Shake, rattle, and roll: Impact of the dynamics of flavivirus particles on their interactions with the host

Abstract

Remarkable progress in structural biology has equipped virologists with insight into structures of viral proteins and virions at increasingly high resolution. Structural information has been used extensively to address fundamental questions about virtually all aspects of how viruses replicate in cells, interact with the host, and in the design of antiviral compounds. However, many critical aspects of virology exist outside the snapshots captured by traditional methods used to generate high-resolution structures. Like all proteins, viral proteins are not static structures. The conformational flexibility and dynamics of proteins play a significant role in protein-protein interactions, and in the structure and biology of virus particles. This review will discuss the implications of the dynamics of viral proteins on the biology, antigenicity, and immunogenicity of flaviviruses.

Keywords: Antibody-mediated neutralization; Dengue virus; Flavivirus; Structural dynamics; Viral breathing; West Nile virus.

Figure 1. Structure of the picornavirus capsid and location of the hydrophobic pocket

The left panel shows a color-coded C-alpha backbone model of the basic repeating unit of a picornavirus virion. The four capsid proteins, VP1, 2, 3, and 4 are color coded representing each of the proteins with VP4 being found on the interior of the particle apparently protected from immune surveillance. 60 copies of this repeating unit are organized as an icosahedron (shown as the particle on the left). At each vertex, five copies of VP1 come together and create a depression or “canyon” where many receptors have been shown to bind. The right panel shows a ribbon diagram of a picornavirus VP1 capsid protein showing the location of a small molecule inhibitor binding into the hydrophobic pocket normally occupied by pocket factor. The binding site is underneath the canyon floor and binding of inhibitor limits the flexibility and dynamics of the particle.

Figure 2. The structure, arrangement, and dynamic motion of E proteins on the mature flavivirus virion

(A) Top view of the flavivirus E protein monomer shown as a ribbon diagram, with domains I, II, and III (DI-DIII) colored in red, yellow, and blue, respectively. The conserved fusion loop at the distal tip of domain II (DII-FL) is colored in green. Not shown are the helical stem and transmembrane anchor located at the carboxy terminus of DIII. (B) The surface of the mature virion contains 90 sets of head to tail E protein dimers arranged with pseudo T=3 icosahedral symmetry. Domains are colored as in panel A. The flavivirus virion must strike a balance between protection of the RNA genome during transmission and the ability to disassemble the virus structure during the entry process. The strength of the contacts between E proteins that hold together the virion structure may differ among viral strains due to sequence variation, with the potential to increase or decrease the extent of conformational flexibility. The effects of this may be relevant in terms of amino acid contacts (B) between E protein rafts, (C) between the E homodimers that comprise the rafts, (D) at the E dimer interface, and (E) at the level of an individual E protein.

Figure 3. Flavivirus dynamics results in reversible and non-reversible conformational changes

Flavivirus neutralization is governed by a stoichiometric threshold and requires binding by a critical number of antibody molecules per virion. Of the 180 potential E protein epitopes (x-axis, bottom of figure), estimates suggest that this threshold corresponds to docking on the virion by 30 antibodies (Pierson et al., 2007). In addition to antibody affinity, epitope accessibility is a critical factor that determines whether neutralization occurs. Because of the dense arrangement of E proteins on the surface of the mature virion, not all E protein epitopes are equally accessible for antibody binding. For some characterized antibodies, neutralization cannot be achieved even in the presence of saturating concentrations of antibody due to the lack of accessible epitopes (Nelson et al., 2008). Flavivirus dynamics can modulate the landscape available for antibody binding by transiently exposing otherwise inaccessible epitopes and increasing the absolute number available for binding. Time- and temperature-dependent increases in neutralization are observed when virus and antibody are incubated for increasing lengths of time (left panel; Ref= reference curve obtained after pre-incubation of virus with serial dilutions of antibody for 1 hour; additional antibody virus complexes were further incubated at 37°C for 5–26 hours before infecting target cells). This particular antibody binds an epitope shown to be inaccessible on the mature form of the virus. Time-dependent increases in neutralization potency reflect exposure of this otherwise ‘cryptic’ epitope through the process of virus breathing. In addition to reversible changes in virus structure (green arrows), we hypothesize that a subset of conformational transitions sample non-infectious structures that can no longer return to an infectious state (red arrow). These ‘dead-end’ structures are detected experimentally as a loss of virus infectivity over time in solution, referred to as intrinsic decay. Differences in the rate of intrinsic decay (shown for WNV and DENV, right panel) among virus strains indicate that sequence variation dictates the steady state virus structures and/or the ensembles available for virus breathing.