Mayumi Imahashi

The APOBEC3C crystal structure and the interface for HIV-1 Vif binding.

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.

Antiviral mechanism and biochemical basis of the human APOBEC3 family

The human APOBEC3 (A3) family (A, B, C, DE, F, G, and H) comprises host defense factors that potently inhibit the replication of diverse retroviruses, retrotransposons, and the other viral pathogens. HIV-1 has a counterstrategy that includes expressing the Vif protein to abrogate A3 antiviral function. Without Vif, A3 proteins, particularly APOBEC3G (A3G) and APOBEC3F (A3F), inhibit HIV-1 replication by blocking reverse transcription and/or integration and hypermutating nascent viral cDNA. The molecular mechanisms of this antiviral activity have been primarily attributed to two biochemical characteristics common to A3 proteins: catalyzing cytidine deamination in single-stranded DNA (ssDNA) and a nucleic acid-binding capability that is specific to ssDNA or ssRNA. Recent advances suggest that unique property of A3G dimer/oligomer formations, is also important for the modification of antiviral activity. In this review article we summarize how A3 proteins, particularly A3G, inhibit viral replication based on the biochemical and structural characteristics of the A3G protein.

Lack of association between intact/deletion polymorphisms of the APOBEC3B gene and HIV-1 risk.

Objective The human APOBEC3 family of proteins potently restricts HIV-1 replication APOBEC3B, one of the family genes, is frequently deleted in human populations. Two previous studies reached inconsistent conclusions regarding the effects of APOBEC3B loss on HIV-1 acquisition and pathogenesis. Therefore, it was necessary to verify the effects of APOBEC3B on HIV-1 infection in vivo. Methods Intact (I) and deletion (D) polymorphisms of APOBEC3B were analyzed using PCR. The syphilis, HBV and HCV infection rates, as well as CD4+ T cell counts and viral loads were compared among three APOBEC3B genotype groups (I/I, D/I, and D/D). HIV-1 replication kinetics was assayed in vitro using primary cells derived from PBMCs. Results A total of 248 HIV-1-infected Japanese men who have sex with men (MSM) patients and 207 uninfected Japanese MSM were enrolled in this study. The genotype analysis revealed no significant differences between the APOBEC3B genotype ratios of the infected and the uninfected cohorts (p = 0.66). In addition, HIV-1 disease progression parameters were not associated with the APOBEC3B genotype. Furthermore, the PBMCs from D/D and I/I subjects exhibited comparable HIV-1 susceptibility. Conclusion Our analysis of a population-based matched cohort suggests that the antiviral mechanism of APOBEC3B plays only a negligible role in eliminating HIV-1 in vivo.

Association of APOBEC3G genotypes and CD4 decline in Thai and Cambodian HIV-infected children with moderate immune deficiency

IntroductionHuman APOBEC3G is a host defense factor that potently inhibits HIV replication. We hypothesize that HIV-infected children with a genetic variant of APOBEC3G will have a more rapid disease progression.MethodsAntiretroviral therapy (ART)-naïve children, aged 1–12 years old with CD4 15-24% and without severe HIV-related symptoms were enrolled. The children had CD4% and absolute CD4 counts every 12 weeks and HIV-RNA every 24 weeks until 144 weeks. ART was started when CD4% declined to < 15% or AIDS-related events developed.APOBEC3G genetic variants were performed by PCR-based restriction fragment length polymorphism techniques from peripheral blood mononuclear cells. Random-effect linear regression analysis was performed to correlate APOBEC3G genotypes and disease progression.Results147 children, 35% male, with a median (IQR) age of 6.5 (4.3-8.8) years were enrolled. CDC N:A:B were 1:63:36%. Median baseline values were 20% for CD4% 605 cells/mm3 for CD4 count and 4.7 log10copies/mL for HIV-RNA.The frequencies of APOBEC3G genotypes AA (186H/H), AG (186H/R), GG (186R/R) were 86%, 12%, and 2% respectively. The APOBEC3G genotype GG was associated with a significant decline in CD4% -5.1% (−8.9 to −1.2%), p<0.001, and CD4 counts −226 (−415 to −34) cells/mm3, p<0.001 by random-effect liner regression analysis. No significant associations of APOBEC3G genotypes with HIV-RNA changes overtime (p=0.16) or progression to CDC B and C (p=0.49) were observed.ConclusionsAPOBEC3G genotype GG was significantly associated with a more rapid decline in CD4. APOBEC3G’s antiviral effects on HIV disease progression in children should be further explored.

Crystal structure of the Vif-interaction domain of the anti-viral APOBE3F

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 Vifbinding 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 Vifinteraction 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.