Characterization of Membrane Topology and Retention Signal of Pestiviral Glycoprotein E1

Abstract

Pestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1 and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface, but localizes predominantly in the ER. Using engineered chimeric TM domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labelling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C-terminus whereas it adopts a hairpin-like structure with the C-terminus located in the ER lumen when the pre-cleavage situation is mimicked by blocking the cleavage site between E1 and E2. Importance The shortage of specific antibodies against E1 making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the TM domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy-terminus located on the luminal side of the ER in the pre-cleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.

Interaction of Pestiviral E1 and E2 Sequences in Dimer Formation and Intracellular Retention

“…However, secretion of the truncated polypeptides was not detectable (Figure 1C). For E1 (construct pYM-93), a low amount of secreted protein could be observed after prolonged exposure time [23], whereas the C-terminally truncated E1 lacking residues 179 to 195 or 166 to 195 (constructs pYM91 or pYM-92, respectively) was not detectable at all in our experimental system (Figure 1C). The lower amounts detected for constructs pYM-92 and pYM-93 might therefore result from instability of the truncated proteins.…”

“…We have shown that there is a dynamic membrane topology change of the transmembrane region of E1 after signal sequence cleavage with a relocation of the E1 Carboxyterminus from the ER to the cytosolic side [23]. This is also true for E2 of pestiviruses [22] and the closely related HCV envelope proteins [32].…”

“…Because of the absence of robustly reacting specific antibodies against E1, it is still unknown whether E1 of pestiviruses forms homooligomers or not. Therefore, we expressed E1 carrying a N-terminal HA-tag (construct pYM-13, Figure 1A and [23]) and analyzed the expression products under non-reducing conditions. Upon transient expression and western blot analysis, three predominant bands were detected by SDS-PAGE under nonreducing conditions (Figure 1B, left part).…”

“…As a supplement, we provide a file with the original western blot results to demonstrate that the two parts were derived from one gel. (C) The HA-tagged E1 expression plasmids were expressed in BHK-21 cells using the Vaccinia virus MVA T7 expression system [23,30,31], and the empty pCI empty vector served as a mock control. Expressed proteins were labelled with 35 S amino acids.…”

“…Viruses carrying these mutations were also viable [22]. Moreover, we recently demonstrated that E1 indeed contains an independent retention signal [23]. Nevertheless, this leaves us with an incomplete understanding of the requirements in E1 for heterodimerization.…”