Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins - PubMed (original) (raw)
Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins
Pei-Gang Wang et al. PLoS One. 2009.
Abstract
Background: Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E.
Methodology/principal findings: We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant.
Conclusions/significance: Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Optimization of codon usage of DV1 prME gene increases its expression level in mammalian cells.
HeLa cells were transfected with either native prME gene (A) or prME-opt gene (B). Dotted lines represent the mock control (HeLa cells transfected with pcDNA empty vector). Transfected cells were permeabilized, stained with the 4E11 anti-E monoclonal antibody and FITC-conjugated secondary antibody and analyzed by flow cytometry. The mean fluorescence intensity (MFI) and percentage of positive cells are indicated.
Figure 2. Subcellular localization of the DV1 E protein in HeLa-prME cells.
HeLa-prME cells were fixed, permeabilized, and stained for E protein (green) and for cellular marker antigens (red). DAPI staining was used to label cell nuclei (blue). Erp72, ERGIC-53 and Golgin-97 are proteins of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and Golgi apparatus, respectively. The scale bar represents 10 µm.
Figure 3. Electron microscopy analysis of the HeLa-prME cell line.
HeLa-prME (DV1) cells were fixed and either prepared for epon embedding (A), or for immuno labeling on thawed cryosections (B–D). A), Round particles are found aligned in the lumen of the ER (arrows) together with tubular structures (arrowheads). B) Labeling for the E protein is present in the lumen of cisternae. It is localized to electron lucent, round particles (arrows) and tubular structures (arrowheads). In addition E protein is also found on amorphous, electron dense material present in these cisternae (asterix). C) Double labeling of E protein (10 nm gold, black arrows) and the ER marker calreticulin (15 nm gold, white arrows). Calreticulin is present on the limiting membrane of the cisternae, which contain round particles positive for E protein labeling. D) Label for the E protein is also found within the Golgi stack (G) and on vesicles in the Golgi area. N = nucleus, all scale bars represent 200 nm.
Figure 4. Biochemical analysis of DV1 recombinant subviral particles.
The cell lysate (CL) and supernatant (SN) of the HeLa-prME stable cell line were analyzed by Western blot using the 4E11 anti-E monoclonal antibody (A, B, F, G), anti-DV1 mouse IgG (C, D) or by silver-staining (E). A) Detection of E protein in CL and SN of HeLa-prME cell line. B) Homodimer of E could not be detected when samples were heated in presence of dithiothreitol. C) The prM protein could be detected with the anti-DV1 serum in CL but not SN samples. D) In the supernatant of HeLa-prME cells cultured in the presence of NH4Cl, the prM protein was also detected. E) The E protein, prM protein and M protein could be observed with silver staining in supernatant from HeLa-prME cell but not in parental HeLa cells (CSN). F, G) E protein in SN bears complex sugar N-glycans. SN and CL samples were treated with PNGase F (panel F) or EndoH (panel G) and subjected to Western blot analysis using 4E11 antibody. E protein in SN but not CL is resistant to EndoH treatment, indicating acquisition of complex sugars in the Golgi apparatus.
Figure 5. Dynamic study of RSPs secretion.
HeLa-prME cells were starved overnight, pulsed with medium containing S35-methionine for one hour and then cultured for 0, 4, 8 and 24 hours in complete medium. Cell supernatants (A) and lysates (B) were subjected to immunoprecipitation with anti-E 4E11 antibody. Proteins were separated on SDS-PAGE, and revealed by X-ray autoradiography (3 days exposure).
Figure 6. Sucrose gradient analysis of DV1 RSPs.
The supernatant from HeLa-prME cells was concentrated and resuspended in PBS or 0.5% Triton-X 100 containing PBS. RSPs were then centrifuged in a 20 to 60% sucrose gradient at 28,000 rpm (Beckman SW-41Ti rotor) for 2.5 hours at 4°C. Fractions of 0.5 ml were collected and E content was measured using CLDB. The percentage of E protein in each fraction is displayed on the Y axis.
Figure 7. Application of the DV RSP-producing HeLa-prME cell line and CLDB to screen a small library of siRNA which targets 122 genes involved in membrane trafficking.
A) The correlation between luminescence density and amount of E protein on PVDF membrane in CLDB assay. B) The screen results of the siRNA library which targets 122 genes involved in membrane trafficking. Non-targeting siRNA (NT) and siRNA targeting DV1 prME were used as controls. Level of E protein in cell supernatant of each siRNA was expressed as its ratio to that of NT controls. Two-sided Student's t test was used to assess the statistical significance of differences between each sample and NT. Differences were considered statistically significant when P<0.05. Genes inducing either a significant decrease or increase in RSPs production are shown in gray and black columns, respectively.
Figure 8. Production of RSPs for all four serotypes of DV.
Production of RSPs by 293T cells transiently transfected with optimized prME genes of DV1-DV4. At 48 hours post transfection, the RSPs in supernatant were analyzed by Western blot using anti-E mAb 4E11 (A) or mixture of four sera from patients infected by DV1-DV4, respectively (B). C) Production of RSPs by 293T-prME and HeLa-prME stable cell lines. DV1-DV3 RSPs in supernatants were analyzed by SDS-PAGE and silver staining of polyacrylamide gels. Bands corresponding to the approximate molecular weight of E monomers and dimers, as well as prM and M are indicated by arrows. Supernatants from parental 293T and HeLa cells were used as controls. D) Analysis of RSPs of 4 serotypes by sucrose gradient. RSPs were centrifuged in a 20 to 60% sucrose gradient at 28,000 rpm for 2.5 hours in 4°C. Fractions of 0.5 ml were collected and measured using CLDB. The percentage of E protein in each fraction is displayed on the Y axis.
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