Based on structural data of the RNA-dependent RNA polymerase, rational targeting of key residues, and screens for Coxsackievirus B3 fidelity variants, we isolated nine polymerase variants with mutator phenotypes, which allowed us to probe the effects of lowering fidelity on virus replication, mutability, and in vivo fitness. These mutator strains generate higher mutation frequencies than WT virus and are more sensitive to mutagenic treatments, and their purified polymerases present lower-fidelity profiles in an in vitro incorporation assay. Whereas these strains replicate with WT-like kinetics in tissue culture, in vivo infections reveal a strong correlation between mutation frequency and fitness. Variants with the highest mutation frequencies are less fit in vivo and fail to productively infect important target organs, such as the heart or pancreas. Furthermore, whereas WT virus is readily detectable in target organs 30 d after infection, some variants fail to successfully establish persistent infections. Our results show that, although mutator strains are sufficiently fit when grown in large population size, their fitness is greatly impacted when subjected to severe bottlenecking, which would occur during in vivo infection. The data indicate that, although RNA viruses have extreme mutation frequencies to maximize adaptability, nature has fine-tuned replication fidelity. Our work forges ground in showing that the mutability of RNA viruses does have an upper limit, where larger than natural genetic diversity is deleterious to virus survival.T hirty years ago, our regard of RNA viruses as simple, uniform organisms changed with the demonstration of the genetically heterogeneous composition of Qβ-phage populations (1), a concept that has been extended to all RNA viruses. Indeed, as early as 1965, the error-prone nature of RNA virus replication had been observed (2). Since that time, significant progress has been made in describing the highly polymorphic mutational distributions within RNA virus populations, the mechanisms responsible for their generation, and their implication in virus adaptability, evolution, and fitness (3). Important progress was made in recent studies using antimutator strains of RNA viruses presenting higher-fidelity polymerases that generate less diverse populations (4-6). These studies revealed that, although mutation rates can be reduced for these viruses, nature has seemingly selected for error-prone replication to maximize adaptability. This results in virus populations with extreme mutation frequencies approaching a maximum beyond which the likelihood of lethal mutations greatly diminishes virus viability (7). In recent years, the study of lethal mutagenesis as an antiviral approach, based on the accumulation of lethal mutations through treatment with mutagenic compounds, was pivotal in showing that RNA viruses are particularly sensitive to even moderate increases in their already elevated mutation frequencies (8-13). The strong correlation between decreased mutation frequencies and comp...

Viral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNAdependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity. IMPORTANCEPositive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects on in vitro nucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth in vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness. P ositive-strand RNA viruses replicate by using virally encoded RNA-dependent RNA polymerases (RdRPs) that provide a direct RNA-to-RNA replication function not found in host cells. The structures of many RdRPs, primarily from picornaviruses, caliciviruses, and flaviviruses, have been solved to reveal a core structure composed of the classic right-hand arrangement of finger, palm, and t...

Membrane-targeting domains play crucial roles in the recruitment of signalling molecules to the plasma membrane. For most peripheral proteins, the protein-to-membrane interaction is transient. After proteins dissociate from the membrane they have been observed to rebind following brief excursions in the bulk solution. Such membrane hops can have broad implications for the efficiency of reactions on membranes. We study the diffusion of membrane-targeting C2 domains using single-molecule tracking in supported lipid bilayers. The ensemble-averaged mean square displacement (MSD) exhibits superdiffusive behaviour. However, traditional time-averaged MSD analysis of individual trajectories remains linear and does not reveal superdiffusion. Our observations are explained in terms of bulk excursions that introduce jumps with a heavy-tail distribution. These hopping events allow proteins to explore large areas in a short time. The experimental results are shown to be consistent with analytical models of bulk-mediated diffusion and numerical simulations.

The measurement of nucleic acid polymerase elongation rates is often done via a lengthy experimental process involving radiolabeled substrates, quenched elongation experiments, electrophoretic product separation, and band quantitation. In this work we describe an alternative real-time stopped-flow assay for obtaining kinetic parameters for elongation of extended sequences. The assay builds on our earlier PETE assay designed for high-throughput screening purposes (Anal. Biochem. 365,(194)(195)(196)(197)(198)(199)(200) and relies of measuring how long it takes a polymerase to reach the end of a defined length template. Using poliovirus polymerase and self-priming hairpin RNA substrates with 6 to 26 nucleotide long templating regions, we demonstrate that the assay can be used to determine V max rates for elongation and apparent K m values for NTP utilization. Modeling the reaction kinetics as a series of irreversible steps allows us to numerically fit the entire time-based dataset by properly accounting for the temporal distribution of intermediate species. This enables us to determine average elongation rates over heterogeneous templating regions that mimic viral genome substrates. The assay is easily extendable to other RNA and DNA polymerases, can accommodate secondary structures in the template, and can in principle be used for any enzyme traversing along an extended substrate.

The crystal structure of the coxsackievirus B3 polymerase has been solved at 2.25-Å resolution and is shown to be highly homologous to polymerases from poliovirus, rhinovirus, and foot-and-mouth disease viruses. Together, these structures highlight several conserved structural elements in picornaviral polymerases, including a proteolytic activation-dependent N-terminal structure that is essential for full activity. Interestingly, a comparison of all of the picornaviral polymerase structures shows an unusual conformation for residue 5, which is always located at a distortion in the ␤-strand composed of residues 1 to 8. In our earlier structure of the poliovirus polymerase, we attributed this conformation to a crystal packing artifact, but the observation that this conformation is conserved among picornaviruses led us to examine the role of this residue in further detail. Here we use coxsackievirus polymerase to show that elongation activity correlates with the hydrophobicity of residue 5 and, surprisingly, more hydrophobic residues result in higher activity. Based on structural analysis, we propose that this residue becomes buried during the nucleotide repositioning step that occurs prior to phosphoryl transfer. We present a model in which the buried N terminus observed in all picornaviral polymerases is essential for stabilizing the structure during this conformational change.The picornaviruses are a family of small positive-strand RNA enteroviruses responsible for a wide range of ailments, including poliomyelitis, the common cold, and hepatitis A in humans and foot-and-mouth disease in livestock. The coxsackieviruses are a group of picornaviruses that commonly cause mild fever, aches, and conjunctivitis, as well as the more severe hand, foot, and mouth disease in children and viral heart disease in adults. These viruses are closely related to poliovirus based on phylogenetic analysis indicating that poliovirus emerged from type A coxsackieviruses and the observation that chimeric viruses with poliovirus capsid and coxsackievirus nonstructural proteins replicate efficiently (11).The small ϳ7.5-kb picornaviral genomes encode a ϳ250-kDa polyprotein that is cleaved to produce several individual proteins, the last of which is an RNA-dependent RNA polymerase (RdRp). Known as 3D pol , this enzyme carries out both initial negative-strand and subsequent positive-strand RNA synthesis and is essential for viral viability. The structures of polymerases from several families of viruses have been solved, and they show a common overall fold where the enzyme has palm, thumb, and fingers domains arranged as if in a right hand (6). In RdRps the tip of the fingers domain makes contact with the tip of the thumb, enclosing the active site located in the middle of the palm domain. This creates a channel through which NTPs enter the active site, while the RNA is located at the front of the polymerase with the template entering from the down along the fingers domain and the product duplex exiting along the thumb (6).One unique aspect ...