Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein.

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalysed DNA elongation. Here, we show that APOBEC3G initially binds ssDNA with rapid on-off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription.

HIV-1 Vif-mediated ubiquitination/degradation of APOBEC3G involves four critical lysine residues in its C-terminal domain.

During coevolution with the host, HIV-1 developed the ability to hijack the cellular ubiquitin/proteasome degradation pathway to counteract the antiviral activity of APOBEC3G (A3G), a host cytidine deaminase that can block HIV-1 replication. Abrogation of A3G function involves the HIV-1 Vif protein, which binds A3G and serves as an adapter molecule to recruit A3G to a Cullin5-based E3 ubiquitin ligase complex. Structure-guided mutagenesis of A3G focused on the 14 most surface-exposed Lys residues allowed us to identify four Lys residues (Lys-297, 301, 303, and 334) that are required for Vif-mediated A3G ubiquitination and degradation. Substitution of Arg for these residues confers Vif resistance and restores A3G's antiviral activity in the presence of Vif. In our model, the critical four Lys residues cluster at the C terminus, opposite to the known N-terminal Vif-interaction region in the protein. Thus, spatial constraints imposed by the E3 ligase complex may be an important determinant in Vif-dependent A3G ubiquitination.

Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G.

APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single-stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse transcription. Here we report the interplay between A3G, NC and reverse transcriptase (RT) and the effect of highly purified A3G on individual reactions that occur during reverse transcription. We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage. In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC. In the case of (-) strong-stop DNA synthesis, the inhibition was independent of A3G's catalytic activity. Fluorescence anisotropy and single molecule DNA stretching analyses indicated that NC has a higher nucleic acid binding affinity than A3G, but more importantly, displays faster association/disassociation kinetics. RT binds to ssDNA with a much lower affinity than either NC or A3G. These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.

Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses.

Trichosanthin (TCS) is a type I ribosome-inactivating protein that inactivates ribosome by enzymatically depurinating the A(4324) at the alpha-sarcin/ricin loop of 28S rRNA. We have shown in this and previous studies that TCS interacts with human acidic ribosomal proteins P0, P1 and P2, which constitute the lateral stalk of eukaryotic ribosome. Deletion mutagenesis showed that TCS interacts with the C-terminal tail of P2, the sequences of which are conserved in P0, P1 and P2. The P2-binding site on TCS was mapped to the C-terminal domain by chemical shift perturbation experiments. Scanning charge-to-alanine mutagenesis has shown that K173, R174 and K177 in the C-terminal domain of TCS are involved in interacting with the P2, presumably through forming charge-charge interactions to the conserved DDD motif at the C-terminal tail of P2. A triple-alanine variant K173A/R174A/K177A of TCS, which fails to bind P2 and ribosomal stalk in vitro, was found to be 18-fold less active in inhibiting translation in rabbit reticulocyte lysate, suggesting that interaction with P-proteins is required for full activity of TCS. In an analogy to the role of stalk proteins in binding elongation factors, we propose that interaction with acidic ribosomal stalk proteins help TCS to locate its RNA substrate.

Structural basis for the interaction of [E160A-E189A]-trichosanthin with adenine.

Trichosanthin is a ribosome-inactivating protein that cleaves specifically the N-glycosidic bond of A-4324 of 28S rRNA. Trichosanthin and its variant [E160A-E189A]-trichosanthin were found to bind an adenine base with a K(d) value of approximately 0.2mM. To determine how this doubly mutated variant of trichosanthin interacts with adenine, the co-crystal structure of [E160A-E189A]-trichosanthin and adenine was resolved to 0.193nm which revealed that the active site conformation of the doubly mutated variant is isomorphous to wild-type trichosanthin. Water molecules were found at locations corresponding to the eliminated side chain of Glu-160 and Glu-189. On the other hand, the adenine base interacted with [E160A-E189A]-trichosanthin in a manner similar to that in wild-type trichosanthin. Our structural analysis illustrates that Glu-160 and Glu-189 in trichosanthin do not play an important role in maintaining the active site conformation and binding adenine, an essential step for substrate-enzyme interaction. On the other hand, removal of two glutamate residues changed a large patch of negatively charged surface to a positive charge, which may account for the destabilization of the oxocarbenium-like transition-state and the significant decrease in ribosome-inactivating activity in [E160A-E189A]-trichosanthin.