Secretion of noninfectious dengue virus-like particles and identification of amino acids in the stem region involved in intracellular retention of envelope protein.
DNA plasmids that express flavivirus premembrane/membrane (prM/M) and envelope (E) proteins in the form of virus-like particles (VLPs) have an excellent potential as DNA vaccine candidates against virus infection. The plasmid-expressed VLPs are also useful as safe, noninfectious antigens in serodiagnostic assays. We have constructed plasmids containing the prM/M and E gene regions for DENV-1, -3, and -4 that express and secrete VLPs when electroporated into Chinese hamster ovary cells. Constructs containing the full-length DENV-1 E protein gene did not secrete VLPs into tissue culture fluid effectively. However, a 16-fold increase in ELISA titers of DENV-1 VLPs was achieved after replacing the carboxy-terminal 20% region of DENV-1 E protein gene with the corresponding sequence of Japanese encephalitis virus (JEV). DENV-3 plasmids containing either the full-length DENV-3 E protein gene or the 20% JEV sequence replacement secreted VLPs to similarly high levels. Whereas DENV-4 VLPs were secreted to high levels by plasmids containing the full-length DENV-4 E protein gene but not by the chimeric plasmid containing 20% JEV E replacement. Domain substitutions by replacing prM/M protein stem-anchor region with the corresponding prM/M stem-anchor region of JEV or DENV-2 in the chimeric DENV-4 construct failed to promote the secretion of DENV-4 VLPs. Using the DENV-2 chimeric plasmid with carboxy-terminal 10% of JEV E gene, the sequence responsible for intracellular localization of E protein was mapped onto the E-H1 alpha-helix domain of DENV-2 E protein. Substitution of three amino acids from the DENV-2 sequence to the corresponding amino acids in the JEV sequence (I398L, M401A, and M412L) in the E-H1 was sufficient to promote extracellular secretion and resulted in detectable titers of DENV-2 VLP secretion.
Purdy
David E
DE
Arbovirus Diseases Branch, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, PO Box 2087, Fort Collins, CO 80522, USA.
Chang
Gwong-Jen J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002678:Chimera / Q000235:genetics; D006224:Cricetinae; D004279:DNA, Viral / Q000235:genetics; D003716:Dengue Virus / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000502:physiology; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D004279:DNA, Viral; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins