After transcription of the HIV-1 provirus, the RNA genome of HIV-1 must perform two distinct functions for the development of an infectious virion: the genome must be translated and packaged. The HIV genome fate is believed to be regulated by dimerization of the 5? untranslated region (UTR). The role of dimerization in genome packaging has been extensively characterized. The role of structural dynamics in 5?UTR translation is not as well-defined. An IRES has been identified in a region overlapping with the packaging signal of the 5?UTR. I hypothesize that the monomeric structure of the 5?UTR is competent for translation by exposure of an internal ribosome entry site (IRES) in the 5?UTR, which stimulates 5? cap-independent translation initiation. The role of 5?UTR structural dynamics in viral replication is poorly understood. I seek to specifically investigate a possible mechanism by which structural dynamics may regulate viral translation. To determine the impact of 5?UTR structure on translation during viral replication, I will utilize structural mutations and monitor their effects on 5?UTR protein production in a cell-free translation assay. These samples will be analyzed by polysome profiling to determine factors necessary for translation in different 5?UTR conformations. A bicistronic reporter assay will be used to determine the effect of conformation on IRES based translation. To directly observe how HIV-1 5?UTR conformation modulates recruitment of translational machinery, I will combine single-molecule Frster resonance energy transfer and single molecule co-localization microscopy to determine the conformation of the 5?UTR when interacting with eukaryotic translation machinery. Host RNA helicases are essential to HIV-1 replication, but the mechanism by which they promote translation remains unclear. The role of host components in modulating HIV-1 5?UTR conformation on translation will be elucidated using enhancers and repressors of HIV-1 translation. I will investigate the role of host components in regulating translation by imaging the conformation of the 5?UTR in the presence of Asp- Glu-Ala-Asp (DEAD)-box polypeptide 3 (DDX3), RNA helicase A (RHA), and human antigen R (HuR). DDX3 and RHA have been shown to promote translation at the 5?UTR, while HuR has demonstrated activity as a repressor of IRES dependent translation at the HIV-1 5?UTR. These experiments will illustrate the role of host factors in modulating the conformation of the HIV-1 5?UTR and how this in turn affects IRES based translation. Here, in the context of this model system, I will investigate the dynamics of this RNA switch, how host protein factors can remodel RNA structure to regulate function, and the role of structural dynamics in regulating viral translation. I will leverage the power of smFRET imaging to visualize the structural dynamics of individual HIV-1 5?UTR molecules, and elucidate their role in translation regulation. This will allow us to visualize how a RNA structural switch regulates a biochemical process in a clinically relevant model.
This application proposes to elucidate the conformational dynamics of individual RNA molecules corresponding to the 5? untranslated region of the HIV-1 genome using single-molecule fluorescence resonance energy transfer imaging, and to probe the role of these dynamics in nucleating virion assembly. These experiments will lead to a better understanding of the mechanism by which the HIV-1 virion incorporates a dimeric genome, and ways in which viral proteins regulate the structure of the genome to promote replication. This information will aid in the discovery of novel means of inhibiting HIV-1 replication, which have thus far been unutilized.
