title: Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  We 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
  Our 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.
authors.affiliation: Hong Kong University-Pasteur Research Centre, The University of Hong Kong, Hong Kong, China. pgwang@hkucc.hku.hk
authors.forename: Pei-Gang
authors.initials: PG
authors.lastname: Wang
authors.forename: Mateusz
authors.initials: M
authors.lastname: Kudelko
authors.forename: Joanne
authors.initials: J
authors.lastname: Lo
authors.forename: Lewis Yu Lam
authors.initials: LY
authors.lastname: Siu
authors.forename: Kevin Tsz Hin
authors.initials: KT
authors.lastname: Kwok
authors.forename: Martin
authors.initials: M
authors.lastname: Sachse
authors.forename: John M
authors.initials: JM
authors.lastname: Nicholls
authors.forename: Roberto
authors.initials: R
authors.lastname: Bruzzone
authors.forename: Ralf M
authors.initials: RM
authors.lastname: Altmeyer
authors.forename: Béatrice
authors.initials: B
authors.lastname: Nal
mesh_terms: D002499:Centrifugation, Density Gradient; D003062:Codon / Q000235:genetics; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000821:virology; D005814:Genes, Viral / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D014176:Protein Biosynthesis; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D055571:Secretory Pathway; D012703:Serotyping; D013347:Subcellular Fractions / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism*; D019065:Virus Assembly / Q000502:physiology*
chemical_list: D003062:Codon; D034741:RNA, Small Interfering; D014764:Viral Proteins