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.
Highlights
Dengue is one of the most important vector-borne viral diseases in humans
To generate dengue virus (DV) recombinant subviral particles (RSPs), we first transfected HeLa cells with native DV1 prME gene derived from the DV1 FGA/NA d1d [24] strain, which contained the signal sequence of vesicular stomatitis virus G (VSV-G) envelope glycoprotein inserted in frame upstream of the prME cDNA. 48 hours post-transfection, expression of dengue E protein was monitored by flow cytometry on permeabilized transfected HeLa cells with the 4E11 monoclonal antibody against dengue E protein
Analysis of the prME gene sequence revealed that 15% of nucleotide triplets were rarely used in mammalian cells so that its codon usage was not adapted for efficient expression in this cell system
Summary
The interaction between dengue virus (DV) and host cells is only partly understood. There are four serotypes of dengue virus (DV1-4). Their genome encodes a polyprotein precursor of at least seven non-structural proteins and three structural proteins which are the capsid protein (C), the membrane protein (M) and the envelope glycoprotein (E) [1]. Flavivirus 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
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