Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging

The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous regions within the 5' end of the gRNA that assumes a higher order structure harboring several structural motifs. To better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ sequences using genetic, biochemical, and structure-function relationship approaches. Our mutational analysis revealed that the unpaired U⁸⁵CUG⁸⁸ stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput selective 2' hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences, with substitutions in the U⁸⁵CUG⁸⁸ stretch, revealed that these mutations had limited structural impact and maintained nucleotides 80-92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our data suggest that the single-stranded U⁸⁵CUG⁸⁸ sequence is important during FIV RNA packaging. Filter-binding assays performed using purified FIV Pr50^Gag on WT and mutant U⁸⁵CUG⁸⁸ ψ RNAs led to reduced levels of Pr50^Gag binding to mutant U⁸⁵CUG⁸⁸ ψ RNAs, indicating that the U⁸⁵CUG⁸⁸ stretch is crucial for ψ RNA-Pr50^Gag interactions. Delineating sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of FIV-based vectors for human gene therapy.