Related Experiment Videos
Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for
J Schalich1, S L Allison, K Stiasny
1Institute of Virology, University of Vienna, Austria.
Abstract:
Recombinant subviral particles (RSPs) obtained by coexpression of the envelope (E) and premembrane (prM) proteins of tick-borne encephalitis virus in COS cells (S. L. Allison, K. Stadler, C. W. Mandl, C. Kunz, and F. X. Heinz, J. Virol. 69:5816-5820, 1995) were extensively characterized and shown to be ordered structures containing envelope glycoproteins with structural and functional properties very similar to those in the virion envelope. The particles were spherical, with a diameter of about 30 nm and a buoyant density of 1.14 g/cm3 in sucrose gradients. They contained mature E proteins with endoglycosidase H-resistant glycans as well as fully cleaved mature M proteins. Cleavage of prM, which requires an acidic pH in exocytic compartments, could be inhibited by treatment of transfected cells with ammonium chloride, implying a common maturation pathway for RSPs and virions. RSPs incorporated [14C]choline but not [3H]uridine, demonstrating that they contain lipid but probably lack nucleic acid. The envelope proteins of RSPs exhibited a native antigenic and oligomeric structure compared with virions, and incubation at an acidic pH (pH <6.5) induced identical conformational changes and structural rearrangements, including an irreversible quantitative conversion of dimers to trimers. The RSPs were also shown to be functionally active, inducing membrane fusion in a low-pH-dependent manner and demonstrating the same specific hemagglutination activity as whole virions. Tick-borne encephalitis virus RSPs thus represent an excellent model system for investigating the structural basis of viral envelope glycoprotein functions.
Insights
Recombinant subviral particles (RSPs) of tick-borne encephalitis virus mimic virions, offering a model to study viral envelope glycoprotein functions. These particles demonstrate similar structural and functional properties to the virus itself.
Area of Science:
- Virology
- Structural Biology
- Biochemistry
Background:
- Tick-borne encephalitis virus (TBEV) poses a significant public health threat.
- Understanding TBEV envelope glycoprotein function is crucial for developing antiviral strategies.
- Previous studies have focused on whole virions, limiting detailed analysis of specific protein functions.
Purpose of the Study:
- To characterize recombinant subviral particles (RSPs) of TBEV.
- To assess the structural and functional similarity of RSPs to native TBEV virions.
- To establish RSPs as a model system for studying viral envelope glycoprotein functions.
Main Methods:
- Coexpression of TBEV E and prM proteins in COS cells to generate RSPs.
- Characterization of RSPs using techniques such as buoyant density centrifugation and protein analysis.
- Assessment of RSPs' antigenic and oligomeric structure, and their response to acidic pH.
- Functional assays including membrane fusion and hemagglutination.
Main Results:
- RSPs were spherical (30 nm diameter) with a buoyant density of 1.14 g/cm3.
- RSPs contained mature E and M proteins, incorporated lipids, and lacked nucleic acid.
- RSP envelope proteins exhibited native antigenic and oligomeric structures, similar to virions.
- Low pH induced conformational changes in RSPs, mirroring virion behavior.
- RSPs demonstrated low-pH-dependent membrane fusion and hemagglutination activity.
Conclusions:
- TBEV RSPs are ordered structures with properties highly similar to native virions.
- RSPs provide a valuable model system for investigating the structural basis of viral envelope glycoprotein functions.
- The maturation pathway of RSPs is comparable to that of infectious virions.