scholarly journals Diagnostic Potential and Antigenic Properties of Recombinant Tick-Borne Encephalitis Virus Subviral Particles Expressed in Mammalian Cells from Semliki Forest Virus Replicons

2013 ◽  
Vol 52 (3) ◽  
pp. 814-822 ◽  
Author(s):  
L. Levanov ◽  
S. Kuivanen ◽  
A. Matveev ◽  
S. Swaminathan ◽  
A. Jaaskelainen-Hakala ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Semliki Forest Virus ◽  
Encephalitis Virus ◽  
Diagnostic Potential ◽  
Tick Borne Encephalitis ◽  
Antigenic Properties ◽  
Tick Borne Encephalitis Virus ◽  
Subviral Particles

scholarly journals Two Distinct Size Classes of Immature and Mature Subviral Particles from Tick-Borne Encephalitis Virus

2003 ◽  
Vol 77 (21) ◽  
pp. 11357-11366 ◽  
Author(s):  
Steven L. Allison ◽  
Yizhi J. Tao ◽  
Gabriel O'Riordain ◽  
Christian W. Mandl ◽  
Stephen C. Harrison ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Size Class ◽  
Encephalitis Virus ◽  
E Proteins ◽  
Virus Particles ◽  
Mature Form ◽  
Size Classes ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Subviral Particles

ABSTRACT Flaviviruses assemble in the endoplasmic reticulum by a mechanism that appears to be driven by lateral interactions between heterodimers of the envelope glycoproteins E and prM. Immature intracellular virus particles are then transported through the secretory pathway and converted to their mature form by cleavage of the prM protein by the cellular protease furin. Earlier studies showed that when the prM and E proteins of tick-borne encephalitis virus are expressed together in mammalian cells, they assemble into membrane-containing, icosahedrally symmetrical recombinant subviral particles (RSPs), which are smaller than whole virions but retain functional properties and undergo cleavage maturation, yielding a mature form in which the E proteins are arranged in a regular T = 1 icosahedral lattice. In this study, we generated immature subviral particles by mutation of the furin recognition site in prM. The mutation resulted in the secretion of two distinct size classes of particles that could be separated by sucrose gradient centrifugation. Electron microscopy showed that the smaller particles were approximately the same size as the previously described mature RSPs, whereas the larger particles were approximately the same size as the virus. Particles of the larger size class were also detected with a wild-type construct that allowed prM cleavage, although in this case the smaller size class was far more prevalent. Subtle differences in endoglycosidase sensitivity patterns suggested that, in contrast to the small particles, the E glycoproteins in the large subviral particles and whole virions might be in nonequivalent structural environments during intracellular transport, with a portion of them inaccessible to cellular glycan processing enzymes. These proteins thus appear to have the intrinsic ability to form alternative assembly products that could provide important clues about the role of lateral envelope protein interactions in flavivirus assembly.

scholarly journals Impact of Quaternary Organization on the Antigenic Structure of the Tick-Borne Encephalitis Virus Envelope Glycoprotein E

2009 ◽  
Vol 83 (17) ◽  
pp. 8482-8491 ◽  
Author(s):  
Stefan Kiermayr ◽  
Karin Stiasny ◽  
Franz X. Heinz
Keyword(s):  
Neutralizing Antibodies ◽  
Encephalitis Virus ◽  
Antigenic Structure ◽  
Icosahedral Symmetry ◽  
Virus Envelope ◽  
E Protein ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Virion Surface ◽  
Subviral Particles

ABSTRACT The envelope protein E of flaviviruses mediates both receptor-binding and membrane fusion. At the virion surface, 180 copies of E are tightly packed and organized in a herringbone-like icosahedral structure, whereas in noninfectious subviral particles, 60 copies are arranged in a T=1 icosahedral symmetry. In both cases, the basic building block is an E dimer which exposes the binding sites for neutralizing antibodies at its surface. It was the objective of our study to assess the dependence of the antigenic structure of E on its quaternary arrangement, i.e., as part of virions, recombinant subviral particles, or soluble dimers. For this purpose, we used a panel of 11 E protein-specific neutralizing monoclonal antibodies, mapped to distinct epitopes in each of the three E protein domains, and studied their reactivity with the different soluble and particulate forms of tick-borne encephalitis virus E protein under nondenaturing immunoassay conditions. Significant differences in the reactivities with these forms were observed that could be related to (i) limited access of certain epitopes at the virion surface; (ii) limited occupancy of epitopes in virions due to steric hindrance between antibodies; (iii) differences in the avidity to soluble forms compared to the virion, presumably related to the flexibility of E at its domain junctions; and (iv) modulations of the external E protein surface through interactions with its stem-anchor structure. We have thus identified several important factors that influence the antigenicity of the flavivirus E protein and have an impact on the interaction with neutralizing antibodies.

scholarly journals N-linked glycan in tick-borne encephalitis virus envelope protein affects viral secretion in mammalian cells, but not in tick cells

2013 ◽  
Vol 94 (10) ◽  
pp. 2249-2258 ◽  
Author(s):  
Kentaro Yoshii ◽  
Natsumi Yanagihara ◽  
Mariko Ishizuka ◽  
Mizuki Sakai ◽  
Hiroaki Kariwa
Keyword(s):  
Envelope Protein ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Consensus Sequence ◽  
Encephalitis Virus ◽  
Secretory Process ◽  
Virus Envelope ◽  
Tick Borne Encephalitis ◽  
Virus Envelope Protein ◽  
Tick Borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV) is a zoonotic disease agent that causes severe encephalitis in humans. The envelope protein E of TBEV has one N-linked glycosylation consensus sequence, but little is known about the biological function of the N-linked glycan. In this study, the function of protein E glycosylation was investigated using recombinant TBEV with or without the protein E N-linked glycan. Virion infectivity was not affected after removing the N-linked glycans using N-glycosidase F. In mammalian cells, loss of glycosylation affected the conformation of protein E during secretion, reducing the infectivity of secreted virions. Mice subcutaneously infected with TBEV lacking protein E glycosylation showed no signs of disease, and viral multiplication in peripheral organs was reduced relative to that with the parental virus. In contrast, loss of glycosylation did not affect the secretory process of infectious virions in tick cells. Furthermore, inhibition of transport to the Golgi apparatus affected TBEV secretion in mammalian cells, but not in tick cells, indicating that TBEV was secreted through an unidentified pathway after synthesis in endoplasmic reticulum in tick cells. These results increase our understanding of the molecular mechanisms of TBEV maturation.

Development of a serodiagnostic IgM-ELISA for tick-borne encephalitis virus using subviral particles with strep-tag

2018 ◽  
Vol 9 (6) ◽  
pp. 1391-1394 ◽  
Author(s):  
Miki Nakayasu ◽  
Minato Hirano ◽  
Memi Muto ◽  
Shintaro Kobayashi ◽  
Hiroaki Kariwa ◽  
...  
Keyword(s):  
Encephalitis Virus ◽  
Tick Borne Encephalitis ◽  
Igm Elisa ◽  
Tick Borne Encephalitis Virus ◽  
Subviral Particles

scholarly journals Intracellular Assembly and Secretion of Recombinant Subviral Particles from Tick-Borne Encephalitis Virus

2003 ◽  
Vol 77 (7) ◽  
pp. 4370-4382 ◽  
Author(s):  
Ivo C. Lorenz ◽  
Jürgen Kartenbeck ◽  
Anna Mezzacasa ◽  
Steven L. Allison ◽  
Franz X. Heinz ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Recombinant Expression ◽  
Side Chain ◽  
Virus Envelope ◽  
Membrane Anchor ◽  
Tick Borne Encephalitis ◽  
Tbe Virus ◽  
Tick Borne Encephalitis Virus ◽  
Subviral Particles

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.

scholarly journals Membrane Fusion Activity of Tick-Borne Encephalitis Virus and Recombinant Subviral Particles in a Liposomal Model System

Virology ◽  
2000 ◽  
Vol 269 (1) ◽  
pp. 37-46 ◽  
Author(s):  
Jeroen Corver ◽  
Antonio Ortiz ◽  
Steven L. Allison ◽  
Juliane Schalich ◽  
Franz X. Heinz ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Model System ◽  
Encephalitis Virus ◽  
Fusion Activity ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Subviral Particles

scholarly journals Development of a serodiagnostic multi-species ELISA against tick-borne encephalitis virus using subviral particles

2016 ◽  
Vol 7 (5) ◽  
pp. 723-729 ◽  
Author(s):  
Eri Inagaki ◽  
Mizuki Sakai ◽  
Minato Hirano ◽  
Memi Muto ◽  
Shintaro Kobayashi ◽  
...  
Keyword(s):  
Encephalitis Virus ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Subviral Particles
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