This project links three broadly important problems in biomedical science. First, the human immunodeficiency virus (HIV) infects 33 million individuals with roughly 3 million new infections per year. Our laboratory has recently developed a model for the overall architecture and secondary structure of the entire RNA genome for one HIV-1 virus. This work revealed numerous previously unrecognized, but evolutionarily conserved, RNA structures. Further refinement of complete HIV genome structures and identification of protein-RNA interactions in the genome hold significant promise for identifying novel targets and pathways for antiretroviral drug development. Second, viruses usurp the genetic instructions present in all living organisms. Structured RNA regulatory elements that we have discovered in the HIV-1 genome appear to influence splicing, protein translation, evasion of host cell defenses, and accessibility towards drug binding. The extraordinary density of information encoded in the HIV-1 RNA genome appears to represent a key component of the genetic code, one that we understand poorly at present. The proposed research program will therefore enhance our understanding of the fundamental principles that govern gene regulation in all of biology. Third, essentially all RNA molecules function in biology only after they fold into specific secondary (and tertiary) structures. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) technology, invented in the PI's laboratory, now makes it possible to analyze the local nucleotide environment at nearly every position for RNAs of arbitrary size and as bound by proteins. However, data analysis remains labor-intensive. We seek to streamline the data processing steps to make possible facile and comprehensive functional analyses of arbitrary intact large RNAs. In collaboration with experts in HIV biology and in bioinformatics, we will therefore tackle the following Aims: (1) Analyze the structures of the complete genomes of two additional HIV-1 related viruses, the simian immunodeficiency virus from chimpanzees (SIVcpz) and from the rhesus macaque (SIVmac). (2) Analyze the complete HIV-1 genome, as it exists inside authentic HIV-1 virions in both immature and mature particles. (3) Create a platform-independent, user-friendly software for fully automated analysis of high-throughput SHAPE information. All technology advances and HIV genome data sets will be made freely available to the biomedical science community.
This work will determine structures for complete HIV-related genomes, using innovative technology created in the project laboratory. This work is signficant because HIV RNA genome structure is a key componenet of the viral genetic code, one we understand poorly at present. Over the long term, nucleotide resolution information about HIV genome structure has significant potential to identify multiple novel frameworks for designing new anti-retroviral therapeutics.
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|Siegfried, Nathan A; Busan, Steven; Rice, Greggory M et al. (2014) RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nat Methods 11:959-65|
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|Leonard, Christopher W; Hajdin, Christine E; Karabiber, Fethullah et al. (2013) Principles for understanding the accuracy of SHAPE-directed RNA structure modeling. Biochemistry 52:588-95|
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