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. 2003 Apr;77(7):4370-82.
doi: 10.1128/jvi.77.7.4370-4382.2003.

Intracellular assembly and secretion of recombinant subviral particles from tick-borne encephalitis virus

Ivo C Lorenz  1 , Jürgen Kartenbeck, Anna Mezzacasa, Steven L Allison, Franz X Heinz, Ari Helenius
Affiliations

Intracellular assembly and secretion of recombinant subviral particles from tick-borne encephalitis virus

Ivo C Lorenz et al. J Virol. 2003 Apr.

Abstract

It is believed that flavivirus assembly occurs by intracellular budding of the nucleocapsid into the lumen of the endoplasmic reticulum (ER). Recombinant expression of tick-borne encephalitis (TBE) virus envelope proteins prM and E in mammalian cells leads to their incorporation into enveloped recombinant subviral particles (RSPs), which have been used as a model system for studying assembly and entry processes and are also promising vaccine candidates. In this study, we analyzed the formation and secretion of TBE virus RSPs and of a membrane anchor-free E homodimer in mammalian cells. Immunofluorescence microscopy showed that E was accumulated in the lumen of the ER. RSPs were observed by electron microscopy in the rough and smooth ER and in downstream compartments of the secretory pathway. About 75% of the particles appeared to be of the size expected for RSPs (about 30 nm in diameter), but a number of larger particles and tubular structures were also observed in these compartments. Secretion of membrane anchor-free E dimers was detected 30 min after synthesis of prM and E, and secretion of RSPs was detected 1 h after synthesis of prM and E. We also found that the presence of the single N-linked oligosaccharide side chain on the E protein and its trimming by glucosidases was necessary for secretion of RSPs and truncated E dimers. Our results suggest that incorporation of prM and E into RSPs occurs at the ER membrane without other viral elements being required, followed by rapid transport along the compartments of the secretory pathway and secretion. Moreover, the carbohydrate side chain of E is involved in at least one assembly or transport step.

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Figures

FIG. 1.
FIG. 1.
Intracellular localization of TBE virus envelope proteins. COS-1 cells transfected with SV-PEwt plasmid DNA were fixed and subjected to indirect immunofluorescent costaining with primary antibodies that recognize TBE virus envelope proteins and antibodies against a marker protein of a cellular organelle. The proteins were then labeled with secondary antibodies conjugated to red or green light-emitting fluorophores. Shown are cells stained with a monoclonal anti-E antibody (A and D), cells labeled with a polyclonal antiserum recognizing both prM and E (G and J), immunofluorescent staining of the ER with polyclonal anti-calnexin (CNX) (B) and anti-protein disulfide isomerase (PDI) antisera (E), cells stained with a monoclonal antibody against a protein in the ERGIC (H), and immunostaining of the Golgi with a monoclonal anti-giantin antibody (K). Also shown is colocalization of viral envelope proteins with the organelle markers, which is represented by the yellow regions within each cell in the merged images (C, F, I, and L).
FIG. 2.
FIG. 2.
Intracellular localization of prM and membrane anchor-free E. COS-1 cells transfected with SV-PE400 plasmid DNA were fixed and subjected to indirect immunofluorescent costaining with primary antibodies that recognize TBE virus envelope proteins and antibodies against a marker protein of a cellular organelle, as shown in Fig. 1 for SV-PEwt. The legend for the panels, indicating the antibodies used, is analogous to that for Fig. 1.
FIG. 3.
FIG. 3.
Electron micrographs of COS-1 cells transfected with SV-PEwt. RSPs of two different sizes are seen in the lumen of the rough and smooth ER (A to G), in transitional elements (TE) (F and G), the Golgi complex (H and I), the trans-Golgi network (TGN) (J), and in a large vesicle, probably representing a secretory vesicle (SV) (K). Short solid and long solid arrows point to small and large particles, respectively. The open arrow in panel C points to a possible late-budding intermediate at the ER membrane. The inset in panel G shows a long tubular structure (diameter, 50 nm) found inside a cisterna of the rough ER. The insets in panel K show magnifications of RSPs of the large (*) and small (○) size. Cl, clathrin-coated vesicle; Co, COP-coated vesicle; MT, microtubule. Magnification is identical (see the bar in panel A) for all panels (including the inset in panel G) except panels C and K and the inset in panel K. All bars are 0.2 μm except in the inset to panel K (0.1 μm).
FIG. 4.
FIG. 4.
Blockage of the secretory pathway by temperature shifts and brefeldin A. COS-1 cells expressing recombinant E and prM were pulse-labeled for 5 min and chased for 2 h. For temperature shift experiments, the cells were chased at 37°C for 45 min to ensure proper protein folding, followed by a 75-min chase at 15 or 20°C to block transport at the ERGIC or Golgi level, respectively. Brefeldin A (BFA) was added to another sample during starvation, pulse, and chase, and the cells were kept at 37°C during the entire pulse and chase period. TBE virus envelope proteins from total cellular extracts (upper panel) and chase media (lower panel) were immunoprecipitated with a polyclonal antiserum recognizing prM and E, analyzed by SDS-PAGE, and detected by autoradiography. Positions of the individual proteins are indicated; molecular size standards are on the right. Aggr, aggregates.
FIG. 5.
FIG. 5.
Kinetics of secretion of TBE virus, RSPs, and membrane anchor-free E dimers. COS-1 cells infected with TBE virus (A) or transfected with plasmids expressing recombinant prM and full-length E (B) or prM and E lacking the two transmembrane segments (C) were pulse-labeled with [35S]Cys/Met for 2 min and chased between 30 min and 4 h, as indicated above the lanes. Postnuclear supernatants (upper panels) and chase media (lower panels) were subjected to immunoprecipitation with a polyclonal anti-prM/E antiserum recognizing both TBE virus envelope proteins, followed by reducing SDS-PAGE and autoradiography. Positions of the individual proteins are marked on the side. The nonspecific band at 45 kDa seen in all lanes in virus-infected cells (A, upper panel) probably corresponds to labeled actin. Molecular weight standards are indicated on the right.
FIG. 6.
FIG. 6.
Effect of glycosylation inhibition on secretion of TBE virus envelope proteins. COS-1 cells transfected with SV-PEwt, SV-PE400, or SV-PE156A (Ser156Ala; i.e., prM and E carrying an amino acid point mutation which abolishes glycosylation of E) were pulse-labeled for 5 min and chased for 2 h in the presence (+) or absence (−) of tunicamycin (Tun). The intracellular and secreted fractions of E (upper and lower panels, respectively) were immunoprecipitated with a polyclonal anti-prM/E antiserum (A) or with a mouse monoclonal anti-E antibody (B) and analyzed by reducing SDS-PAGE as described above, followed by autoradiography. Molecular weight standards are indicated on the right.
FIG. 7.
FIG. 7.
Secretion of TBE virus envelope proteins after inhibition of glucose trimming with bDNJ. COS-1 cells transfected with SV-PEwt (A) or SV-PE400 (B) were pulse-labeled for 5 min and chased for 2 h. To prevent glucose trimming on the carbohydrate side chains, the glucosidase inhibitor N-butyl-deoxynojirimycin (bDNJ) was added to starvation, pulse, and chase media (+) or to the chase medium after 15 or 30 min as indicated. −, no bDNJ added. Immunoprecipitates of postnuclear supernatants and chase media (upper and lower panels, respectively) were analyzed by reducing SDS-PAGE, followed by autoradiography. Molecular weight standards are indicated on the right.
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