The higher-order structure of HIV genomic RNA may be important for formation of a mature core. Results from a number of studies suggest that condensation of the core is essential for infectious viruses. The goal is to study and understand, in detail, the structure of the HIV-1 genomic RNA A phylogenetic comparison of the full-length RNAs from various isolates will provide the framework for a secondary structure model of the entire genome. in addition, structure-specific chemicals and enzymatic probes will be used along with primer extension for further characterization of the structure. Nuclease digestion and chemical modification patterns of several HIV isolates will be compared to identify the conserved structural features of the RNA. Folding properties of in vitro transcribed HIV RNA fragments will be extensively studied. A comparison of structure probing data from in vitro transcripts of HIV RNAs that vary in sizes ranging from approximately 200 to 3000 nucleotides, all with mature 5' ends, will be helpful in understanding how the RNA folds. To determine how RNA conformation influences the structure of the capsid core, it will be necessary to study the structure of genomic RNA inside the core. The core is permeable to nucleases and parts of the RNA inside are susceptible to cleavage (J. Coffin, personal communication). Mild digestion of encapsidated RNA will determine if there are RNase hypersensitive sites. Gradual increase in nuclease concentration and/or denaturating condition, will reveal sites that are protected by higher-order RNA structures or by interaction with core protein(s). Regions of RNA that are resistant to high concentrations of nucleases under denaturing conditions may prove to be structurally interesting and functionally important. Results from the digestion experiments will help to determine if the RNA is folded into major domains. If distinct domains exist, structure probing will be used to determine whether or not the in vitro transcribed RNAs, corresponding to these domains, are able to fold into correct structures independently, or if core proteins are needed to aid in folding the RNA. In order to determine sites on the RNA that are in contact with proteins, the structure of the encapsidated RNA will be compared with phenol extracted RNA. Finally, an attempt will be made to identify structural differences in RNAs from viruses harboring mutations that prevent condensation of the core versus condensed-core RNAs.
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