The ganglioside GM1a functions as a coreceptor/attachment factor for dengue virus during infection.

Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routes by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. We studied the interaction of the virus with GM1a using fluorescence correlation spectroscopy, fluorescence crosscorrelation spectroscopy, imaging fluorescence correlation spectroscopy, amide hydrogen/deuterium exchange mass spectrometry, and isothermal titration calorimetry. Additionally, we explored the effect of this interaction on infectivity and movement of the virus during infection was explored using plaque assay and fluorescence-based imaging and single particle tracking. GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a 10-fold and 7-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems, respectively. We determined that the interaction of the virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection. Collectively, our results suggest that GM1a functions as a coreceptor/attachment factor for DENV during infection in mammalian systems.

Hyperosmotic Stress Allosterically Reconfigures Betaine Binding Pocket in BetP.

The transporter BetP in C. glutamicum is essential in maintaining bacterial cell viability during hyperosmotic stress and functions by co-transporting betaine and Na+ into bacterial cells. Hyperosmotic stress leads to increased intracellular K+ concentrations which in turn promotes betaine binding. While structural details of multiple end state conformations of BetP have provided high resolution snapshots, how K+ sensing by the C-terminal domain is allosterically relayed to the betaine binding site is not well understood. In this study, we describe conformational dynamics in solution of BetP using amide hydrogen/deuterium exchange mass spectrometry. These reveal how K+ alters conformation of the disordered C- and N-terminal domains to allosterically reconfigure transmembrane helices 3, 8, and 10 to enhance betaine interactions. A map of the betaine binding site, at near single amino acid resolution, reveals a critical extrahelical H-bond mediated by TM3 with betaine.

Aurora-A Breaks Symmetry in Contractile Actomyosin Networks Independently of Its Role in Centrosome Maturation.

Cell polarity is facilitated by a rearrangement of the actin cytoskeleton at the cell cortex. The program triggering the asymmetric remodeling of contractile actomyosin networks remains poorly understood. Here, we show that polarization of Caenorhabditis elegans zygotes is established through sequential downregulation of cortical actomyosin networks by the mitotic kinase, Aurora-A. Aurora-A accumulates around centrosomes to locally disrupt the actomyosin contractile activity at the proximal cortex, thereby promoting cortical flows during symmetry breaking. Aurora-A later mediates global disassembly of cortical actomyosin networks, which facilitates the initial polarization through suppression of centrosome-independent cortical flows. Translocation of Aurora-A from the cytoplasm to the cortex is sufficient to interfere with the cortical actomyosin networks independently of its roles in centrosome maturation and cell-cycle progression. We propose that Aurora-A activity serves as a centrosome-mediated cue that breaks symmetry in actomyosin contractile activity, and facilitates the initial polarization through global suppression of cortical actomyosin networks.

Infectivity of Dengue Virus Serotypes 1 and 2 Is Correlated with E-Protein Intrinsic Dynamics but Not to Envelope Conformations.

Dengue is a mosquito-borne virus with dire health and economic impacts. Dengue is responsible for an estimated 390 million infections per year, with dengue 2 (DENV2) being the most virulent strain among the four serotypes. Interestingly, it is also in strains of this serotype that temperature-dependent large-scale morphological changes, termed "breathing," have been observed. Although the structure of these morphologies has been solved to 3.5-Å resolution, the dynamics of the viral envelope are unknown. Here, we combine fluorescence and mass spectrometry with molecular dynamics simulations to provide insights into DENV2 (NGC strain) structural dynamics in comparison with DENV1 (PVP 159). We observe hitherto unseen conformational changes and structural dynamics of the DENV2 envelope that are influenced by both temperature and divalent cations. Our results show that for DENV2 and DENV1 the intrinsic dynamics, but not the specific morphologies, are correlated with viral infectivity.

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.