As anyone who has renovated a home knows, the process of remodeling often requires an elaborate orchestration of timing and materials. Remodeling at the molecular level is likewise an intricate process in which macromolecules must adopt particular structures at specific times in order to ensure a successful outcome. How is this coordination achieved, and how are large, stable macromolecules disassembled and then reassembled to form new structures in an energetically efficient manner? We answered these questions for a fascinating and medically relevant set of macromolecules: retroviral RNA and protein species.
As anomalies of the central dogma, retroviruses like HIV harbor RNA genomes that must be reverse transcribed to form DNA in order for each virus to replicate itself. Prior to reverse transcription, a specific, highly structured region of the retroviral RNA genome must be disassembled in order to affix the tRNA molecule that serves as a primer for the reverse transcriptase enzyme. This primer annealing process also requires the disassembly of the stably folded tRNA primer. Both of these RNA remodeling events are accomplished by a small (~5-6 kD) retroviral protein known as nucleocapsid. To investigate the structural and energetic consequences of this RNA remodeling process, we used NMR spectroscopy to construct a “renovation blueprint” detailing the structures present during each step of the remodeling. Our work focused on a prototypical retrovirus: Moloney murine leukemia virus (MLV).
We found that the MLV nucleocapsid proteins comprise a highly efficient remodeling crew. Though many chaperone proteins disassemble RNA by swamping the entire nucleic acid segment with excess protein molecules, we found that in the case of RNA remodeling prior to reverse transcription, the MLV nucleocapsid binds in a step-wise fashion to just a few, selected sites in the tRNA primer and the corresponding region of the viral genome. These sites are strategically positioned such that nucleocapsid binding triggers release of the RNA residues that are important for the primer annealing process. Furthermore, these specific sites all bind to the nucleocapsid protein with high affinity (in the nanomolar range), suggesting a mechanism by which these retroviral proteins can specifically select their targets out of the sea of cellular RNAs.
Unlike in the case of many chaperone proteins, RNA remodeling by nucleocapsid does not require energy from ATP hydrolysis. Our structural and biophysical findings explain how the highly favorable intramolecular interactions within the MLV genome and tRNA primer are disassembled by nucleocapsid without ATP. It is the specific and high-affinity binding of nucleocapsid binding that provides the energy for selective destabilization of critical RNA residues. Furthermore, the positioning of the nucleocapsid binding sites adjacent to, but not overlapping with, the RNA sequences required for primer annealing means that the nucleocapsid proteins may stay associated with the RNA even as the new, intermolecular RNA contacts form. Thus, unlike for many other known nucleic remodeling events, ATP input is not required during or after the nucleocapsid chaperone-mediated primer annealing process, making nucleocapsid a tiny but highly efficient remodeling machine.
Thus, in addition to providing the first three-dimensional structure of this critical region of the retroviral RNA genome, our findings detail the structural and energetic mechanisms by which the ATP-independent nucleocapsid chaperone proteins facilitate RNA remodeling. Our work highlights how deceptively simple retroviruses can, with just a tiny toolkit of machinery, orchestrate elaborate molecular events that ultimately allow these particles to overtake entire cells and organisms.
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