A strong endoplasmic reticulum retention signal in the stem–anchor region of envelope glycoprotein of dengue virus type 2 affects the production of virus-like particles

Abstract

Recombinant virus-like particles (VLPs) of flaviviruses have been shown to be produced efficiently by co-expressing the precursor membrane (PrM) and envelope (E) proteins with few exceptions, such as dengue virus type 2 (DENV2). It was reported previously that chimeric DENV2 PrM/E construct containing the stem-anchor region of E protein of Japanese encephalitis virus (JEV) produced VLPs efficiently (Chang, G. J., Hunt, A. R., Holmes, D. A., Springfield, T., Chiueh, T. S., Roehrig, J. T., and Gubler, D. J. 2003. Enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis virus. Virology 306, 170-180.). We investigated the mechanisms involved and reported that compared with authentic DENV2 PrM/E-expressing cells, E protein in chimeric DENV2 PrM/E-expressing cells was also present in an endoglycosidase H (endo H)-resistant compartment and has shifted more to the pellets of the soluble fraction. Replacement of the transmembrane and cytoplasmic domains of CD4 with the stem-anchor of DENV2 (CD4D2) or JEV (CD4JEV) rendered the chimeric CD4 retained predominantly in the endoplasmic reticulum (ER). Flow cytometry revealed higher proportion of CD4JEV than CD4D2 expressed on the cell surface. Together, these findings suggested that the stem-anchor of DENV2 contained an ER retention signal stronger than that of JEV, which might contribute to the inefficient production of DENV2 VLPs. Moreover, co-expression of C protein can enhance the production of DENV2 VLPs, suggesting a mechanism of facilitating viral particle formation during DENV2 replication.

The Helical Domains of the Stem Region of Dengue Virus Envelope Protein Are Involved in both Virus Assembly and Entry

“…The prM/E expression construct of DENV4 (pCB-CD4) was described previously (15,16). To generate mutants of the stem region, a two-step PCR mutagenesis was performed using pCB-D4 as template and primers d4EI398P-A2, d4EI398P-B1, d4EM401P-A2, d4EM401P-B1, d4ET405P-A2, d4ET405P-B1, d4EG408P-A2, d4EG408P-B1, d4EM412P-A2, d4EM412P-B1, d4EL415P-A2, d4EL415P-B1, d4ET418P-A2, d4ET418P-B1, d4EF422P-A2, d4EF422P-B1, d4EV425P-A2, d4EV425P-B1, d4EF429P-A2, d4EF429P-B1, d4EL432P-A2, d4EL432P-B1, d4EV436P-A2, d4EV436P-B1, d4EV439P-A2, d4EV439P-B1, d4EV443P-A2, d4EV443P-B1, d4ET446P-A2, and d4ET446P-B1, as described previously (16).…”

“…293T cells, prepared in a 10-cm culture dish at 5 ϫ 10 5 cells per dish 1 day earlier, were transfected with 10 g of plasmid DNA by the calcium phosphate method. At 48 h posttransfection, culture supernatants were collected (see below) and cells were washed with phosphate-buffered saline (PBS) and treated with 1% NP-40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000 ϫ g and 4°C for 30 min to obtain cell lysates (15). Culture supernatants were clarified by centrifugation at 1,250 ϫ g for 20 min, filtered through a 0.22-m-pore-sized membrane (Sartorius), layered over a 20% sucrose buffer, and ultracentrifuged at 65,000 ϫ g and 4°C for 5 h to obtain pellets, which were resuspended in 30 l TNE (Tris-NaCl-EDTA) buffer (15).…”

“…At 48 h posttransfection, culture supernatants were collected (see below) and cells were washed with phosphate-buffered saline (PBS) and treated with 1% NP-40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000 ϫ g and 4°C for 30 min to obtain cell lysates (15). Culture supernatants were clarified by centrifugation at 1,250 ϫ g for 20 min, filtered through a 0.22-m-pore-sized membrane (Sartorius), layered over a 20% sucrose buffer, and ultracentrifuged at 65,000 ϫ g and 4°C for 5 h to obtain pellets, which were resuspended in 30 l TNE (Tris-NaCl-EDTA) buffer (15). For Western blot analysis, cell lysates or pellets were added to nonreducing buffer (2% SDS, 0.5 M Tris [pH 6.8], 20% glycerol, 0.001% bromophenol blue [final concentrations]) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by transfer to a nitrocellulose membrane, blocking, and incubation with primary antibody, serum from a confirmed human dengue case or anticalnexin mouse monoclonal antibody (MAb) E-10 (Santa Cruz), and secondary antibody (15,55).…”

“…Culture supernatants were clarified by centrifugation at 1,250 ϫ g for 20 min, filtered through a 0.22-m-pore-sized membrane (Sartorius), layered over a 20% sucrose buffer, and ultracentrifuged at 65,000 ϫ g and 4°C for 5 h to obtain pellets, which were resuspended in 30 l TNE (Tris-NaCl-EDTA) buffer (15). For Western blot analysis, cell lysates or pellets were added to nonreducing buffer (2% SDS, 0.5 M Tris [pH 6.8], 20% glycerol, 0.001% bromophenol blue [final concentrations]) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by transfer to a nitrocellulose membrane, blocking, and incubation with primary antibody, serum from a confirmed human dengue case or anticalnexin mouse monoclonal antibody (MAb) E-10 (Santa Cruz), and secondary antibody (15,55). After the final wash, the signals were detected by enhanced chemiluminescence reagents (Perkin Elmer Life Sciences).…”

Production of single-round infectious chimeric flaviviruses with DNA-based Japanese encephalitis virus replicon

“…In contrast, production of DENV-and YFV-SRIPs was less efficient. The low infectious titre of SRIPs containing at least dengue prM-E may be explained by the low specific infectivity of particles encapsidated in DENV envelope protein (van der Schaar et al, 2007;Winkelmann et al, 2011), although we were unable to exclude the possibility that the viral assembly and/or secretion with dengue prM-E is not efficient (Chang et al, 2003;Hsieh et al, 2008). Adaptive mutations in structural and NS proteins could possibly enhance the production of infectious particles by improving the specific infectivity of the resulting particles (Winkelmann et al, 2011).…”