Partial Nucleotide Sequence and Deduced Amino Acid Sequence of the Structural Proteins of Dengue Virus Type 2, New Guinea C and PUO-218 Strains

We constructed two recombinant Autographa californica nuclear polyhedrosis baculoviruses. Spodoptera frugiperda (Sf9) cells containing these constructs produce carboxy-terminally truncated envelope E proteins representing dengue (DEN) virus serotypes 2 and 3. The two recombinant proteins contained their homologous signal sequences at the N terminus and were truncated by 71 and 74 amino acids at the C terminus, respectively. This allowed the translocation of the recombinant proteins to the endoplasmic reticulum followed by glycosylation processing and secretion into the extracellular medium. An additional unglycosylated form which was not secreted was detected inside the infected Sf9 cells. Sera from Swiss mice immunized with the infected St9 cell lysates gave a DEN cross-reactive response in ELISA and substantial amounts of neutralizing antibodies to the homologous virus. Similar antibody titres were obtained when the two recombinant proteins were inoculated concomitantly. BALB/c mice were vaccinated with three doses of the recombinant E proteins, taken as monovalent or bivalent immunogens, and challenged with mouse-adapted DEN-2 virus. DEN-2 E protein induced a good protection (90 %) against lethal encephalitis and recombinant DEN-3 E protein gave a substantial cross-protection (54%). Eighty-two percent of the mice immunized with a mixture of both recombinant E proteins survived the DEN-2 virus challenge.

Section: Discussionmentioning

confidence: 99%

Analysis of C-terminally truncated dengue 2 and dengue 3 virus envelope glycoproteins: processing in insect cells and immunogenic properties in mice

Delenda¹,

Staropoli²,

Frenkiel³

et al. 1994

“…Cells were radiolabelled for 1 h as above. After a 20 h chase the medium was harvested, clarified by a low speed (Gruenberg et al, 1988) inserted into pRIT2T. Numbers indicate the first nucleotide of a gene and correspond to those of Irie et al (1989).…”

Section: Cells and Virusmentioning

confidence: 99%

“…Construction of expression plasmids, cDNA fragments of the New Guinea C strain of DEN-2 (Gruenberg et al, 1988) were inserted into the unique BamHI site of the plasmid expression vector pRIT2T (Nilsson et al, 1985;Lowenadler et al, 1986) purchased from Pharmacia LKB Biotechnology (Fig. 1).…”

Section: Cells and Virusmentioning

confidence: 99%

Processing of the dengue virus type 2 proteins prM and C-prM

Murray

Aaskov

Wright

1993

Self Cite

A glycoprotein C-prM of 35000 M r was immunoprecipitated from lysates ofAedes albopictus cells infected with dengue virus type 2 (DEN-2) using antisera directed against the C protein or an amino-terminal fragment of the prM glycoprotein. C-prM was not detected in infected Vero cells. The prM glycoprotein synthesized in infected A. albopictus and Vero cells was cleaved to produce the membrane-associated virion protein (M) and the non-M fragment (pr) immediately preceding or occurring simultaneously with the release of viral particles from cells. The cleavage was less efficient in mosquito cells. The pr fragment was found only in the medium and was not rapidly degraded. To obtain prspecific and M-specific antisera for these studies, proteins containing fragments of DEN-2 prM fused with staphylococcal Protein A were synthesized in Escherichia coli using the expression vector pRIT2T. The fusion proteins were stable and were used to raise antisera in rabbits for immunoprecipitation of radiolabelled cell extracts and culture medium. This is the first report of the detection of a C-prM protein in flavivirus-infected cells and the identification of the pr component of prM.

“…The primary structure of the E protein for 10 flaviviruses has been deduced from cloning and sequencing of viral cDNAs [YF: Rice et al, 1985;West Nile (WN) : Wengler et al, 1985;MVE: Dalgarno et al, 1986;JE: Sumiyoshi et al, 1986;McAda et al, 1987;DEN-l: Mason et al, 1987b;DEN-2: Deubel et al, 1986;Hahn et al, 1988;Gruenberg et al, 1988;DEN-4: Zhao et al, 1986;SLE : Trent et al, 1987;Kunjin (KUN): Coia et al, 1988 ;TBE : Mandl et al, 1988]. There is significant heterogeneity in the E protein sequences among these viruses, as indicated by overall relatedness values of 45 to 80~.…”

Section: Introductionmentioning

confidence: 99%

Molecular Characterization of a Neutralizing Domain of the Japanese Encephalitis Virus Structural Glycoprotein

Mason

Dalrymple

Gentry³

et al. 1989

SUMMARYExpression of antigenic fragments of the Japanese encephalitis virus envelope protein (E) in Escherichia coli has been used to define the boundaries of an antigenic domain that contains the binding sites for 10 anti-E monoclonal antibodies (MAbs). All of these antibodies neutralized the virus in vitro and some of them passively protected mice from a fatal virus challenge. We have shown previously that nine of these antibodies react with the antigenic determinants encoded by a 405 bp fragment of viral cDNA. To determine the amino acid sequences of specific determinants, truncated polypeptides were expressed as fusion proteins in E. coli following progressive Bal 31 exonuclease digestion of the 5' and 3' ends of the cDNA fragment. Examination of the immunoreactivity of these polypeptides revealed that the region from methionine 303 to tryptophan 396 was the shortest sequence capable of reacting with any of the I0 MAbs or with a polyclonal, antiviral hyperimmune mouse ascitic fluid. Biochemical tests showed that an intramolecular disulphide cross-linkage between cysteine 304 and cysteine 335 of the E protein sequence was required for presentation of the binding site(s) for these MAbs. Although this 95 amino acid antigenic domain appeared to be capable of forming several conformational neutralizing epitopes, it was not an effective immunogen for inducing neutralizing or protective antibodies in mice.