Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form

Abstract

A quantitative study was performed to investigate the requirements for secretion of recombinant soluble and particulate forms of the envelope glycoprotein E of tick-borne encephalitis (TBE) virus. Full-length E and a carboxy terminally truncated anchor-free form were expressed in COS cells in the presence and absence of prM, the precursor of the viral membrane protein M. Formation of a heteromeric complex with prM was found to be necessary for efficient secretion of both forms of E, whereas only low levels of anchor-free E were secreted in the absence of prM. The prM-mediated transport function could also be provided by coexpression of prM and E from separate constructs, but a prM-toE ratio of greater than 1:1 did not further enhance secretion. Full-length E formed stable intracellular heterodimers with prM and was secreted as a subviral particle, whereas anchor-free E was not associated with particles and formed a less stable complex with prM, suggesting that prM interacts with both the ectodomain and anchor region of E.

Molecular Interactions in the Assembly of Coronaviruses

“…The observations demonstrate the unique budding mechanism of coronaviruses, which is dependent solely on the envelope proteins M and E but independent of a nucleocapsid. Somewhat similar observations have been described for the flavivirus tick-borne encephalitis virus and for hepatitis B virus, which also produce proteolipid particles on expression of their envelope proteins preM and E (Allison et al, 1995;Mason et al, 1991) and S (Patzer et al, 1986;Simon et al, 1988), respectively, but these particles are much smaller than the corresponding virions. In contrast, particles with the typical, large size of coronaviruses are acquired by the concerted action of just the proteins M and E (Baudoux et al, 1998b;Vennema et al, 1996).…”

Structures and mechanisms in flavivirus fusion

“…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.…”

A Zika virus vaccine expressing premembrane-envelope-NS1 polyprotein

“…The E protein is a type II fusion protein which mediates cellular attachment and membrane fusion, and is the target for most neutralizing antibodies (Abs). Flavivirus prM protein typically associates with E to form heterodimers and is important for proper folding of E [13][14][15][16] . Co-expression of prM and E of several flaviviruses including ZIKV results in the secretion of virus-like particles (VLPs) termed recombinant subviral particles [17][18][19] .…”