Browsing by Subject "restriction"

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    Regulation of APOBEC3B and the Restriction of HIV-1 in Myeloid Cells
    (2018-07) Molan, Amy
    The APOBEC3 (A3) DNA cytosine deaminase family comprises a fundamental arm of the innate immune response and is best known for retrovirus restriction. Several A3 enzymes restrict HIV-1 and related retroviruses by deaminating viral cDNA cytosines to uracils compromising viral genomes. Human APOBEC3B (A3B) and APOBEC3G (A3G) show strong virus restriction activities in a variety of experimental systems. A3B and A3G are also subject to tight post-translational regulation evidenced by cell-specific HIV-1 restriction activity of A3B and HIV-1 Vif-mediated degradation of A3G. After observing several potential acetylations on A3B in a mass spectrometry screen, we asked whether lysines and/or lysine post-translational modifications are required for these A3B activities. A lysine-free derivative of human A3B was constructed and shown to be indistinguishable from the wild-type enzyme in DNA cytosine deamination, HIV-1 restriction, and nuclear localization activities. However, lysine loss did render the protein resistant to degradation by SIV Vif. Taken together, we conclude that lysine side chains and modifications thereof are unlikely to be central to A3B function or regulation in human cells. HIV-1 replication in CD4-positive T lymphocytes requires counteraction of multiple different innate antiviral mechanisms. Many studies have combined to demonstrate roles for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H in HIV-1 restriction and mutation in CD4-positive T lymphocytes, whereas the APOBEC enzymes (if any) involved in HIV-1 restriction in macrophages has yet to be delineated. We show that multiple APOBEC3 genes are expressed in myeloid cell lines including THP-1. Vif-deficient HIV-1 produced from THP-1 is less infectious than Vif-proficient virus indicating the presence of at least one functional APOBEC3 enzyme. Proviral DNA resulting from such infections shows strong G-to-A mutation biases in the dinucleotide motif preferred by APOBEC3G. Moreover, Vif mutant viruses selectively sensitive to APOBEC3G show Vif-null virus-like infectivity levels and similarly strong APOBEC3G-biased G-to-A mutation spectra. These studies combine to indicate that APOBEC3G is the main HIV-1 restricting APOBEC3 family member in THP-1 cells. Overall, my thesis research provides new insights into the post-translational regulation of A3B as well as uncovering a novel restriction mechanism in myeloid lineage cells. This research provides a great backbone to build on in understanding how the HIV-1 replication cycle works in myeloid cells as well as contributing to the understanding of the post-translational regulation of A3B.