| The global health and economic burden presented by the AIDS epidemic demands the continuing pursuit of improved therapies and a preventative vaccine. Over the past 30 years of HIV research, the improved understanding of Protease, Reverse Transcriptase and Integrase viral enzymes, which facilitate cellular infection has yielded the multitude of viral enzymatic inhibitors that make up antiretroviral therapies. However, the cellular factors that facilitate or inhibit HIV in early events of cellular infection have only recently begun to be identified. These early events including cytoplasmic entry, trafficking, reverse transcription, capsid uncoating and nuclear translocation remain incompletely understood. However, the advancements in cell and molecular biology within recent years have allowed the identification of hundreds of potentially required cellular factors, while the characterization of restriction factors continues to reveal the novel mechanisms that cells utilize to respond to intracellular viral pathogens. In these studies, we used siRNAs to screen a series of previously identified HIV dependency factors with documented or predicted roles in cytoskeletal function, and characterized the nuclear-cytoplasmic shuttling of the restriction factor rhTRIM5alpha. Utilizing viruses pseudotyped with HIV-1 and VSV-G envelopes, and assaying for reverse transcription and nuclear translocation, we determined that knockdown of MAP4 and DNAL1, two microtubule associated proteins, impaired HIV-1 infection after fusion and prior to integration. Through use of a series of characterized capsid mutant viruses, we determine that MAP4 and DNAL1 knockdown do not distinctly impact infection of viruses with altered core stabilities. Furthermore, we determined that these observed effects on the HIV-1 viral life cycle are not the result of general cellular impairment. In studying the cellular localization of rhTRIM5alpha, we determined that cellular treatment with Leptomycin B, results in the nuclear retention of rhTRIM5alpha. Through generation of GFP-fusion constructs and targeted amino acid mutations, we determined that rhTRIM5alpha is actively shuttled to the nucleus, contains two CRM1-dependent nuclear export signals, and harbors a multi-partite CRM1-independent nuclear export motif. Furthermore, inhibition of nuclear export of rhTRIM5alpha leads to the generation of nuclear bodies at distinct nuclear foci. Ongoing experiments will determine the effects of nuclear-cytoplasmic shuttling of rhTRIM5alpha on HIV-1 restriction. |
