Current funding cycle
Project: Structural, functional and dynamic differences between the 3CD precursor and its fully processed counterparts, the 3C protease and the 3D polymerase
Objective: To better understand how viruses maximize their informational content by differentiating functions between polyprotein precursors and their fully proteolyzed counterparts
Grant: R01 AI104878 “Watching conformational rearrangements in picornavirus replication proteins
Overview:
Some of the most important new and (re)emerging pathogens are positive-strand RNA viruses, including the picornaviruses Enterovirus D68, Enterovirus A71 and even poliovirus. These viruses directly use their RNA genome to guide the synthesis of a large polyprotein, which must then be proteolyzed into its component parts, including the capsid proteins and enzymes important for genome replication. Virus RNA genomes are rather small, and so these viruses have evolved strategies to essentially expand their functional proteomes. For example, the picornavirus 3C protein is a multi-functional protein that has protease activity, binds RNA control sequences important for coordinating replication and translation processes, and binds phosphoinositide lipids found in virus “replication organelles”, which act to protect the virus from host cell defenses. All of these activities are encoded within its small 18 kDa structure. Another strategy to expand functional protein content is for proteolytic precursors to have different functions than their fully processed counterparts. For example, 3C is also found as part of the 3CD protein, but the 3CD protein has different protease specificity, and different RNA and lipid binding affinities. The 3CD protein also has a 3D domain; the 3D protein is the RNA-dependent RNA polymerase but 3CD does not possess polymerase activity. By itself, 3CD also upregulates phosphoinositide lipid production and induces membrane proliferation, events important for replication organelle biogenesis. How the different and emergent functions of 3CD arise is poorly understood; X-ray crystal structures indicate that 3CD is merely a composite of the 3C and 3D proteins joined together by a small flexible linker. We propose that structural dynamics, that is, the ability to sample multiple structural conformations, is the missing ingredient in understanding virus protein function. We propose that 3C fluctuates among many conformations, providing 3C the ability to access and coordinate its many functions, and we propose that 3CD fluctuates into different conformations, providing it with alternative functions. These dynamic excursions can be further modified by interactions with RNA, lipids and protein binding partners to coordinate virus protein function. We will evaluate these protein structural dynamics through solution-state nuclear magnetic resonance spectroscopy, which provide atomic-level detail of protein motions from the picosecond to second timescales, and complement these studies with mutagenesis studies, functional assays and cell-based approaches to better understand the roles of protein structural dynamics in the virus life cycle. The completed work will provide new opportunities for rational anti-viral strategies, for example, by finding molecules that bind to alternative protein conformations and/or disrupt functionally-important motions, as already validated for 3D.
Selected Publications:
Winston, D.S. and D.D. Boehr. 2021. The picornavirus precursor 3CD has different conformational dynamics compared to 3Cpro and 3Dpol in functionally relevant regions. Viruses, 13, 442.
Viruses have evolved numerous strategies to maximize the use of their limited genetic material, including proteolytic cleavage of polyproteins to yield products with different functions. The poliovirus polyprotein 3CD is involved in important protein-protein, protein-RNA and protein-lipid interactions in viral replication and infection. It is a precursor to the 3C protease and 3D RNA-dependent RNA polymerase, but has different protease specificity, is not an active polymerase, and participates in other interactions differently than its processed products. These functional differences are poorly explained by the known X-ray crystal structures. It has been proposed that functional differences might be due to differences in conformational dynamics between 3C, 3D and 3CD. To address this possibility, we conducted nuclear magnetic resonance spectroscopy experiments, including multiple quantum relaxation dispersion, chemical exchange saturation transfer and methyl spin-spin relaxation, to probe conformational dynamics across multiple timescales. Indeed, these studies identified differences in conformational dynamics in functionally important regions, including enzyme active sites, and RNA and lipid binding sites. Expansion of the conformational ensemble available to 3CD may allow it to perform additional functions not observed in 3C and 3D alone despite having nearly identical lowest-energy structures.
Previous funding cycle
Project: The fidelity mechanism of the viral RNA-dependent RNA polymerase
Objective: To leverage our understanding of the structural dynamics underpinning function and fidelity of the RNA-dependent RNA polymerase towards anti-viral strategies, including vaccine and drug design
Grant: R01 AI104878 “Watching conformational rearrangements in poliovirus RNA-dependent RNA polymerase”
Overview:
The genomes of RNA viruses are replicated by the virally-encoded RNA-dependent RNA polymerase (RdRp). The principles governing the fidelity of nucleotide addition by nucleic acid polymerases have yet to be fully established in any system. The importance of RdRp fidelity is underscored by the fact that it is a determinant of virulence with great potential for manipulation in the rational design of attenuated virus vaccine strains. The conserved nature of the RdRp active site and the mechanism of nucleotidyl transfer also make this enzyme a superb target for development of broad-spectrum, antiviral therapeutics. Kinetic studies of poliovirus (PV) RdRp have established the existence of conformational-change steps before, during and after the nucleotide addition that are required for efficient and faithful nucleotidyl transfer. However, the structural mechanisms governing these conformational changes are still poorly understood. Our central objective is to exploit our ability to use NMR to elucidate the conformational states governing each step of the nucleotide-addition cycle, and to determine how each state is perturbed by RdRp derivatives displaying a fidelity phenotype distinct from that of wild-type. These studies can then be leveraged towards generating RdRp enzymes with altered fidelity, which can be combined with other methods to generate potential vaccine candidates.
Selected Publications:
The 2’-C-methyl ribonucleosides are nucleoside analogs representing an important class of antiviral agents, especially against positive-strand RNA viruses. Their value is highlighted by the highly successful anti-hepatitis C drug sofosbuvir (aka Sovaldi). Once appropriately phosphorylated, these nucleotides are successfully incorporated into RNA by the virally-encoded RNA-dependent RNA polymerase (RdRp). This activity prevents further RNA extension, but the mechanism is poorly characterized. Previously, we had identified NMR signatures characteristic of formation of RdRp-RNA binary and RdRp-RNA-NTP ternary complexes for the poliovirus (PV) RdRp, including an open-to-closed conformational change necessary to prepare the active site for catalysis of phosphoryl transfer. Here, we used these observations as a framework for interpreting the effects of 2’-C-methyl adenosine analogs on RNA chain extension in solution-state NMR spectroscopy experiments, enabling us to gain additional mechanistic insights into 2’-C-methyl ribonucleoside-mediated RNA chain termination. Contrary to what has been previously proposed, PV RdRp that was bound to RNA with an incorporated 2’-C-methyl nucleotide still could bind to the next incoming NTP. Our results also indicated that incorporation of the 2’-C-methyl nucleotide does not disrupt RdRp-RNA interactions and does not prevent translocation. Instead, incorporation of the 2’-C-methyl nucleotide blocked closure of the RdRp active site upon binding of the next correct incoming NTP, which prevented further nucleotide addition. We propose that other nucleotide analogs that act as nonobligate chain terminators may operate through a similar mechanism.
The conserved structural motif D is an important determinant of the speed and fidelity of viral RNA-dependent RNA polymerases (RdRps). Structural and computational studies have suggested that conformational changes in the motif-D loop that help to reposition the catalytic lysine represent critical steps in nucleotide selection and incorporation. Conformations of the motif-D loop in the poliovirus RdRp are likely controlled in part by noncovalent interactions involving the motif-D residue Glu364. This residue swivels between making interactions with Lys228 and Asn370 to stabilize the open and closed loop conformations, respectively. We show here that we can rationally control the motif-D loop conformation by breaking these interactions. The K228A variant favors a more active closed conformation, leading to increased nucleotide incorporation rates and decreased nucleotide selectivity, and the N370A variant favors a less active open conformation, leading to decreased nucleotide incorporation rates and increased nucleotide selectivity. Similar competing interactions likely control nucleotide incorporation rates and fidelity in other viral RdRps. Rational engineering of these interactions may be important in the generation of live, attenuated vaccine strains, considering the established relationships between RdRp function and viral pathogenesis.