PT - JOURNAL ARTICLE AU - Rachel A. Knoener AU - Edward L. Evans III AU - Jordan T. Becker AU - Mark Scalf AU - Bayleigh E. Benner AU - Nathan M. Sherer AU - Lloyd M. Smith TI - Identifying HIV-1 RNA splice variant protein interactomes using HyPR-MS<sub>SV</sub> AID - 10.1101/2020.09.15.298190 DP - 2020 Jan 01 TA - bioRxiv PG - 2020.09.15.298190 4099 - http://biorxiv.org/content/early/2020/09/16/2020.09.15.298190.short 4100 - http://biorxiv.org/content/early/2020/09/16/2020.09.15.298190.full AB - HIV-1 generates unspliced (US), partially spliced (PS), and completely spliced (CS) classes of RNAs; each playing distinct roles in viral replication. Elucidating their host protein “interactomes” is crucial to understanding virus-host interplay. Here, we present HyPR-MSSV for isolation of US, PS, and CS transcripts from a single population of infected CD4+ T-cells and mass spectrometric identification of their in vivo protein interactomes. Analysis revealed 212 proteins differentially associated with the unique RNA classes; including, preferential association of regulators of RNA stability with US- and PS-transcripts and, unexpectedly, mitochondria-linked proteins with US-transcripts. Remarkably, &gt;80 of these factors screened by siRNA knock-down impacted HIV-1 gene expression. Fluorescence microscopy confirmed several to co-localize with HIV-1 US RNA and exhibit changes in abundance and/or localization over the course of infection. This study validates HyPR-MSSV for discovery of viral splice variant protein interactomes and provides an unprecedented resource of factors and pathways likely important to HIV-1 replication.