Assembly in vitro of a spanning membrane protein of the endoplasmic reticulum: the E1 glycoprotein of coronavirus mouse hepatitis virus A59.

Abstract

The El glycoprotein of coronavirus mouse hepatitis virus A59 was synthesized in vitro by translation of viral mRNA in the presence of dog pancreatic microsomes. Its disposition in the membrane was investigated by digestion with proteases and by selective NH2-terminal labeling. The protein spans the membrane, but only small portions from the NH2 and COOH terminus are exposed respectively in the lumenal and cytoplasmic domains; the bulk of the molecule is apparently buried in the membrane. The protein lacks a cleavable leader sequence and does not acquire its characteristic 0-linked oligosaccharides in rough microsomes. It may enter the membrane at any stage during synthesis of the first 150 amino acid residues. These unusual features of the protein might help to explain why it is not transported to the cell surface in vivo but remains in intracellular membranes, causing the virus to bud there.

Characterization of the Coronavirus Mouse Hepatitis Virus Strain A59 Small Membrane Protein E

“…Figure 7B shows that treatment of 35 S-labeled virions eliminated the epitope present in the amino terminus of the M protein recognized by the monoclonal J1.3 (␣M N ). It also reduced the size of the polypeptide by about 2.5 kDa as shown previously (33). The E protein seemed unaffected.…”

“…In this assay cells were permeabilized selectively in their plasma membrane by using SLO to allow intracellular access of antibodies. The assay was first tested with cells expressing the M protein whose membrane topology has been well established (33,35): its aminoand carboxy-terminal domains are exposed luminally and cy- The results are compiled in panel C. For each protein the radioactivity in the chase samples was related to that observed after the pulse labeling, which was set at 100.…”

Sequence and N-terminal Processing of the Transmembrane Protein E1 of the Coronavirus Transmissible Gastroenteritis Virus

“…Our results provide substantial evidence that TGEV E1 undergoes Nterminal processing and that its exposed NH2 extremity may be significantly larger and possibly more complex in secondary structure than those of IBV and MHV. Protease digestion has been shown to remove an external glycopeptide of nine residues (IBV; Cavanagh et al, 1986) and 2.5K (MHV; Rottier et al, 1984). In comparison, the TGEV E 1 external segment may approach 30 residues, including four cysteines (see Fig.…”

The Role of a 21-kDa Viral Membrane Protein in the Assembly of Vaccinia Virus from the Intermediate Compartment

“…The pellet was resuspended in 450 l of RM buffer and the A 280 measured against the untreated microsomes. As a control we used the mouse hepatitis M protein, which has been shown to associate with efficiencies of up to 95% with rough microsomal membranes (Rottier et al, 1984(Rottier et al, , 1985. The M and p21 protein were translated in vitro according to the instructions of the manufacturer using a 25-l system.…”