Biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells

Coronavirus envelope (E) protein is a small integral membrane protein with multi‐functions in virion assembly, morphogenesis and virus–host interaction. Different coronavirus E proteins share striking similarities in biochemical properties and biological functions, but seem to adopt distinct membrane topology. In this report, we study the membrane topology of the SARS‐CoV E protein by immunofluorescent staining of cells differentially permeabilized with detergents and proteinase K protection assay. It was revealed that both the N‐ and C‐termini of the SARS‐CoV E protein are exposed to the cytoplasmic side of the membranes (N(cyto)C(cyto)). In contrast, parallel experiments showed that the E protein from infectious bronchitis virus (IBV) spanned the membranes once, with the N‐terminus exposed luminally and the C‐terminus exposed cytoplasmically (N(exo(lum))C(cyto)). Intriguingly, a minor proportion of the SARS‐CoV E protein was found to be modified by N‐linked glycosylation on Asn 66 and inserted into the membranes once with the C‐terminus exposed to the luminal side. The presence of two distinct membrane topologies of the SARS‐CoV E protein may provide a useful clue to the pathogenesis of SARS‐CoV.