Low‐resolution structure of HIV‐1 genomic RNA regions obtained by small angle x‐ray scattering analysis

The 5′ untranslated region (5′UTR) of the human immunodeficiency virus type 1 (HIV‐1) RNA genome (vRNA) controls many aspects of virus replication, including vRNA dimerization, tRNA priming, and virus assembly. While the secondary structure of the 5′UTR is well annotated, a model for the tertiary structure of the entire 5′UTR remains elusive. In this work, using a divide‐and‐conquer approach, various portions of the 5′ UTR of the vRNA were examined using small angle x‐ray scattering (SAXS). SAXS requires homogeneous sample preparation, which was accomplished through a combination of optimizing construct design, buffer/refolding optimization, size‐exclusion chromatography, and point mutations. As expected, primer tRNALys3 adopts a compact L‐shaped fold, consistent with crystallographic data. The 2‐helix 104‐nt TAR/polyA RNA exists in an elongated conformation, with apparently co‐axially stacked helices. In contrast, the 95‐nt 3‐helix Psi RNA packaging signal adopts a less compact conformation, likely due to the flexible linker connecting stemloops SL2 and SL3. These preliminary models represent the first step toward our goal of building a low‐resolution model of the entire 5′UTR in the absence and presence of interacting partners. This research was supported by Ohio State University and by a fellowship to CPJ from the OSU Center for RNA Biology.