Folding and Dimerization of Tick-Borne Encephalitis Virus Envelope Proteins prM and E in the Endoplasmic Reticulum

Abstract

Flavivirus envelope proteins are synthesized as part of large polyproteins that are co-and posttranslationally cleaved into their individual chains. To investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (TBE) virus envelope glycoproteins prM and E by using a recombinant plasmid expression system and virus-infected cells. When expressed in their polyprotein context, prM and E achieved their native folded structures with half-times of approximately 4 min for prM and about 15 min for E. They formed heterodimeric complexes within a few minutes after synthesis that were required for the final folding of E but not for that of prM. Heterodimers could also be formed in trans when these proteins were coexpressed from separate constructs. When expressed without prM, E could form disulfide bonds but did not express a specific conformational epitope and remained sensitive to reduction by dithiothreitol. This is consistent with a chaperone-like role for prM in the folding of E. PrM was able to achieve its native folded structure without coexpression of E, but signal sequence cleavage at the N terminus was delayed. Our results show that prM is an especially rapidly folding viral glycoprotein, that polyprotein cleavage and folding of the TBE virus envelope proteins occurs in a coordinated sequence of processing steps, and that proper and efficient maturation of prM and E can only be achieved by cosynthesis of these two proteins.

Post-translational regulation and modifications of flavivirus structural proteins

“…1b) Lorenz et al, 2002;Setoh et al, 2012). Liberation of prM from this signal sequence by host cell signallase is tightly co-ordinated and is dependent on prior cleavage of C by viral NS2B/3 protease (see 'Polyprotein processing' below and Fig.…”

“…The prM protein is believed to primarily mediate this heterodimerization, as it folds more rapidly than E and is required to be present for E to achieve complete antigenic conformation (Lorenz et al, 2002). As such, detailed investigations have been undertaken to determine specific residues or domains in prM that may be responsible for efficient assembly and secretion of virus particles.…”

“…Heterodimerization between the flavivirus prM and E proteins is an important first step in the generation of the envelope, with prM purported to play a role in the chaperone-mediated folding of E (Konishi & Mason, 1993;Lorenz et al, 2002). Heterodimerization may facilitate the arrangement of prM/E at the ER membrane forming heterotrimeric spikes (Zhang et al, 2007a).…”

“…Heterodimerization may facilitate the arrangement of prM/E at the ER membrane forming heterotrimeric spikes (Zhang et al, 2007a). In turn, the ordered arrangement of these prM/E spikes may induce curvature of the ER membrane, resulting in budding of nascent virions into the ER lumen (Zhang et al, 2013a).The prM protein is believed to primarily mediate this heterodimerization, as it folds more rapidly than E and is required to be present for E to achieve complete antigenic conformation (Lorenz et al, 2002). As such, detailed investigations have been undertaken to determine specific residues or domains in prM that may be responsible for efficient assembly and secretion of virus particles.…”

“…All three domains appear to be involved in the dimerization of the E protein through hydrophilic interactions in mature virions (Zhang et al, 2004). The E protein is liberated from the genomic polyprotein at its N and C termini via cleavage with host signal peptidase in the ER lumen.The E protein is synthesized immediately downstream of prM with which it rapidly heterodimerizes, an important interaction associated with the correct folding of E (Lorenz et al, 2002). These heterodimers coalesce on the lumenal face of the ER membrane forming trimeric spikes and ultimately bud off to give rise to either empty prM/E particles, or nucleocapsidcontaining immature virions (Roby et al, 2012).…”