Processing of dengue virus type 2 structural proteins containing deletions in hydrophobic domains

Abstract

The 5' end of the genome of the dengue virus type 2 encoding the structural proteins was expressed using recombinant vaccinia virus. Three additional recombinants derived by deletion of selected dengue sequences within the parental construct were also expressed. They were designed to assess the role of hydrophobic domains in the processing of the viral polyprotein in intact cells. The first construct contained a deletion of nucleotides encoding most of the C protein; nucleotides encoding the hydrophobic domain at the carboxy terminus were retained. The second and third constructs contained smaller deletions of 72 bp and 129 bp encoding hydrophobic domains at the carboxy termini of C and prM respectively. Indirect immunofluorescence and radioimmunoprecipitation were used to detect prM and E in cells infected with recombinant viruses. The results showed that deletion of 90% of C had no apparent effect on the processing of prM and E, and that the signal sequence for E at the carboxy terminus of prM was active in the absence of the upstream signal sequence for prM at the carboxy terminus of C. Deletion of the hydrophobic sequences preceding the amino terminus of E prevented cleavage at the prM-E junction. These results obtained using infected cells were consistent with the published findings for the translation of flavivirus RNA in vitro, and indicated the importance of membrane association in the cleavage of structural proteins from the flavivirus polyprotein. In addition, cells infected with the recombinant virus containing the large deletion in the C coding region released the E glycoprotein into the culture medium.

Histidine 39 in the dengue virus type 2 M protein has an important role in virus assembly

“…The culture medium and cell lysate samples were centrifuged at 15 000 g for 8 min at 4 uC to remove cellular debris and stored at 270 uC until required for further analysis. Proteins contained in radiolabelled samples were analysed by radioimmunoprecipitation (RIP) using a mixture of anti-E mAbs followed by SDS-PAGE, as described previously (Gruenberg & Wright, 1992;Lin et al, 1994;Pryor et al, 1998). Antigen-antibody complexes were allowed to form overnight at 4 uC and then captured by using protein A-Sepharose CL-4B (Amersham Biosciences).…”

Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication

“…Approximately 7 to 10 g of transcript RNA and 50 g of carrier tRNA were electroporated into BHK-21 cells, which were then incubated at 33 or 37°C. The cells were examined for immunofluorescence 4 to 6 days later using anti-E monoclonal antibodies (20). At 7 days the culture medium was used to infect C6/36 cells.…”

Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA.

“…These clones were pDS419, a chimeric DEN-2 construct which had been produced by ligation of cDNA derived from NGC DEN-2 (nt 31-402) and PUO-218 DEN-2 (nt 403-2477) (Gruenberg & Wright, 1992), and pD1046, pDEN34 and pDEN75, corresponding to nt 2341-6345, 6859-8060 and 7881-9236 of NGC DEN-2, respectively. RT-PCR was used to synthesize cDNA fragments corresponding to regions of the NGC DEN-2 genome not represented by the pre-existing cDNA clones.…”

“…At 4-6 days post-electroporation, virus replication was assayed by monitoring the production of E using a mix of anti-E monoclonal antibodies by indirect immunofluorescence as described previously (Gruenberg & Wright, 1992).…”