Evolution on the Biophysical Fitness Landscape of an RNA Virus.

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.

A high-throughput drop microfluidic system for virus culture and analysis.

High mutation rates and short replication times lead to rapid evolution in RNA viruses. New tools for high-throughput culture and analysis of viral phenotypes will enable more effective studies of viral evolutionary processes. A water-in-oil drop microfluidic system to study virus-cell interactions at the single event level on a massively parallel scale is described here. Murine norovirus (MNV-1) particles were co-encapsulated with individual RAW 264.7 cells in 65 pL aqueous drops formed by flow focusing in 50 µm microchannels. At low multiplicity of infection (MOI), viral titers increased greatly, reaching a maximum 18 h post-encapsulation. This system was employed to evaluate MNV-1 escape from a neutralizing monoclonal antibody (clone A6.2). Further, the system was validated as a means for testing escape from antibody neutralization using a series of viral point mutants. Finally, the replicative capacity of single viral particles in drops under antibody stress was tested. Under standard conditions, many RNA virus stocks harbor minority populations of genotypic and phenotypic variants, resulting in quasispecies. These data show that when single cells are encapsulated with single viral particles under antibody stress without competition from other virions, the number of resulting infectious particles is nearly equivalent to the number of viral genomes present. These findings suggest that lower fitness virions can infect cells successfully and replicate, indicating that the microfluidics system may serve as an effective tool for isolating mutants that escape evolutionary stressors.

Flexibility in surface-exposed loops in a virus capsid mediates escape from antibody neutralization.

UNLABELLED: New human norovirus strains emerge every 2 to 3 years, partly due to mutations in the viral capsid that allow escape from antibody neutralization and herd immunity. To understand how noroviruses evolve antibody resistance, we investigated the structural basis for the escape of murine norovirus (MNV) from antibody neutralization. To identify specific residues in the MNV-1 protruding (P) domain of the capsid that play a role in escape from the neutralizing monoclonal antibody (MAb) A6.2, 22 recombinant MNVs were generated with amino acid substitutions in the A'B' and E'F' loops. Six mutations in the E'F' loop (V378F, A382K, A382P, A382R, D385G, and L386F) mediated escape from MAb A6.2 neutralization. To elucidate underlying structural mechanisms for these results, the atomic structure of the A6.2 Fab was determined and fitted into the previously generated pseudoatomic model of the A6.2 Fab/MNV-1 virion complex. Previously, two distinct conformations, A and B, of the atomic structures of the MNV-1 P domain were identified due to flexibility in the two P domain loops. A superior stereochemical fit of the A6.2 Fab to the A conformation of the MNV P domain was observed. Structural analysis of our observed escape mutants indicates changes toward the less-preferred B conformation of the P domain. The shift in the structural equilibrium of the P domain toward the conformation with poor structural complementarity to the antibody strongly supports a unique mechanism for antibody escape that occurs via antigen flexibility instead of direct antibody-antigen binding. IMPORTANCE: Human noroviruses cause the majority of all nonbacterial gastroenteritis worldwide. New epidemic strains arise in part by mutations in the viral capsid leading to escape from antibody neutralization. Herein, we identify a series of point mutations in a norovirus capsid that mediate escape from antibody neutralization and determine the structure of a neutralizing antibody. Fitting of the antibody structure into the virion/antibody complex identifies two conformations of the antibody binding domain of the viral capsid: one with a superior fit and the other with an inferior fit to the antibody. These data suggest a unique mode of antibody neutralization. In contrast to other viruses that largely escape antibody neutralization through direct disruption of the antibody-virus interface, we identify mutations that acted indirectly by limiting the conformation of the antibody binding loop in the viral capsid and drive the antibody binding domain into the conformation unable to be bound by the antibody.

Functional TRPM7 channels accumulate at the plasma membrane in response to fluid flow.

Many cells are constantly exposed to fluid mechanical forces generated by flowing blood, and wall shear stresses modulate aspects of their structure and function. However, the mechanisms for mechanotransduction of flow are not well understood. Here we report that TRPM7, which is both an ion channel and a functional kinase, is translocated within cells in response to laminar flow. After exposure of cells to physiological values of laminar fluid flow, the number of TRPM7 molecules localized at or near the plasma membrane increased up to 2-fold, in less than 100 seconds. This increase in membrane-localized GFP-TRPM7, as seen by total internal reflection fluorescence microscopy, closely correlated with increases in TRPM7 current. Both endogenous and heterologously expressed TRPM7 was found in tubulovesicular structures that were translocated to the region of the plasma membrane on induction of shear stress. In vascular smooth muscle cells, but not in several types of endothelial cells, fluid flow increased endogenous native TRPM7 current amplitude. We hypothesize that TRPM7 plays a role in pathological response to vessel wall injury.

Whole-cell voltage clamp measurements of anthrax toxin pore current.

Protective antigen (PA) of anthrax toxin binds cellular receptors and forms pores in target cell membranes, through which catalytic lethal factor (LF) and edema factor (EF) are believed to translocate to the cytoplasm. Using patch clamp electrophysiological techniques, we assayed pore formation by PA in real time on the surface of cultured cells. The membranes of CHO-K1 cells treated with activated PA had little to no electrical conductivity at neutral pH (7.3) but exhibited robust mixed ionic currents in response to voltage stimuli at pH 5.3. Pore formation depended on specific cellular receptors and exhibited voltage-dependent inactivation at large potentials (>60 mV). The pH requirement for pore formation was receptor-specific as membrane insertion occurs at significantly different pH values when measured in cells specifically expressing tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2), the two known cellular receptors for anthrax toxin. Pores were inhibited by an N-terminal fragment of LF and by micromolar concentrations of tetrabutylammonium ions. These studies demonstrated basic biophysical properties of PA pores in cell membranes and served as a foundation for the study of LF and EF translocation in vivo.

A mechanism for the direct regulation of T-type calcium channels by Ca2+/calmodulin-dependent kinase II.

Low-voltage-activated (LVA) Ca2+ channels are widely distributed throughout the CNS and are important determinants of neuronal excitability, initiating dendritic and somatic Ca2+ spikes that trigger and shape the pattern of action potential firing. Here, we define a molecular mechanism underlying the dynamic regulation of alpha1H channels (Cav3.2), by Ca2+/CaM-dependent protein kinase II (CaMKII). We show that channel regulation is selective for the LVA alpha1H Ca2+ channel subtype, depends on determinants in the alpha1H II-III intracellular loop, and requires the phosphorylation of a serine residue absent from unregulated alpha1G (Cav3.1) channels. These studies identify the alpha1H channel as a new substrate for CaMKII and provide the first molecular mechanism for the direct regulation of T-type Ca2+ channels by a protein kinase. Our data suggest a novel mechanism for modulating the integrative properties of neurons.

T-type calcium channel regulation by specific G-protein betagamma subunits.

Low-voltage-activated (LVA) T-type calcium channels have a wide tissue distribution and have well-documented roles in the control of action potential burst generation and hormone secretion. In neurons of the central nervous system and secretory cells of the adrenal and pituitary, LVA channels are inhibited by activation of G-protein-coupled receptors that generate membrane-delimited signals, yet these signals have not been identified. Here we show that the inhibition of alpha1H (Ca(v)3.2), but not alpha(1G) (Ca(v)3.1) LVA Ca2+ channels is mediated selectively by beta2gamma2 subunits that bind to the intracellular loop connecting channel transmembrane domains II and III. This region of the alpha1H channel is crucial for inhibition, because its replacement abrogates inhibition and its transfer to non-modulated alpha1G channels confers beta2gamma2-dependent inhibition. betagamma reduces channel activity independent of voltage, a mechanism distinct from the established betagamma-dependent inhibition of non-L-type high-voltage-activated channels of the Ca(v)2 family. These studies identify the alpha1H channel as a new effector for G-protein betagamma subunits, and highlight the selective signalling roles available for particular betagamma combinations.

Stimulation of recombinant Ca(v)3.2, T-type, Ca(2+) channel currents by CaMKIIgamma(C).

Molecular cloning of low-voltage activated (LVA) T-type calcium channels has enabled the study of their regulation in heterologous expression systems. Here we investigate the regulation of Ca(v)3.2 alpha(1)-subunits (alpha1H) by calcium- and/or calmodulin-dependent protein kinase II (CaMKII). 293 cells stably expressing alpha1H were transiently transfected with CaMKIIgamma(C). Using the whole-cell recording configuration, we observed that elevation of pipette free Ca(2+) (1 microM) in the presence of CaM (2 microM) increases T-type channel activity selectively at negative potentials, evoking an 11 mV hyperpolarizing shift in the half-maximal potential (V(1/2)) for activation. The V(1/2) of channel inactivation is not altered by Ca(2+)/CaM. These effects reproduced modulation observed in adrenal zona glomerulosa cells. The potentiation by Ca(2+)/CaM was dependent on the co-expression of CaMKIIgamma(C) and required Ca(2+)/CaM-dependent kinase activity. Peptide (AIP) and lipophilic (KN-62) protein kinase inhibitors prevented the Ca(2+)/CaM-induced changes in channel gating without altering basal Ca(v)3.2 channel activity (27 nM free Ca(2+)) as did replacing pipette ATP with adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable analogue. CaMKII-dependent potentiation of channel opening resulted in significant increases in apparent steady-state open probability (P(o)) and sustained channel current at negative voltages. Under identical conditions, CaMKII activation did not regulate the activity of Ca(v)3.1 channels, the first cloned member (alpha1G) of the T-type Ca(2+) channel family. Our results provide the first evidence for the differential regulation of two members of the Ca(v)3 family by protein kinase activation and the first report reconstituting CaMKII-dependent regulation of any cloned Ca(2+) channel.