Research Article: Different Mutagenic Potential of HIV-1 Restriction Factors APOBEC3G and APOBEC3F Is Determined by Distinct Single-Stranded DNA Scanning Mechanisms

Date Published: March 20, 2014

Publisher: Public Library of Science

Author(s): Anjuman Ara, Robin P. Love, Linda Chelico, Christopher Aiken.

http://doi.org/10.1371/journal.ppat.1004024

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.

Partial Text

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]. Research has been highly focused on primarily A3G and secondarily A3F for a number of years since they appeared to be the most efficient restrictors of HIV replication [3], [4], [5], [6], [7]. Although there are documented restrictive effects of A3G, and possibly A3F, at an individual level (reviewed in [8]), the suppression of HIV by A3G and A3F at a population level is lost due to the HIV protein Vif (viral infectivity factor) [6], [9]. Vif forms an E3 ubiquitin ligase with host proteins and causes A3G and A3F polyubiquitination and degradation through the proteasome [6], [10], [11], [12], [13], [14].

Reports have demonstrated that A3F is less effective than A3G at restricting HIV replication and leaves less of a mutational footprint [22], [23], [24], [29]. This could be due to many reasons such as differences in mRNA/protein expression levels [22], [31], virion encapsidation levels [4], [33], deamination site preference [4], [18], or the inherent biochemical characteristics of the enzymes that govern deamination activity during proviral DNA synthesis. There is no consensus in the literature regarding whether any of the variables determined by cellular conditions, e.g., mRNA expression levels, create disparity between A3F and A3G HIV restriction activities. In addition, other reports have found an equal capacity of A3F and A3G to restrict HIV [3], [5], [6], [7], [20], [21], [30]. To account for these differences in the literature we undertook a biochemical characterization of A3F in comparison to A3G. The data have enabled us to form a biochemical model to account for cell-based observations and propose that the processive DNA scanning mechanism and the preferred deamination motif of A3 deoxycytidine deaminases are determinants of HIV restriction efficiency.

 

Source:

http://doi.org/10.1371/journal.ppat.1004024

 

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