Retroviral genomes are assembled from two sense-strand RNAs by non-covalent interactions at their 5' ends, forming a dimer. Gag, a viral polyprotein, recruits and packages the retroviral dimer in a nascent virion. After export from the infected cell, the virus and its RNA dimeric genome undergo maturation, a process that renders the virus infective. Viral maturation is initiated by proteolytic cleavage of Gag into a set of constituent proteins including nucleocapsid (NC). Gag and nucleocapsid proteins both bind the RNA dimer at different stages of viral development with exquisite specificity. Genomic RNA dimerization and packaging represent elegant, but poorly characterized, examples of molecular recognition and are potential targets for antiretroviral therapy. The fundamental dimerization unit for the Moloney murine sarcoma and leukemia gamma retroviruses (MuLV and MuSV, respectively) spans a 170-nt minimal dimerization active sequence (MiDAS). The MiDAS domain also contains sequences implicated in specific RNA dimer recognition and packaging. In the dimer, two self-complementary (palindromic) sequences, PAL1 and PAL2, form intermolecular duplexes and an SL1-SL2 (stem-loop) domain forms loop-loop base pairs and extensive tertiary interactions. I first develop a framework for assembly of the retroviral RNA dimer, by analyzing the thermal stabilities and secondary structures of MiDAS mutants in which PAL1, PAL2 heteroduplexes or the SL1-SL2 interactions were successively disrupted. The well-conserved SL1-SL2 domain makes the first and most stable contacts in the dimer (10 kcal/mol), while PAL1 and PAL2 heteroduplexes make much smaller contributions to dimer stability (0.5 to 2 kcal/mol). As an important step towards a common retroviral dimerization mechanism, I next demonstrate that the monomer and dimer MiDAS structures for two murine retroviruses (MuLV and MuSV), while not identical, are similarly organized. I then show that the structure of the in vitro-generated dimer robustly reflects the structure of the mature dimeric RNA extracted from infectious MuLV virions. In the genomic RNA, I identify a long-range interaction between the MiDAS domain and a sequence ∼300 nucleotides downstream. Finally, I provide an explanation for the ability of a retrovirus to exclusively package only genomic RNA dimers. I characterize specific protein binding sites within the MiDAS domain directly inside authentic virions and confirm these sites in vitro. Both Gag and nucleocapsid interact specifically with the first and last nucleotides in UCUG sequences through precise, zinc finger-mediated, contacts. I define a minimal packaging signal for the retroviral RNA dimer as a tandem array of UCUG sequences, flanked by stable base-paired regions.
|Advisor:||Weeks, Kevin M.|
|Commitee:||Erie, Dorothy A., Fried, Howard M., Redinbo, Matthew R., Thompson, Nancy L.|
|School:||The University of North Carolina at Chapel Hill|
|School Location:||United States -- North Carolina|
|Source:||DAI-B 68/06, Dissertation Abstracts International|
|Keywords:||RNA dimerization, Retroviruses|
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