Cleavage of Group 1 Coronavirus Spike Proteins: How Furin Cleavage Is Traded Off against Heparan Sulfate Binding upon Cell Culture Adaptation

Abstract

A longstanding enigmatic feature of the group 1 coronaviruses is the uncleaved phenotype of their spike protein, an exceptional property among class I fusion proteins. Here, however, we show that some group 1 coronavirus spike proteins carry a furin enzyme recognition motif and can actually be cleaved, as demonstrated for a feline coronavirus. Interestingly, this feature can be lost during cell culture adaptation by a single mutation in the cleavage motif; this, however, preserves a heparan sulfate binding motif and renders infection by the virus heparan sulfate dependent. We identified a similar cell culture adaptation for the human coronavirus OC43.Enveloped viruses use different types of fusion proteins to realize the membrane fusion by which they initiate their infection. For coronaviruses, it is the spike (S) protein that is responsible for cell entry, and this S protein has been shown to belong to the class I fusion proteins (4). These proteins typically occur in virions as homotrimeric complexes primed for fusion through cleavage by furin-like enzymes. Membrane fusion by these activated proteins can then be triggered upon receptor binding (e.g., human immunodeficiency virus type 1) or by conditions such as low pH after endosomal uptake (e.g., influenza A virus) (for a recent review, see reference 50).One of the puzzling questions about coronavirus S proteinmediated membrane fusion regards the cleavage requirement of the S protein. Coronaviruses have been assigned to different groups based on antigenic and genetic criteria (41). Interestingly, while the group 1 coronaviruses carry uncleaved S proteins, the S proteins of almost all viruses from groups 2 and 3 are furin activated (10) by processing at a characteristic multibasic motif (often RRXRR) present in these proteins. The importance of cleavage for infectivity was underscored recently by the revelation that the two prominent group 2 viruses lacking such a furin recognition site, and hence carrying uncleaved spikes, appeared to depend on a different, new processing mechanism. Thus, the severe acute respiratory syndrome coronavirus (SARS-CoV) and the murine hepatitis virus strain 2 (MHV-2) were both shown to require proteolytic cleavage in their target cell, which is mediated by cathepsin enzymes (23,36,42). The cathepsin cleavage site of the SARS-CoV spike protein was mapped to the same region as that in which, in other viruses, the S protein is activated by furin (B. J. Bosch and P. J. M. Rottier, unpublished observations), hence similarly generating an amino-terminal, receptor binding domain (S1) and a membrane-anchored carboxy-terminal domain (S2) responsible for membrane fusion (for reviews, see references 3 and 9).When looking closer into the enigmatic lack of cleavage of the group 1 coronavirus spike proteins, we established that the infection of cells by two of those viruses, human coronavirus (HCoV) NL63 (23) and feline infectious peritonitis virus strain 79-1146 (our unpublished observations), is insensitive to cathepsin inhibitors. However, w...

Full-length genome analysis of canine coronavirus type I

“…This stretch had been also observed in the sequence of the S protein of Elmo/02 (position 801-805), but in this strain an Ala residue has replaced Val at position 803 (Pratelli et al, 2003). With few exceptions (de Haan et al, 2008), other alphacoronaviruses do not share this finding. The E protein is 82-aa long and does not present any Nglycosylation sites, whereas three N-glycosylated residues have been detected in the 264-aa long M protein, which is in agreement with what has been observed in other FCoV/CCoV strains, with the exception of CCoV-II isolate BGF10 that shows only two glycosylated Asn residues.…”

The cholesterol transport inhibitor U18666A inhibits type I feline coronavirus infection

“…For example, type II FCoV S protein binds to the virus receptor, aminopeptidase N (APN), but type I FCoV S protein does not bind to APN (Hohdatsu et al, 1998). In addition, type I FCoV S protein binds to heparin, but type II FCoV S protein does not (de Haan et al, 2008). The differences in these S protein properties between the serotypes may also influence the differences in the effects of U18666A.…”

Intriguing interplay between feline infectious peritonitis virus and its receptors during entry in primary feline monocytes

“…According to , subsequent escape from endosomes is mediated by cathepsin B for FIPV strains 79-1146 and DF2, while for the enteric strain 79-1683 low pH in endocytic compartments and cathepsin L need to assist cathepsin B. In contrast, de Haan et al (2008) observed that infection with FIPV 79-1146 appears to be insensitive to cathepsin inhibitors.…”