Browsing by Subject "Genetics, Cell Biology, and Development BS"

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    Regulation of the Human Cell Cycle by HIV-1 Accessory Protein Vif
    (2017) Freeman, Madeline;
    HIV-1 is transmissible lentivirus that infects millions of people worldwide and causes progressive immunodeficiency. The accessory proteins Nef, Vpr, Vpu, and Vif set HIV-1 apart from other retroviruses. Each promotes HIV survival and propagation by counteracting one or more host-encoded antiviral proteins. In its canonical role, Vif functions by recruiting the cellular CUL5-ELOB/C ubiquitin ligase complex to degrade members of the APOBEC3 protein family of DNA cytosine dreaminess. Vif also induces G2 cell cycle arrest by an unclear, only partially understood mechanism. This research takes a comprehensive approach, via a large semi-randomized mutant Vif library, containing ~100,000-200,000 unique Vif sequences, to identify and clarify the amino acid residues in Vif necessary for the interaction with host factors that lead to host G2 cell cycle arrest. Here we show a-helix 1 may be a key structure in Vif’s role in G2 cell cycle arrest. A cell cycle selection screen was performed in CD4+ T cells (SupT) using the large semi-randomized mutant Vif library over a 6-week time course to enrich for vif sequences with a loss in ability to induce G2 arrest. Sanger sequencing on a partial sample showed enrichment of frameshift mutations and depletion of wild type Vif. A subset of selected mutagenic Vif clones were functionally validated for loss of arrest phenotype. Whole population Illumina sequencing showed enrichment for non-functional mutants in the a-helix 1 region. We hypothesize helix 1 is a docking station for a host cell factor, enabling the host cell factor to interact with two soluble loop domains. Single amino acid substation Vif clones were generated for a-helix 1 amino acid residues. Preliminary results showed each single amino acid substitution Vif clones had reduced ability to induce G2 cell cycle arrest. This finding has led to current work to isolate the precise amino acids in a-helix 1 responsible for a possible interaction. We anticipate this work to contribute to the effort to isolate and identify the key host cell proteins that interact with Vif to induce G2 cell cycle arrest.