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Single-Molecule Studies Probe Critical Steps in the HIV-1 Life Cycle
Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play distinct roles in the viral life cycle, controlling the timing of reverse transcription and capsid uncoating. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structures, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a complementary RNA hairpin. In contrast, during viral assembly, NC, as a domain of the group-specific antigen (Gag)polyprotein, binds the genomic RNA with surprising specificity and facilitates RNA packaging into new virions. We wish to understand how the same protein, alone or as part of Gag, performs such different nucleic acid binding functions in the viral life cycle. Combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium stability and unfolding barrier for TAR RNA. We find that, while both NC and Gag destabilize the TAR hairpin, Gag binding is localized to two sites in the stem, while NC targets sites near the top loop. Unlike Gag, NC destabilizes this loop, shifting the location of the reaction barrier toward the folded state and increasing the natural rate of hairpin opening by x10 4. Thus, Gag cleavage and NC release is an essential prerequisite for reverse transcription within the virion. New experiments with our Lumicks C-Trap combine single-molecule trapping with fluorescence. We observe NC-induced toroid formation on double stranded DNA, which may induce capsid uncoating. Finally, we visualize the kinetics of Gag cluster formation on single stranded DNA, which may drive viral packaging.

Collaborators:
Micah J. McCauley, Ioulia Rouzina, Jasmine Li, Michael Morse, Megan E. Núñez, Robert J. Gorelick, Karin Musier-Forsyth, and Mark C. Williams

Instructor:
Micah McCauley, PhD
Northeastern University

Cost: Free of charge
Duration: 45 minutes
Language: English
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