STRUCTURAL AND FUNCTIONAL STUDIES OF HIV-1 5?-LEADER RNAS

Abstract

HIV-1 produces multiple types of unspliced and spliced RNA transcripts with functions and fates that are established by the structure of their 5?leader regions. Extensive studies have found that the unspliced transcript that adopts a dimeric 5?leader structure is selectively packaged as the viral genome, while the full-length transcript with a monomeric 5?leader is used for splicing and translation. Although the two conformations of unspliced 5?leader have been extensively studied, the mechanisms by which they direct distinct RNA functions and fates are still not fully understood. The first part of this research focused on identifying the structural elements in the unspliced dimeric 5?leader that are crucial for genome packaging. These studies revealed that a previously identified coaxially stacked 5?tandem hairpin structure, which sequesters the 5?cap, is essential for selective RNA packaging, presumably by preventing the dimeric genome from being captured by the cellular RNA processing and translation machinery. In contrast, the 5?cap is exposed in the translation-promoting monomeric 5?leader due to a structural rearrangement involving destabilization of the PolyA hairpin. However, the structural features of the monomeric leader, including the PolyA region, were not probed under native-like conditions due to technical limitations. Thus, the second part of this work studied the structures of monomeric and dimeric unspliced 5?leaders in two strains of HIV-1 under physiological-like conditions by chemical probing. The results support that the PolyA region does not form a stable hairpin structure in the monomeric 5?leader, which results in an exposed 5?cap for translation initiation and leads to its inefficient packaging. The third part of this research focused on an also packaging incompetent spliced RNA of HIV-1, specifically the structure of the 5?leader of env spliced RNA that encodes the envelope protein. The NMR and biochemical studies revealed that the 5?leader of the env RNA adopts a similar structure with the monomeric form of the unspliced RNA with an exposed 5?cap. These structure features explain the translational role of spliced RNAs rather than being packaged into progeny virions. In summary, this work provides insights into the structure-function relationship of the 5?leader RNAs in multiple HIV-1 transcripts.