Physiological Role of APOBEC3
Final Report Abstract
HIV-1 and other lentiviruses have developed accessory proteins that counterattack the cellular antiviral defenses. The original aim of the study was to study the physiological role of APOBEC3, a cellular antiviral protein targeted by the viral accessory protein Vif. APOBEC3G is a cytidine deaminase that is encapsidated in Δvif HIV-1 virions during virus assembly and blocks its replication after viral infection. The study was designed to investigate the physiological role of APOBEC3 using knock-out mice. Progress of the project was delayed due to technical difficulties in breeding and characterizing the knockout mice which made additional preliminary experiments necessary. APOBEC3 knockout mice were backcrossed to generation N7 to ensure a uniform genetic background and new, highly purified recombinant APOBEC3 protein was produced and used to immunize rabbits in order to generate a highly specific anti-APOBEC3 serum to characterize the mice. To ensure scientific progress and success I also focused on a different cellular restriction to lentiviral infection counteracted by another viral accessory protein, Vpx. In contrast to HIV-1 lentiviruses of the HIV-2/SIVmac lineage encode in addition to Vpr the closely related, non-structural protein Vpx. Both proteins are packaged into virions and mediate their effects in the target cell through an interaction with an E3 ubiquitin ligase containing DCAF1. Vpx enhances virus replication in macrophages and dendritic cells. When introduced into primary macrophages and dendritic cells in virus-like particles, Vpx can enhance the efficiency of a subsequent infection. Here, we confirm the ability of Vpx to enhance SIV and HIV-1 infection of macrophages up to 100-fold using single-cycle reporter viruses and by pretreatment of the cells with Vpx-containing virus-like particles. Vpx was also active in the monocytic THP-1 cells but not in other cell lines. Induction of an antiviral state in macrophages with type I interferon significantly magnified the effect of Vpx on HIV-1 infection, suggesting that Vpx helps the virus to overcome an inducible intracellular restriction. PCR quantitation of SIV and HIV-1 reverse transcripts in newly infected macrophages showed that the block was at an early step in reverse transcription. In spite of its structural similarity, Vpr was inactive. This difference allowed us to map the functional domains of Vpx with a panel of Vpr/Vpx chimeras. Analysis of the chimeras demonstrated that the amino-terminal domain of Vpx is important for the enhancement of infection. Fine mapping of the region indicated that amino acids at positions 9, 12, and 15-17 were required. Although the mutants failed to enhance infection, they retained their ability to interact with DCAF1. These findings suggest that the Vpx amino-terminus contains an activation domain that serves as the binding site for a cellular restriction factor.
Publications
- Evidence for an activation domain at the amino terminus of simian immunodeficiency virus Vpx., J Virol. 2010 Feb; 84(3):1387-96
Gramberg et al.
