STUDIES ON THE MECHANISMS OF ANTI-HIV-1 FUNCTIONS OF APOBEC3F AND APOBEC3G
Date
2017-07-12
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Thesis
Degree Level
Doctoral
Abstract
The seven human cytidine deaminases in the APOBEC3 (A3) family deaminate cytosine in single-stranded DNA to form uracil. The enzymes recognize specific di- and tri-nucleotide sequences and deaminate cytosines within them. The A3 proteins are potent antiviral restriction factors capable of inhibiting retrotransposons and both exogenous and endogenous retroviruses. The overall goal of my Ph.D. research was to biochemically characterize the A3 enzymes, APOBEC3F (A3F) and APOBEC3G (A3G) and identify the biochemical determinants of their anti-HIV-1 function. I characterized these enzymes alone and how they may act in concert to restrict HIV-1 replication. In order to inhibit HIV-1, A3 enzymes must become encapsidated into budding virions and upon infection of the next target cell, the enzymes can deaminate cytosines in HIV-1 single-stranded DNA generated during reverse transcription. The promutagenic uracils formed act as a template for second strand synthesis and result in numerous transition mutations in the double-stranded proviral DNA. These mutations inactivate the virus.
To understand how these deaminations take place, I characterized A3F and A3G biochemically. I found that like A3G, A3F is a processive enzyme that can deaminate at least two cytosines in a single enzyme-substrate encounter. Processivity is achieved through diffusional mechanisms termed sliding, jumping, and intersegmental transfer. Unlike A3G, which scans ssDNA using both sliding and jumping movements, A3F solely relies on jumping movements. Further, A3F jumping movements are distinct from A3G. We discovered that a 190NPM192 motif in A3F prevents its sliding movement since insertion of 195NPM197 into A3G decreased its sliding movements. Our data demonstrated that A3G is a more potent inhibitor of HIV-1 owing primarily to its unique DNA scanning mechanism and secondly to its deamination motif specificity. The data support a model in which the processive DNA scanning mechanism of an A3 enzyme can predict its mutagenic potential.
Since A3F and A3G are coexpressed in the CD4+ T cells that HIV-1 infects, we undertook a study to determine if A3F and A3G were coencapsidated and could be concurrently deaminating viral DNA. First, we found that an A3F/A3G hetero-oligomer can form in cells and in vitro, in the absence of RNA. This hetero-oligomer has unique biochemical properties and more efficiently deaminates cytosines compared to each A3 alone. Namely, the A3F in the A3F/A3G hetero-oligomer enhances A3G-mediated deamination. Moreover, A3F and A3F/A3G caused the accumulation of shorter reverse transcripts due to decreasing the reverse transcriptase efficiency, which would leave single-stranded (-)DNA exposed for longer periods of time enabling more deamination events to occur.
Overall my thesis research identified and characterized the mechanisms by which A3F, A3G, and A3F/A3G hetero-oligomer act as inhibitors of HIV-1. Future studies on whether hetero-oligomers of other A3s involving A3D, A3F, A3G, and A3H will be very interesting.
Description
Keywords
APOBEC3F, APOBEC3G, HIV-1, Restriction factors
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Microbiology and Immunology
Program
Microbiology and Immunology