Single-molecule studies of flavivirus envelope dynamics: Experiment and computation.

Flaviviruses are simple enveloped viruses exhibiting complex structural and functional heterogeneities. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology approaches. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. Here, we review different single-molecule sensitive fluorescence methods that are employed to investigate flaviviruses. In particular, we review how (i) time-resolved Förster resonance energy transfer (trFRET) was applied to probe dengue envelope conformations; (ii) FRET-fluorescence correlation spectroscopy to investigate dengue envelope intrinsic dynamics and (iii) single particle tracking to follow the path of dengue viruses in cells. We also discuss how such methods may be supported by molecular dynamics (MD) simulations over a range of spatio-temporal scales, to provide complementary data on the structure and dynamics of flaviviral systems. We describe recent improvements in multiscale MD approaches that allowed the simulation of dengue particle envelopes in near-atomic resolution. We hope this review is an incentive for setting up and applying similar single-molecule studies and combine them with MD simulations to investigate structural dynamics of entire flavivirus particles over the nanosecond-to-millisecond time-scale and follow viruses during infection in cells over milliseconds to minutes.