APOBEC3 Multimerization Correlates with HIV-1 Packaging and Restriction Activity in Living Cells

Abstract

APOBEC3G belongs to a family of DNA cytosine deaminases that are involved in the restriction of a broad number of retroviruses including HIV-1. Prior studies have identified two distinct mechanistic steps in Vif-deficient HIV-1 restriction: packaging into virions and deaminating viral cDNA. APOBEC3A, for example, although highly active, is not packaged and is therefore not restrictive. APOBEC3G, on the other hand, although having weaker enzymatic activity, is packaged into virions and is strongly restrictive. Although a number of studies have described the propensity for APOBEC3 oligomerization, its relevance to HIV-1 restriction remains unclear. Here, we address this problem by examining APOBEC3 oligomerization in living cells using molecular brightness analysis. We find that APOBEC3G forms high-order multimers as a function of protein concentration. In contrast, APOBEC3A, APOBEC3C and APOBEC2 are monomers at all tested concentrations. Among other members of the APOBEC3 family, we show that the multimerization propensities of APOBEC3B, APOBEC3D, APOBEC3F, and APOBEC3H (haplotype II) bear more resemblance to APOBEC3G than to APOBEC3A/3C/2. Prior studies have shown that all of these multimerizing APOBEC3 proteins, but not the monomeric family members, have the capacity to package into HIV-1 particles and restrict viral infectivity. This correlation between oligomerization and restriction is further evidenced by two different APOBEC3G mutants, which are each compromised for multimerization, packaging, and HIV-1 restriction. Overall, our results imply that multimerization of APOBEC3 proteins may be related to the packaging mechanism and ultimately to virus restriction.

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

“…Furthermore, in an in vitro RT model system, the presence of APOBEC3C cause a higher mutation frequency than APOBEC3C S188. A previous study correlated multimerization of APOBEC3s with the capacity to restrict lentiviruses [51], and our finding that the monomeric variant (S188) was less antivirally active than the dimer-forming, more active variant (I188), is consistent with this conclusion. Therefore, our model is that isoleucine at position 188 of APOBEC3C enhances lentiviral restriction by improving dimerization and in turn, the enzymatic activity of the protein.…”

“…Previous studies have reported that the S188 variant of APOBEC3C is a monomeric protein, both in solution [149] and in cells [51]. Indeed, by size exclusion chromatography we also found that baculovirus/Sf9-produced APOBEC3C S188 (the common variant) is monomeric ( Figure 3.7A, apparent molecular weight 17 kDa).…”

“…To mediate encapsidation the A3 enzymes bind viral or cellular RNAs with the same sequence affinity as the NC domain of Gag that packages the HIV-1 RNA [49,50]. Encapsidation is also facilitated by A3 oligomerization on RNA although the mechanism is not entirely understood [51]. It could simply be that oligomerization is a way of A3s hedging bets for encapsidation through multiple mechanisms, since there is no guarantee of an A3 in each virion.…”

“…A3H is the only member that has a Z3-domain; A3D and A3F have two Z2-domains, A3G and A3B have a Z1-(CTD) and Z2-(NTD) domain [23]. A3H is also the only single domain enzyme to oligomerize in solution and similar to A3G and A3F, A3H hap II is able to interact with cellular RNA and become encapsidated into virions [51]. As both virion encapsidation and the biochemical properties of the enzymes are important for the efficient restriction of HIV, biochemical characterization of A3H has demonstrated that it is a processive enzyme.…”

Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain

“…1D). Our data combine to indicate that the A3Bctd is active as a monomer, although we have not addressed the possibility that it might oligomerize upon substrate engagement, and it is notable that full-length protein is capable of forming higher order oligomers in a cellular context, most likely through its insoluble N-terminal domain (54). Closed Conformation of the A3B Active Site-In the structure of A3Bctd-QM⌬loop3, a single zinc ion is bound between the N-terminal ends of ␣2 and ␣3, coordinated by two cysteines (Cys-284 and Cys-289) and a histidine (His-253; Fig.…”