transcription factors, but the exact mechanism remains to be determined and warrants further study.