137. Viral Hitchhiking to Increase Therapeutic Potential: Autonomously Packaged Elements (APEs)

Abstract

Human Immunodeficiency Virus (HIV) preferentially packages its own RNA genome and various RNA transcripts (tRNA-Lys3, 7SL, aptamers, etc.) with a broad range of consequences. For example, genome and tRNA are required for viral life cycle (positive impact on viral fitness), various cellular RNAs such as 7SL appear to “hitchhike” with no obvious essential roles (neutral), and aptamers that bind RT exert their influence by co-packaging with the viral proteins (inhibitory). While the mechanisms that promote selective incorporation of these transcripts have not been fully described, it is generally accepted that RNA secondary structures allow the RNA to associate with viral proteins during packaging. Identification of these RNA secondary structures would allow for exploitation of a range of packaging mechanisms to drive RNA incorporation for therapeutic purposes. We therefore applied a novel in vivo selection to identify RNAs specifically associated with incorporation into virus particles. We have previously demonstrated that multiple RNA aptamers selected against HIV Reverse Transcriptase (RT) are incorporated into the budding virus– potentially through interactions with the RT– and that they effectively reduce infectivity. To identify conserved features of highly efficient aptamer packagers, we have developed a cell-based protocol for the selection of Autonomously Packaging Elements (APEs). Pre-enriched RNA aptamer libraries were transfected into virus producing cells where we exploit the intracellular viral life cycles to perform the selection of APEs. We inserted an aptamer library downstream of a CMV promoter and co-transfected the library with HIV proviral plasmids. This generated pseudotyped virus particles containing the packaged RNA aptamers, while the cells retain the non-packaged RNA. By reverse transcribing the packaged RNA component, then amplifying and re-inserting it into the expression vector and iteratively transfecting fresh producer cells, we increase packaging competition and aptamer specificity. Subsequent High-Throughput Sequencing and computational analysis will allow us to probe the sequence and structural requirements of APEs. Individual aptamers will be evaluated for their packaging affinity and ability to inhibit viral replication in cell culture. Expanding on this selection scheme by swapping out the RT in the transfection with a panel of divergent RTs is expected to identify aptamer motifs which target evolutionarily conserved RT residues. Application of the APE selection system to an extended viral panel utilizing a range of starting libraries allows for nearly limitless variability in selection output.