Studies on HIV‐1 RNA‐protein interactions.

In the absence of a cure and a vaccine, the human immunodeficiency virus (HIV‐1) continues to affect millions of people worldwide. HIV is a retrovirus, a unique family of viruses that replicate via a DNA intermediate. The newly replicated HIV RNA must be spliced in order for it to exit the nucleus of the host cell and be packaged into new viral particles. Normally, unspliced or partially spliced HIV RNA's are trapped within the nucleus and degraded. To combat this, HIV has evolved an accessory protein, Rev. The protein Rev binds to the RRE (located on the unspliced or partially spliced RNA) in order to create a complex that allows for the unspliced HIV RNA genome to be exported out of the nucleus. The secondary structure of the HIV‐1 Rev‐Response Element contains five unique stems. The protein Rev binds to sites situated on stems I and II. We have used electrophoretic mobility shift assays (EMSAs) to assess the significance of the various stems, Stems I, III, IV and V, in the functionality of the Rev‐RRE complex. In this assay, electrophoresis was used to resolve the Rev‐RRE complexes after titrating the mutants with Rev protein. RRE mutant RNAs were obtained in vitro and purified. Free RNAs and protein‐bound RNAs were resolved on a gel; the effectiveness of RNA mutants to bind Rev provide insight into the role of the RNA structure in Rev binding. Support or Funding Information This project is supported by the Defense Threat Reduction Agency and the US Naval Academy.