Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions

Abstract

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/cm 3 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 [ 14 C]choline but not [ 3 H]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.

Structures and mechanisms in flavivirus fusion

“…The structure and oligomeric arrangement of the E protein on the surface of TBE virus RSPs appear to be very similar to that on the snrface of virions. This conclusion is supported by a variety of different experimental data (Schalich et al, 1996). RSPs and virions are indistinguishable in their reactivity pattern with a panel of E protein-specific monoclonal antibodies, several of which are neutralizing and conformation-specific.…”

“…Therefore, even without exposure to acidic pH, the contacts at 3-fold axes might be strong enough (and those at the 2-fold axes weak enough) to allow the isolation of trimers under certain experimental conditions. In the TBE virus system, however, there is clearly a quantitative oligomeric switch from Triton X-100-stable dimers at neutral pH to trimers at acidic pH, and in all the experiments carried out so far, this change was irreversible (Allison et al, 1995a;Stiasny et al, 1996;Schalich et al, 1996;Stadler et al, 1997).…”

“…Flaviviruses are similar to other enveloped viruses in that their fusion activity apparently depends on triggered conformational A B (Kimura and Ohyama, 1988;Guirakhoo et al, 1989) and by the demonstration that the reactivity with conformation-specific monoclonal antibodies changes after preincubation of viruses at low pH Roehrig et al, 1990;Guirakhoo et al, 1992;Heinz et al, 1994). When the specific location of these epitopes in the three-dimensional structure of the TBE virus E protein was established (Rey et al, 1995), it became clear that these alterations affect the entire molecule, with changes occurring in each of the three domains Heinz et al, 1994;Schalich et al, 1996). The loss or reduction of the reactivity of several conformational epitopes involved in neutralization and the increased reactivity of others suggest an extensive change in the E protein structure after low pH treatment.…”

“…It is a characteristic property of flaviviruses that the coexpression of the prM and E proteins in the absence of the C protein leads to the formation and secretion of a capsidless subviral particle . Recombinant subviral particles (RSPs) have been generated for several different flaviviruses Konishi et al, 1991;Pincus et al, 1992;Konishi et al, 1992a;Konishi et al, 1992b;Yamshchikov and Compans, 1993;Sato et al, 1993;Konishi et al, 1994;Fonseca et al, 1994;Pugachev et al, 1995;Allison et al, 1995b), but they have been the most extensively characterized in the TBE virus system (Schalich et al, 1996). The TBE virus RSPs are smaller than virions (approximate diameter 30 vs. 50 nm), but their assembly pathway is quite similar to that of the virus.…”

“…Consistent with their smaller size, they exhibit a slower sedimentation rate than virions, and because of the lack of a nucleocapsid their buoyant density is lower (1.13 vs. 1.19 g/cm 3 in sucrose). The following evidence suggests that TBE virus RSPs possess a lipid membrane (Allison et al, 1995b;Schalich et al, 1996;Corver et al, 2000): (1) They have been shown in metabolic labeling experiments to incorporate [14C]choline and fluorescently labeled fatty acids, (2) they are readily disrupted by treatment with nonionic detergent, (3) they fuse with liposomes at low pH, and (4) they are capable of inducing cell-cell fusion when applied externally to cells and acidified.…”