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abstracts
Human cells express a family of cytidine deaminases, called APOBEC3 (A3) (A3A, B, C, D, F, G, and H). The family enzymes, especially A3G and A3F potentially inhibit replication of retroviruses including HIV-1. However, HIV-1 overcomes the A3-mediated antiviral system by expressing a virus-encoded antagonist, viral infectivity factor (Vif) protein. In HIV-1-infected cells, Vif specifically binds with A3 followed by proteasomal degradation of A3. Hence, inhibition of the interaction between A3 and Vif is an attractive strategy for developing novel anti-HIV-1 drugs. To date, we have determined the first crystal structure of A3 with Vif-binding interface, A3C (PDB ID: 3VOW). In addition, our extensive mutational analysis, based on the A3C structure, revealed that structural features of the Vif-binding interface are highly conserved among A3C, DE, and F [1]. However, more recently, Bohn et al. and Karen et al. have shown the crystal structures of mutant A3F C-terminal domain (CTD) which is responsible for the Vif interaction, and have predicted more extended area, including our identified residues, for the interface on the A3F CTD [2][3]. To clarify the Vif-binding interface of A3F, we sought to determine the crystal structure of the wild-type A3F CTD and evaluated contributions of the additional residues for the Vif-interaction interface by virological method. First, we have successfully determined the crystal structure of A3F CTD at 2.75 Å resolution. Furthermore, we have identified four additional residues unique on the A3F CTD but not A3C for Vif interaction, which are located in the vicinity of our previously reported interface. These results demonstrated that the structural features of Vif-binding interface are indeed conserved between A3C and A3F. Taken together, these results will provide the fine-tuned structure information to understand the binding between A3 and Vif and to facilitate a development of novel anti-HIV-1 compounds targeting A3 proteins.
abstracts
The HIV-1 full length capsid protein (CA-FL) is increasingly viewed as an attractive therapeutic target since proper capsid formation is required for viral infection. CA-FL is synthesized as a central domain of a structural Gag polyprotein that is involved in both early and late stages of the viral life cycle. During the HIV-1 maturation process, Gag is cleaved by a viral protease to produce several discrete new proteins that include matrix, capsid (CA-FL), and nucleocapsid. After proteolytic cleavage, CA-FL forms hexamers and pentamers that rearrange into a fullerene cone-shaped structure, which surrounds the viral genome at the center of the mature virus. Crystal structures of the native unassembled hexameric CA-FL (without cross-linked residues that might prevent changes in the inter- or intra-subunit interactions) are of great interest, as they may provide insights relevant to the development of drugs that prevent or impede the transition from the preassembled to the assembled capsid states. Recently, we crystallized and solved the crystal structure of the first hexameric HIV-1 CA-FL in its native form (without engineered cross-linking cysteines). There is one molecule per asymmetric unit, and the P6 space group generates the native hexameric assembly. We have also identified a small molecule, 18E8, which exhibits broad anti-HIV activity in cell-based assays, and targets CA-FL. This was demonstrated by experiments that selected for viruses with drug resistance and revealed that an A105T mutation in CA-FL confers resistance to the compound. Time-of-inhibitor addition experiments showed that 18E8 targets an early step in the HIV replication cycle, after reverse transcription and before integration. Electron microscopy experiments suggest that 18E8 does not impart significant morphological changes in CA-FL tubular assemblies. Our structure of CA-FL and our ongoing work with the CA-FL/18E8 complex will provide a system for the investigation of molecular interactions between CA-FL and small molecule antivirals that work with a novel mechanism of action.
