Single-molecule studies on the role of HIV-1 nucleocapsid protein/nucleic acid interaction in the viral replication cycle
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The discovery of the crucial intermediates and pathway in the process of the reverse transcription was reported using single-molecule spectroscopy and related techniques including single-molecule fluorescence resonance energy transfer, fluorescence correlation spectroscopy and confocal imaging. Reverse transcription of the HIV-1 RNA genome involves several complex nucleic acid rearrangement steps that are catalyzed by the HIV-1 nucleocapsid protein (NC), including for example, the annealing of the transactivation response (TAR) region of the viral RNA to the complementary region (TAR DNA) in minus-strand strong-stop DNA. In this dissertation, the research focused on elucidating the mechanism of NC-facilitated TAR DNA/RNA annealing. The single molecule spectroscopic measurements reported that the crucial intermediates as well as the mechanistic insight into the annealing of TAR RNA with TAR DNA mediated by viral NC proteins. The data reveal that NC partially melted the secondary structure of TAR DNA (termed the "YTAR") as well as TAR RNA. In the subsequent studies, various short DNA oligonucleotdies were applied to anneal with the TAR to mimic the initial annealing steps. The data support that the YTAR serves as a nucleation center for the annealing to occur through the multiple sites along the TAR structure. Two major nucleation pathways were observed, which are the annealing through the 3'/5' termini, namely "zipper" pathway and the annealing through the hairpin loop region, namely "kissing" pathway. The annealing mechanism was further explored by performing the annealing of wild-type TAR DNA with wild-type TAR RNA in the presence of NC in vitro. The annealing kinetic data suggest that the nucleation of TAR DNA/RNA annealing occurs in an encounter complex form in which one or two DNA/RNA strands in the "Y" form associated with multiple NC molecules. This encounter complex leads to the multiple nucleation complexes, i.e. zipper or kissing intermediates. The data further indicate that although the two complementary strands nucleate at multiple sites, i.e. any single-strand region of TAR, the annealing of two TAR complements occurs through a common mechanism.