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

Abstract

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 have mapped the 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 employing 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 and mutant FIV ψ sequences with substitutions in the U85CUG88 stretch revealed that these mutations had limited structural impact and maintained nucleotides 80 to 92 unpaired, as in the wild type structure. Since these mutations dramatically affected packaging, our data suggests that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter binding assays performed using purified FIV Pr50Gag on wild type and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA-Pr50Gag 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 development of FIV-based vectors for human gene therapy.