Determinants of HIV-1 Restriction by the A3G Complex
Creators: name: University Of California Office Of The President
Publisher: name: University of California Office of the President
publisherIdentifier: https://ror.org/00dmfq477
publisherIdentifierScheme: ROR
Year: 2024

description: For more than twenty years, HIV biologists have puzzled over why resting CD4 T cells so effectively resist infection by HIV. This block could be due to a lack of a key factor or perhaps an active mechanism of anti-viral defense. Recent studies in Dr. Warner Greene’s laboratory have revealed the presence of an active defense system in these cells involving the antiviral host factor APOBEC3G (A3G). Resting CD4 T cells express a low molecular weight (LMW) form of A3G that displays the ability to mutate viral DNA. However, when T cells are activated, LMW A3G shifts into an enzymatically inactive HMW complex comprised of A3G, host cofactors, and an RNA. The formation of such HMW A3G complexes could explain why activated CD4 T cells are permissive for HIV infection. To formally test the possibility that LMW A3G blocks HIV infection in resting CD4 T cells, short interfering A3G RNAs (siRNAs) were introduced. These siRNAs effectively reduced A3G expression in these cells. Strikingly, this siRNA treatment rendered these resting CD4 T cells permissive for HIV infection squarely implicating LMW A3G as an active shield against HIV infection in these resting cells. LMW A3G acts by slowing down the reverse transcription reaction in the resting CD4 T cells. Yet, it remained unknown whether the DNA-mutator activity of A3G was required for its antiviral action. When reverse transcripts from these cells were sequenced, only low levels of viral mutation were observed raising the possibility that DNA mutation was not the key. A strong second possibility is the ability of A3G to bind RNA. LMW A3G binding to the viral RNA might slow the reverse transcriptase. Alternatively, it is also possible that A3G-mutated viral DNAs are very rapidly degraded in resting CD4 T cells. If so, the sequencing result might underestimate the importance of DNA mutation in the anti-HIV effect of LMW A3G. In this proposal, I will determine precisely how LMW A3G blocks HIV infection in resting CD4 T cells. I will prepare mutants of A3G that lack either DNA mutator activity or RNA binding activity. siRNA-resistant versions of these mutants will be prepared so that they can be used in the presence of the A3G siRNA. These A3G mutants will be expressed in resting CD4 T cells at the same time that endogenous A3G is depleted by the A3G siRNA (Specific Aim 1). These studies should determine whether the RNA binding or DNA mutator activity of A3G is the key to A3G’s antiviral activity in these resting cells. In a second line of investigation, I will prepare and test a broader range of A3G mutations to identify parts of the protein that are essential for recruitment into the inactive HMW RNA-protein complexes (Specific Aim 2). These mutants will also be studied for their ability to form HMW complexes, to dimerize, to be recruited into HIV virions, to mutate DNA, to bind RNA, and to assemble into morphologically interesting subcellular structures termed “cytoplasmic bodies.” Together, these studies should provide key insights into the mechanism of action of LMW A3G and define key structure-function relationships in the A3G protein that are important for its successful recruitment into inactive HMW RNA-protein complexes. By understanding the nature of HMW complex formation, it might be possible to identify agents that promote complex disassembly thereby conferring protection against HIV infection in normally permissive cells by the expression of active LMW forms of A3G.