The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3, referred to as A3) proteins are cellular cytidine deaminases that potently restrict retrovirus replication. However, HIV-1 viral infectivity factor (Vif) counteracts the antiviral activity of most A3 proteins by targeting them for proteasomal degradation. To date, the structure of an A3 protein containing a Vif-binding interface has not been solved. Here, we report a high-resolution crystal structure of APOBEC3C and identify the HIV-1 Vif-interaction interface. Extensive structure-guided mutagenesis revealed the role of a shallow cavity composed of hydrophobic or negatively charged residues between the α2 and α3 helices. This region is distant from the DPD motif (residues 128-130) of APOBEC3G that participates in HIV-1 Vif interaction. These findings provide insight into Vif-A3 interactions and could lead to the development of new pharmacologic anti-HIV-1 compounds.

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response and have broad activity 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-catalyzed 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.

Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins. The mature parts of the Tom70-dependent precursor proteins have the propensity to aggregate, and the presence of the receptor domain of Tom70 prevents their aggregate formation. Therefore Tom70 plays the role of a docking site for not only cytosolic chaperones but also aggregate-prone substrates to maintain their solubility for efficient transfer to downstream components of the mitochondrial import machineries.

The HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1-, F2-, and F3-box motifs are essential for both A3C and A3F degradation, and an additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1-, F2-, and F3-box motifs sterically cluster to form a flexible A3F interaction interface, which represents hydrophobic and positively charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases. IMPORTANCEHIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Three discontinuous sequence motifs of Vif, F1, F2, and F3 boxes, assemble to form an A3F interaction interface. In addition, we determined a crystal structure of the wild-type A3F C-terminal domain responsible for the Vif interaction. These results demonstrated that both electrostatic and hydrophobic interactions are the key force driving Vif-A3F binding and that the Vif-A3F interfaces are larger than the Vif-A3C interfaces. These findings will allow us to determine the configurations of the Vif-A3F complex and to construct a structural model of the complex, which will provide an important basis for inhibitor development. Human cells have evolved intrinsic defense systems against retroviruses, which include the APOBEC3 (A3) family of polynucleotide cytidine deaminases (reviewed in references 1, 2, 3, and 4]). The A3 family comprises seven members that contain either one (A3A, A3C, and A3H) or two (A3B, A3D, A3F, and A3G) Zn 2ϩ coordination domains (Z domains) with conserved HXE(X) 23-28 CXXC motifs (5, 6). Based ...

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Virus replication in the host proceeds by chains of interactions between viral and host proteins. The interactions are deeply influenced by host immune molecules and anti-viral compounds, as well as by mutations in viral proteins. To understand how these interactions proceed mechanically and how they are influenced by mutations, one needs to know the structures and dynamics of the proteins. Molecular dynamics (MD) simulation is a powerful computational method for delineating motions of proteins at an atomic-scale via theoretical and empirical principles in physical chemistry. Recent advances in the hardware and software for biomolecular simulation have rapidly improved the precision and performance of this technique. Consequently, MD simulation is quickly extending the range of applications in biology, helping to reveal unique features of protein structures that would be hard to obtain by experimental methods alone. In this review, we summarize the recent advances in MD simulations in the study of virus–host interactions and evolution, and present future perspectives on this technique.

Human APOBEC3 (A3) proteins are cellular cytidine deaminases that potently restrict the replication of retroviruses by hypermutating viral cDNA and/or inhibiting reverse transcription. There are seven members of this family including A3A, B, C, DE, F, G, and H, all encoded in a tandem array on human chromosome 22. A3F and A3G are the most potent inhibitors of HIV-1, but only in the absence of the virus-encoded protein, Vif. HIV-1 utilizes Vif to abrogate A3 functions in the producer cells. More specifically, Vif, serving as a substrate receptor, facilitates ubiquitination of A3 proteins by forming a Cullin5 (Cul5)-based E3 ubiquitin ligase complex, which targets A3 proteins for rapid proteasomal degradation. The specificity of A3 degradation is determined by the ability of Vif to bind to the target. Several lines of evidence have suggested that three distinct regions of A3 proteins are involved in the interaction with Vif. Here, we review the biological functions of A3 family members with special focus on A3G and base our analysis on the available structural information.

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