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HIV restriction factor APOBEC3G binds in multiple steps and conformations to search and deaminate single-stranded DNA.

ABSTRACT: APOBEC3G (A3G), an enzyme expressed in primates with the potential to inhibit human immunode?ciency virus type 1 (HIV-1) infectivity, is a single-stranded DNA (ssDNA) deoxycytidine deaminase with two domains, a catalytically active, weakly ssDNA binding C-terminal domain (CTD) and a catalytically inactive, strongly ssDNA binding N-terminal domain (NTD). Using optical tweezers, we measure A3G binding a single, long ssDNA substrate under various applied forces to characterize the binding interaction. A3G binds ssDNA in multiple steps and in two distinct conformations, distinguished by degree of ssDNA contraction. A3G stabilizes formation of ssDNA loops, an ability inhibited by A3G oligomerization. Our data suggests A3G securely binds ssDNA through the NTD, while the CTD samples and potentially deaminates the substrate. Oligomerization of A3G stabilizes ssDNA binding but inhibits the CTD's search function. These processes explain A3G's ability to efficiently deaminate numerous sites across a 10,000 base viral genome during the reverse transcription process.

SUBMITTER: Morse M

PROVIDER: S-EPMC6946564 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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HIV restriction factor APOBEC3G binds in multiple steps and conformations to search and deaminate single-stranded DNA.

Morse Michael M Naufer M Nabuan MN Feng Yuqing Y Chelico Linda L Rouzina Ioulia I Williams Mark C MC

eLife 20191218

APOBEC3G (A3G), an enzyme expressed in primates with the potential to inhibit human immunodeﬁciency virus type 1 (HIV-1) infectivity, is a single-stranded DNA (ssDNA) deoxycytidine deaminase with two domains, a catalytically active, weakly ssDNA binding C-terminal domain (CTD) and a catalytically inactive, strongly ssDNA binding N-terminal domain (NTD). Using optical tweezers, we measure A3G binding a single, long ssDNA substrate under various applied forces to characterize the binding interacti ...[more]

PMID: 31850845

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Project description:The APOBEC3 deoxycytidine deaminase family functions as host restriction factors that can block replication of Vif (virus infectivity factor) deficient HIV-1 virions to differing degrees by deaminating cytosines to uracils in single-stranded (-)HIV-1 DNA. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite the uracils, thereby inducing C/G→T/A mutations that can functionally inactivate HIV-1. Although both APOBEC3F and APOBEC3G are expressed in cell types HIV-1 infects and are suppressed by Vif, there has been no prior biochemical analysis of APOBEC3F, in contrast to APOBEC3G. Using synthetic DNA substrates, we characterized APOBEC3F and found that similar to APOBEC3G; it is a processive enzyme and can deaminate at least two cytosines in a single enzyme-substrate encounter. However, APOBEC3F scanning movement is distinct from APOBEC3G, and relies on jumping rather than both jumping and sliding. APOBEC3F jumping movements were also different from APOBEC3G. The lack of sliding movement from APOBEC3F is due to an ¹⁹⁰NPM¹⁹² motif, since insertion of this motif into APOBEC3G decreases its sliding movements. The APOBEC3G NPM mutant induced significantly less mutations in comparison to wild-type APOBEC3G in an in vitro model HIV-1 replication assay and single-cycle infectivity assay, indicating that differences in DNA scanning were relevant to restriction of HIV-1. Conversely, mutation of the APOBEC3F ¹⁹¹Pro to ¹⁹¹Gly enables APOBEC3F sliding movements to occur. Although APOBEC3F ¹⁹⁰NGM¹⁹² could slide, the enzyme did not induce more mutagenesis than wild-type APOBEC3F, demonstrating that the unique jumping mechanism of APOBEC3F abrogates the influence of sliding on mutagenesis. Overall, we demonstrate key differences in the impact of APOBEC3F- and APOBEC3G-induced mutagenesis on HIV-1 that supports a model in which both the processive DNA scanning mechanism and preferred deamination motif (APOBEC3F, 5'TTC; APOBEC3G 5'CCC) influences the mutagenic and gene inactivation potential of an APOBEC3 enzyme.

Dataset Information

HIV restriction factor APOBEC3G binds in multiple steps and conformations to search and deaminate single-stranded DNA.

Publications

HIV restriction factor APOBEC3G binds in multiple steps and conformations to search and deaminate single-stranded DNA.

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