A Model for Oligomeric Regulation of APOBEC3G Cytosine Deaminase-dependent Restriction of HIV

Abstract

APOBEC3G (A3G) restricts HIV-1 infection by catalyzingprocessive C 3 U deaminations on single-stranded DNA (ssDNA) with marked 3 3 5 deamination polarity. Here we show that A3G exists in oligomeric states whose composition is dictated primarily by interactions with DNA, with salt playing an important, yet secondary, role. Directional deaminations correlate with the presence of dimers, tetramers, and larger oligomers observed by atomic force microscopy, and random deaminations appear to correlate mainly with monomers. The presence of a 30-nt weakly deaminated "dead" zone located at the 3-ssDNA end implies the presence of a preferred asymmetric direction for A3G catalysis. Single turnover reaction rates reveal a salt-dependent inhibition of C deamination toward the 3-ssDNA region, offering a molecular basis underlying A3G deamination polarity. Presteady state analysis demonstrates rapid diffusionlimited A3G-ssDNA binding, a slower salt-dependent conformational change, possibly indicative of DNA wrapping, and long (5-15 min) protein-DNA complex lifetimes. We suggest that diverse A3G oligomerization modes contribute to the human immunodeficiency virus, type 1, proviral DNA mutational bias.In 2002, Sheehy et al.(1) determined that APOBEC3G (A3G), originally called CEM15, a proposed cytidine deaminase based on sequence analysis, is the nonpermissive host factor that blocks virion infectivity factor-defective (⌬vif) HIV-1 infection of T cells. The experimental determination that CEM15 was a cytidine deaminase belonging to the APOBEC family followed soon after with experiments demonstrating G 3 A-induced hypermutation of proviral DNA in a ⌬vif HIV-1 virion (2, 3). A3G has a duplicated deaminase domain structure, but only the C-terminal domain is responsible for the single-stranded DNA (ssDNA) 2 deamination activity (4, 5).Apart from A3G-catalyzed deamination, A3G may also have the capacity for blocking reverse transcription, (ϩ)-DNA synthesis, and provirus formation either by interacting with RNA or DNA of HIV-1 (6 -8) or by possibly interacting with HIV-1 proteins (9, 10). However, these noncatalytic effects on HIV inhibition may be attributable to the overexpression of A3G and may not be occurring during normal infection (11-13).It is important to bear in mind that any actions of A3G on DNA, catalytic and possibly noncatalytic, are balanced in vivo against cellular RNA binding, which forms a high molecular mass A3G-RNA complex, which may prevent A3G incorporation into virions (7, 14 -16), and against HIV RNA binding, which forms an intravirion A3G complex in which A3G must be activated by HIV RNase H for DNA deamination to ensue (16).We have shown that A3G-catalyzed deamination occurs processively while exhibiting a 3Ј 3 5Ј polarity favoring deamination toward the 5Ј-region of ssDNA (15). Directional deamination is an intrinsic property of A3G, occurring in the absence of an obvious source of energy (e.g. ATP or GTP) (15) and can in principle contribute to the HIV-1 G 3 A mutational bias, increasing in a 3Ј-direction ...

Stochastic properties of processive cytidine DNA deaminases AID and APOBEC3G

“…APOBEC3G catalysis is exquisitely sensitive to the salt concentration (Chelico et al 2008). For APOBE-C3G, unbiased deaminations are catalysed with low salt (figure 2a(ii)) and directionally biased deaminations with high salt (5 mM MgCl 2 ; figure 2a(iii)).…”

“…this is an intrinsic property of the enzyme. Our current model has correlated monomers, which mainly form on DNA at low salt, with catalysis of unbiased deaminations and oligomers, such as dimers and tetramers, which form on DNA at high salt, with catalysis of directionally biased deaminations (Chelico et al 2008). There is a saltdependent inhibition of the intrinsic C deamination rate towards the 3 0 -ssDNA region, which offers a molecular basis for how oligomers impose a 5 0 -ssDNA end deamination bias (Chelico et al 2008).…”

“…There is a saltdependent inhibition of the intrinsic C deamination rate towards the 3 0 -ssDNA region, which offers a molecular basis for how oligomers impose a 5 0 -ssDNA end deamination bias (Chelico et al 2008). For both unbiased and biased deaminations, there is a 30 nt 'dead' zone at the 3 0 -ssDNA end where deaminations are at least 10-fold less than elsewhere on the substrate (Chelico et al 2008).…”

“…Our current model has correlated monomers, which mainly form on DNA at low salt, with catalysis of unbiased deaminations and oligomers, such as dimers and tetramers, which form on DNA at high salt, with catalysis of directionally biased deaminations (Chelico et al 2008). There is a saltdependent inhibition of the intrinsic C deamination rate towards the 3 0 -ssDNA region, which offers a molecular basis for how oligomers impose a 5 0 -ssDNA end deamination bias (Chelico et al 2008). For both unbiased and biased deaminations, there is a 30 nt 'dead' zone at the 3 0 -ssDNA end where deaminations are at least 10-fold less than elsewhere on the substrate (Chelico et al 2008).…”

“…Biochemical studies of AID and APOBEC3G have indicated that both enzymes act processively on ssDNA, and deaminations occur stochastically at the hot-and cold-spot targets (Pham et al 2003(Pham et al , 2007Bransteitter et al 2004;Chelico et al 2006Chelico et al , 2008. Here, we discuss how processive deaminations of AID and APOBEC3G relate to their actions in vivo with a more in-depth review of AID mutants affecting processivity and deamination specificity.…”