April 25, 2024: Keerthi Gottipati PhD, Indiana University, Bloomington

Cryo-EM structure of HIV-1 Rev Response Element RNA: Structural basis of Rev-RRE interaction
Viral genomic RNA not only codes for proteins but also acts as a regulatory element during virus replication and pathogenesis. While the primary RNA sequence determines viral protein structure and function, the secondary and tertiary structure of RNA are critical to its role as a regulatory element. During HIV infection, several RNA transcripts from overlapping reading frames and alternative splicing, are generated in the nucleus. Cytoplasmic export of unspliced (~9 kb) and partially spliced (~4 kb) HIV viral RNA is required for progression of viral infection. This critical process is mediated by a specific interaction between the HIV Rev (Regulator of viral expression) protein and the Rev response element (RRE) RNA. The architecture of the Rev-RRE multimeric complex involving Rev homo-oligomers and full-length RRE is unknown.
To determine the structure of full-length HIV-1 RRE RNA, we expressed RRE (237 nt) recombinantly with a tRNA-scaffold, a method we previously employed successfully to express and purify highly homogenous and structurally stable chimeric viral RNAs for structure determination using X-ray crystallography. We used several approaches to achieve optimal conditions for cryo-EM sample preparation including RNA specific antibody Fab fragments, multi-blot techniques and finally the ink-jet spray system (Chameleon) at NCCAT. We describe the resultant reconstruction of RRE and the various challenges of RNA cryo-EM we encountered in the process.