Abstract
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.
Highlights
APOBEC3F (A3F) and APOBEC3G (A3G) are members of a family of seven single-strandedDNA cytosine deaminases (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) [1] and play a role in restriction of the retrovirus HIV-1 [2]
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, 59TTC; APOBEC3G 59CCC) influences the mutagenic and gene inactivation potential of an APOBEC3 enzyme
There are particular enzymes, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H, that appear to be most relevant to restricting HIV-1 replication in CD4+ T cells using this mutagenic mechanism, if they can avoid degradation that is induced by the HIV-1 protein Vif
Summary
APOBEC3F (A3F) and APOBEC3G (A3G) are members of a family of seven single-stranded (ss)DNA cytosine deaminases (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) [1] and play a role in restriction of the retrovirus HIV-1 (referred to as HIV) [2]. The general mechanism by which A3G restricts HIV, which has been a paradigm for other A3 enzymes, requires that it be encapsidated with the ribonucleoprotein complex of HIV [9,15]. In the target cells that these virions infect, encapsidated A3G can deaminate cytosines to uracils in (2)DNA reverse transcribed from the RNA genome, after the reverse transcriptase associated RNaseH activity enables ssDNA regions on the (2)DNA to be accessed by the enzyme [18,19]. The deaminases A3D, A3F, and A3H appear to follow this general mechanism of restriction in cell culture, but to differing degrees than A3G [3,4,20,21,22,23,24]. The exceptions are A3A, which inhibits incoming HIV viral particles in myeloid lineage cells [25,26], A3C, which does not appear to become encapsidated or restrict HIV in cell culture [20,27], and A3B, which can restrict HIV in 293T and HeLa cells, but not SupT1 cells [20,28]
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