title: C-terminal helical domains of dengue virus type 4 E protein affect the expression/stability of prM protein and conformation of prM and E proteins.
abstract: BACKGROUND
  The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines.
  
  
  RESULTS
  In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested.
  
  
  CONCLUSIONS
  A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response.
authors.affiliation: Department of Tropical Medicine, Medical Microbiology and Pharmacology, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
authors.forename: Wen-Yang
authors.initials: WY
authors.lastname: Tsai
authors.forename: Szu-Chia
authors.initials: SC
authors.lastname: Hsieh
authors.forename: Chih-Yun
authors.initials: CY
authors.lastname: Lai
authors.forename: Hong-En
authors.initials: HE
authors.lastname: Lin
authors.forename: Vivek R
authors.initials: VR
authors.lastname: Nerurkar
authors.forename: Wei-Kung
authors.initials: WK
authors.lastname: Wang
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry; D000914:Antibodies, Viral / Q000097:blood / Q000737:chemistry; D000915:Antibody Affinity; D003715:Dengue / Q000097:blood / Q000276:immunology; D003716:Dengue Virus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D017433:Protein Structure, Secondary; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D000914:Antibodies, Viral; D014759:Viral Envelope Proteins; C466981:glycoprotein E, dengue virus type 4