A functional conserved intronic G run in HIV-1 intron 3 is critical to counteract APOBEC3G-mediated host restriction

Background: The HIV-1 accessory proteins, Viral Infectivity Factor (Vif) and the pleiotropic Viral Protein R (Vpr) are important for efficient virus replication. While in non-permissive cells an appropriate amount of Vif is critical to counteract APOBEC3G-mediated host restriction, the Vpr-induced G2 arrest sets the stage for highest transcriptional activity of the HIV-1 long terminal repeat. Both vif and vpr mRNAs harbor their translational start codons within the intron bordering the non-coding leader exons 2 and 3, respectively. Intron retention relies on functional cross-exon interactions between splice sites A1 and D2 (for vif mRNA) and A2 and D3 (for vpr mRNA). More precisely, prior to the catalytic step of splicing, which would lead to inclusion of the non-coding leader exons, binding of U1 snRNP to the 5' splice site (5'ss) facilitates recognition of the 3'ss by U2 snRNP and also supports formation of vif and vpr mRNA. Results: We identified a G run localized deep in the vpr AUG containing intron 3 (G(I3)-2), which was critical for balanced splicing of both vif and vpr non-coding leader exons. Inactivation of G(I3)-2 resulted in excessive exon 3 splicing as well as exon-definition mediated vpr mRNA formation. However, in an apparently mutually exclusive manner this was incompatible with recognition of upstream exon 2 and vif mRNA processing. As a consequence, inactivation of G(I3)-2 led to accumulation of Vpr protein with a concomitant reduction in Vif protein. We further demonstrate that preventing hnRNP binding to intron 3 by G(I3)-2 mutation diminished levels of vif mRNA. In APOBEC3G-expressing but not in APOBEC3G-deficient T cell lines, mutation of G(I3)-2 led to a considerable replication defect. Moreover, in HIV-1 isolates carrying an inactivating mutation in G(I3)-2, we identified an adjacent G-rich sequence (G(I3)-1), which was able to substitute for the inactivated G(I3)-2. Conclusions: The functionally conserved intronic G run in HIV-1 intron 3 plays a major role in the apparently mutually exclusive exon selection of vif and vpr leader exons and hence in vif and vpr mRNA formation. The competition between these exons determines the ability to evade APOBEC3G-mediated antiviral effects due to optimal vif expression.