Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins

Abstract

BackgroundFlavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E.Methodology/Principal FindingsWe have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant.Conclusions/SignificanceOur data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus.

Highly Conserved Residues in the Helical Domain of Dengue Virus Type 1 Precursor Membrane Protein Are Involved in Assembly, Precursor Membrane (prM) Protein Cleavage, and Entry

“…4B), suggesting that VLPs derived from 293T cells contain immature particles. This is in agreement with a previous report (54). We then used a rabbit anti-M serum (against M peptide) in the Western blot analysis and found a predominant prM band with some M band in the WT VLPs, suggesting that VLPs derived from 293T cells contain predominant immature or partially immature particles and small proportion of mature particles (54,55).…”

“…This is in agreement with a previous report (54). We then used a rabbit anti-M serum (against M peptide) in the Western blot analysis and found a predominant prM band with some M band in the WT VLPs, suggesting that VLPs derived from 293T cells contain predominant immature or partially immature particles and small proportion of mature particles (54,55). The amounts of M protein relative to prM protein in VLPs derived from most (eight of nine) mutants were lower than those of WT except mutant S112A (Fig.…”

High plasma level of nucleocapsid-free envelope glycoprotein-positive lipoproteins in hepatitis C patients

“…36 Subviral, nucleocapsid-free particles, bearing the envelope glycoprotein, are also frequently found during dengue virus or tick-borne encephalitis virus Flavivirus infections. 37,38 Subviral particles appear to exert biologically relevant properties. For example, HBsAg inhibits TLR9-mediated activation and interferon-a production in plasmacytoid dendritic cells (DCs).…”

COBRA-Based Dengue Tetravalent Vaccine Elicits Neutralizing Antibodies Against All Four Dengue Serotypes

“…For other virus systems, such as HIV or influenza, VLPs expressing the envelope glycoproteins are more effective at eliciting high titer neutralizing antibodies than soluble versions [31,[38][39][40][41][42][43][44][45]. Genomeless viral particle vaccines, unlike single proteins, can bind appropriate surface receptors and enter cells in a manner similar to true virus infection [45].…”