Growth restriction of dengue virus type 2 by site-specific mutagenesis of virus-encoded glycoproteins.

Abstract

The three flavivirus glycoproteins prM, E and NS1 are formed by post-translational cleavage and are glycosylated by the addition of N-linked glycans. NS1 may form homodimers, whereas E may form homodimers, homotrimers or heterodimers (prM-E). Modification of these processes by mutagenesis of the proteins has the potential to generate viruses that are restricted in growth and are possible vaccine candidates. Using an SV40-based expression system, we previously analysed dimerization and secretion of the NS1 protein of dengue virus type 2 (DEN-2) with mutations in the conserved Cys residues, or within hydrophilic or hydrophobic regions, or at glycosylation sites. In this study, mutations which reduce cleavage at the DEN-2 prM/E signalase cleavage site are described. On the

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

“…Growth of BHK-21, Aedes albopictus C6/36 and COS cells has been described previously Pryor et al, 1998). Stocks of DENV-2 viruses were grown and titrated by plaque assay in C6/36 cells at 28 uC as described previously .…”

“…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).…”

“…The plasmid pSVprM-E, containing cDNA derived from pDVWS501 encoding the prM signal sequence and prM and E proteins, was used to transfect COS cells. The kinetics of production, association and secretion of the prM and E proteins were examined by pulse-chase labelling with [ 35 S]methionine/cysteine and RIP using a mixture of anti-E mAbs that had previously been used to coprecipitate the prM protein with the E protein from lysates of DENV-2-infected cells (Lin et al, 1994;Pryor et al, 1998). Proteins with molecular masses of 19-20 and 60-61 kDa, corresponding to the glycosylated forms of the prM and E proteins, were detected in all samples, both intra-and extracellular (Fig.…”

Replication of dengue virus type 2 in human monocyte-derived macrophages: comparisons of isolates and recombinant viruses with substitutions at amino acid 390 in the envelope glycoprotein.

“…The procedures for transcription of RNA from plasmids, electroporation into BHK-21 cells, and passaging of virus in Aedes albopictus C6/36 cells were described previously. 29,33 The culture medium from BHK-21 cells that had been electroporated with transcribed RNA and maintained for 5-7 days was passaged twice in C6/36 cells to prepare virus stocks for infection of MDMs. The stocks were assayed in C6/36 cells and analyzed by reverse transcriptase-polymerase chain reaction and sequencing over a short segment of the E gene to confirm the presence of the desired mutation.…”

Flaviviral Infections and Potential Targets for Antiviral Therapy

“…In course of infection with flaviviruses the polyprotein is cleaved co-and post-translationally by proteases (Pryor et al, 1998). The amino terminus of the polyprotein is processed into 3 structural proteins.…”