Dengue virus (DENV) causes the most prevalent mosquito-borne viral disease with estimated 3.6 billion people living in risk areas. The control of the growing threat of DENV is hindered by the lack of effective therapies and vaccines. Newly assembled DENV particles undergo maturation to become infectious s. The proteins present on the surface of the dengue immature virus are the envelope (E) protein that is responsible for viral entry and premembrane (prM) protein that consists of an N-terminal pr domain followed, via furin cleavage site, by the M protein. Interactions between the pr portion of prM and E-protein prevent premature viral fusion or inactivation in the acidic intracellular compartments through which virions traffic. The mature DENV envelope contains E protein and M protein, a cleaved derivative of prM. This maturation is required for formation of pre-fusion conformation of E-protein. Following E-protein binding to cell surface receptors, the virus is internalized via endocytosis. In our earlier studies, we dissected the pathway of DENV entry and fusion and identified an anionic lipid specific for late endosomes as an essential lipid cofactor of E-protein fusion machinery. Our findings explained why DENV fuses not in early but in late endosomes, where a combination of acidic pH and anionic lipids facilitates fusogenic restructuring of E from homodimer to homotrimer that brings about fusion between viral envelope and endosomal membrane and, thus, effectively delivers viral RNA into cytosol in the vicinity of the translation-replication sites. Our work also explained why earlier attempts of different laboratories to develop fusion assays for DV fusion have failed. We proceeded to develop an arsenal of high-throughput assays for DENV cell surface binding, internalization and intracellular fusion. In our most recent study (collaboration with Dr. L. Margolis lab) we have focused on the surface protein composition of DENV particles and developed an assay for characterization of the maturation status of individual virions. We examined whether there are any DENV particles that are fully matured, i.e., in which all prM molecules are cleaved, or all the virions are either fully immature with all prM molecules in uncleaved form or mosaic, with the same virions carrying both M protein and prM. To answer this question, we analyzed these proteins on the surface of single virions using a novel high throughput flow virometry technique to characterize the degree of maturation of individual virions in a preparation of infectious DENV. We labeled DENV with a fluorescent lipid probe and then with fluorescent antibody to prM protein; captured labeled virions (operationally defined as as a membrane particle that carries E protein), with small MNPs coupled with an antibody against E protein. We then separated the DENV-MNPs complexes from unbound antibodies using magnetic columns; and eluted DENV-MNPs complexes from the columns. Finally we analyzed the eluted complexes with a flow cytometer. We found that in DENV population produced by BHK-21 cells approximately half of virions are fully mature as no prM could be detected on their surface. In contrast, furin-deficient LoVo cells produced DENV population fully mature virions constitute only 15%. Detailed characterization of the maturation status of DENV released by infected cells is especially important since immature DENV has been found to play a significant role in disease pathogenesis. Note that our approach characterizes the variability between virions within the same population and thus, complements the existing bulk techniques, which describe the composition and properties of an average virion in the preparation. Since the heterogeneity of viral particles may impact their biological properties, we expect the high throughput approach developed here to help in developing vaccine and antivirals for DENV and, possibly, other viruses.
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