The antiviral restriction of a chimeric APOBEC3A protein
Renato Aguiar, UC San Francisco
In this proposal, we will study the potential antiviral infectivity of a fused Vpr.APOBEC3A (Vpr.A3A) protein. A3A belongs to APOBEC family of cellular cytidine deaminase proteins that cause hypermutations of nascent retroviral genome by the deamination of cytidine residues. These innate antiretroviral factors can block HIV-1 infectivity. However, this restriction can be counteracted by the HIV-1 virion infectivity factor (Vif) that is able to induce the degradation of APOBEC proteins. The A3A is able to cause cytidine deamination; however, there has been no antiviral activity described. Here, we constructed an A3A chimeric protein that is incorporated into viral particles and able to restrict HIV-1 infectivity, even in the presence of Vif. The incorporation of this protein was promoted by its fusion with the HIV-1 Vpr protein that is packaged into viral cores. The long term objectives of this proposal are to evaluate the use of this chimeric Vpr.A3A in therapeutic strategies to restrict HIV-1 replication. The general strategy is to understand the molecular mechanism involved in this antiviral activity. Three specific aims will be addressed for this proposal. In the first specific aim, we will determine the relevance of Vpr targeting for the antiviral effect of A3A through mutations in the viral motifs involved in the incorporation of Vpr and in the cytidine deaminase domain of A3A. We will also compare the incorporation rates of Vpr.A3A and single A3A in the core of the virus and the distribution of these proteins in the high or low molecular mass complexes that can regulate the antiviral activity of APOBEC proteins. For the second specific aim, we will evaluate the antiviral activity of the Vpr.A3A protein against others retroviruses such as SIV, MLV, MMTV and HERV-Kcon. Viruses harboring the Vpr.A3A will be generated by cotransfection using proviral genomes of each particular retrovirus. The infectivity of these retroviruses will be evaluated by the expression of reporter genes in target cells. In the last aim we will determine the utilization of this chimeric APOBEC protein to restrict incoming viruses by constructing a stable cell line expressing this protein, which will be challenged with HIV-1. Our studies, will not only reveal new aspects of targeting of APOBEC proteins into viral particles, but the potent antiretroviral activity of the Vpr.A3A fusion protein suggests its use as intracellular immunization in AIDS therapy.