title: Characterization of the ectodomain of the envelope protein of dengue virus type 4: expression, membrane association, secretion and particle formation in the absence of precursor membrane protein.
abstract: BACKGROUND
  The envelope (E) of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. After biosynthesis E protein forms a heterodimer with precursor membrane (prM) protein. Recent reports of infection enhancement by anti-prM monoclonal antibodies (mAbs) suggest anti-prM responses could be potentially harmful. Previously, we studied a series of C-terminal truncation constructs expressing DENV type 4 prM/E or E proteins and found the ectodomain of E protein alone could be recognized by all 12 mAbs tested, suggesting E protein ectodomain as a potential subunit immunogen without inducing anti-prM response. The characteristics of DENV E protein ectodomain in the absence of prM protein remains largely unknown.
  
  
  RESULTS
  In this study, we investigated the expression, membrane association, glycosylation pattern, secretion and particle formation of E protein ectodomain of DENV4 in the presence or absence of prM protein. E protein ectodomain associated with membrane in or beyond trans-Golgi and contained primarily complex glycans, whereas full-length E protein associated with ER membrane and contained high mannose glycans. In the absence of prM protein, E protein ectodomain can secrete as well as form particles of approximately 49 nm in diameter, as revealed by sucrose gradient ultracentrifugation with or without detergent and electron microscopy. Mutational analysis revealed that the secretion of E protein ectodomain was affected by N-linked glycosylation and could be restored by treatment with ammonia chloride.
  
  
  CONCLUSIONS
  Considering the enhancement of DENV infectivity by anti-prM antibodies, our findings provide new insights into the expression and secretion of E protein ectodomain in the absence of prM protein and contribute to future subunit vaccine design.
authors.affiliation: Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
authors.forename: Szu-Chia
authors.initials: SC
authors.lastname: Hsieh
authors.affiliation: Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
authors.forename: Wen-Yang
authors.initials: WY
authors.lastname: Tsai
authors.affiliation: Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America; Pacific Center for Emerging Infectious Diseases Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
authors.forename: Vivek R
authors.initials: VR
authors.lastname: Nerurkar
authors.affiliation: Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America; Pacific Center for Emerging Infectious Diseases Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
authors.forename: Wei-Kung
authors.initials: WK
authors.lastname: Wang
mesh_terms: D002460:Cell Line; D003715:Dengue / Q000235:genetics / Q000473:pathology / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D008566:Membranes / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D011498:Protein Precursors; D014759:Viral Envelope Proteins; C466981:glycoprotein E, dengue virus type 4