Structure and Function of Vif and APOBEC3 (A3) Proteins

Division Of Basic Sciences - Nci

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Abstract

We collaborated with Dr. Xiong's group (Yale University, New Haven, CT) and determined the cryo-electron microscopy (cryo-EM) structure of HIV-1 Vif bound to PPP2R5A, one of the 5 regulatory subunits (PPP2R5A-E) of cellular protein phosphatase 2A (PP2A). Recent evidence showed that Vif mediates degradation of PPP2R5A, which induces G2/M cell cycle arrest. Vif uses different interfaces to interact with A3G, A3F, and A3H hap II, and PPP2R5A does not bear any structural resemblance to the A3 proteins, so it is unclear how Vif can recognize these different sets of proteins. The Xiong lab determined the cryo-EM structure of PPP2R5A in complex with HIV-1 Vif-CBFb-elongin B-elongin C at 3.58 Å resolution. The structure showed a large distinct Vif-PPP2R5A interface that partially overlaps with the Vif-A3G and Vif-A3F interfaces. We carried out extensive mutational analysis, including charge-swap mutagenesis of charged amino acids predicted to interact, to validate the structure. The structure also showed that Vif interaction blocks a canonical PPP2R5A substrate-binding site, indicating that it suppresses the PP2A phosphatase activity through both degradation-dependent and degradation-independent mechanisms. Our work identifies critical Vif motifs regulating the recognition of various A3 and PPP2R5A substrates, whereby disruption of these host-virus protein interactions could serve as potential new targets for development of HIV-1 therapeutics. We developed vectors that express A3G-D128K, membrane-associated fusion inhibitor peptide mC46, and O6-methylguanine-DNA-methyltransferase (MGMT) selectable marker for in vivo selection of transduced CD34+ hematopoietic stem and progenitor cells. MGMT-selected T cell lines MT4, CEM, and PM1 expressing A3G-D128K (with or without mC46) potently inhibited NL4-3 infection up to 45 days post infection with no detectable viral replication. Expression of mC46 was sufficient to block infection >80% in a single-cycle assay. Importantly, expression of mC46 provided a selective advantage to the A3G-D128K-modified T cells in the presence of replication competent virus. Higher transduction efficiencies will likely be needed to express Vif-resistant A3G in a sufficient proportion of target cells to achieve therapeutic levels. Improvements in gene transfer efficiency, and combining this approach with other therapeutic strategies, could provide an effective gene therapy treatment and a potential functional cure for HIV-1 infection. To determine whether alterations in the expression of ZNF genes influence expression of host genes that ensure survival of HIV-1 infected cells, we constructed retroviral vectors that express 3XHA-tagged ZNF genes in T cells. We were unable to select stable cell lines that constitutively expressed the ZNF genes, indicating that long-term strong expression of the ZNF genes leads to cytotoxicity. We have found that expressing the ZNF genes at a low level from a doxycycline (dox)-inducible promoter results in minimal cytotoxicity. The impact of exogenous codon-optimized ZNF expression in puromycin-selected T cell lines (CEM, Jurkat, PM1) on expression of other host genes is being investigated by performing next-generation sequencing of host mRNAs. We will determine whether alterations of host gene expression promotes survival of cells expression HIV-1 proteins, which would otherwise be recognized and killed by the host immune system.