The human cytidine deaminase APOBEC3C restricts retroviruses independent of editing – A biochemical and structural analysis

The capability of the Human immunodeficiency virus (HIV) to replicate in cells of different species depends on the presence or absence of interacting or restricting cellular factors. The restriction factors are part of the host’s defense mechanism against pathogens. The family of human APOBEC3 (A3) cytidine deaminases forms part of the intrinsic immunity and protects placental mammals from viral pathogens. A3 proteins have one or two copies of a cytidine deaminase motif and can restrict retroviral replication by deamination of their genomes during reverse transcription. HIV counteracts this inhibition by the Vif-protein, which induces the destruction of A3s and thereby prevents its incorporation into virions. Although one member of this human protein family, APOBEC3C (A3C), is highly expressed in many human tissues and in CD4+ T cells, A3C does not restrict vif-deficient HIV (HIV Δvif) but is active against SIV Δvif. This study aimed to characterize i) the different mechanisms of resistance of HIV and SIV against A3C, ii) the restriction mechanism of A3C against SIV, and iii) residues crucial for the antiviral activity of A3C based on a structural model. With the help of a three- dimensional protein model of A3C, amino acids residues were predicted and finally experimentally identified that are involved in protein dimerization and that mediate the incorporation of A3C into virions. The result showed that dimerization of A3C is essential for antiviral activity against SIV. Beside dimerization, the antiviral activity is regulated by encapsidation into viral particles. Encapsidation of A3C depends on the substrate binding-pocket distal from the zinc-coordinating enzymatic centre. This binding pocket mediates the RNA-dependent incorporation of the protein into budding viral particles. It was found that A3C restricts SIV Δvif without cytidine deamination of viral ssDNA. Therefore, alternative mechanisms were investigated. In this study the experiments showed clearly that A3C does neither deaminates viral RNA nor has a significant inhibitory effect on the reverse transcription of SIV. Thus, it is likely that A3C has a so far unknown restriction mechanism against SIV Δvif. Results of this study also show that the resistance of HIV against A3C can be overcome. An A3C mediated inhibition of HIV was achieved by fusing the Viral Protein R (Vpr) of HIV-1 either to the C- or N- terminus of A3C. Interestingly both, A3C and Vpr.3C were incorporated into viral particles and found to be localized in the same viral compartment, the viral core. These data indicate that HIV encodes a factor additional to vif to counteract the A3C activity. In search for such a viral factor, the viral integrase was identified as a candidate inhibitor of A3C. Additional experiments are required for a better understanding of the interaction of A3C with HIV. However, the results here implicate that enhancing the antiviral activity of the ubiquitously expressed A3C would likely repress HIV-1 replication in patients and thus should be considered as a novel approach for treatment.