Folding, assembly and secretion of tick-borne encephalitis virus envelope proteins in mammalian cells

Abstract

The folding of a newly synthesized protein from a linear amino acid sequence into a three-dimensional functional conformation is a highly coordinated process that involves stringent quality control mechanisms, ensuring that only properly folded proteins are transported to their target destinations inside or outside the cell. Viral envelope proteins are useful models to analyze the principles of protein folding in vivo. Moreover, analyzing the folding of these proteins may also reveal new insights into viral replication strategies. The envelope of tick-borne encephalitis (TBE) virus, a flavivirus, contains two proteins, the envelope protein E and the small membrane protein M, the latter being synthesized as a precursor form, prM. In contrast to many other viral envelope proteins that form orthogonal projections to the viral surface, E forms head-to-tail homodimers lying parallel to the viral membrane. By analyzing the folding of TBE virus prM and E expressed recombinantly and in virus-infected cells, we found that both proteins formed disulfide bonds and acquired their native structure within a few minutes after synthesis. Heterodimerization of newly synthesized prM and E occurred rapidly, and E required prM as a chaperone-like factor to fold properly. Both proteins were continuously localized to the endoplasmic reticulum (ER). This was consistent with the finding that TBE virions assemble intracellularly by budding into the ER lumen, followed by transport to the cell surface via the secretory pathway. Expression of prM and E without other viral proteins leads to the generation of recombinant subviral particles (RSPs), capsidless membrane vesicles containing the envelope proteins, but no nucleocapsid. Secretion of RSPs started one hour after prM and E had been synthesized. Inhibition of glycosylation of E or blockage of glucose trimming on the carbohydrate side chain of E abolished secretion of RSPs. Thus, the carbohydrate side chain of the E protein plays an important role in at least one step of flavivirus oligomerization, assembly and transport. Taken together, TBE virus envelope proteins prM and E do not require other viral elements to fold and to form the viral envelope, but expression of both proteins as well as the presence and proper glucose trimming of the carbohydrate side chain of E is required for virion assembly. Whether cellular factors are involved in virion formation remains to be elucidated. Faltung, Zusammenbau und Sekretion der Hullproteine des Zeckenenzephalitis-Virus 3