Cleavage Inhibition of the Murine Coronavirus Spike Protein by a Furin-Like Enzyme Affects Cell-Cell but Not Virus-Cell Fusion

Abstract

Cleavage of the mouse hepatitis coronavirus strain A59 spike protein was blocked in a concentrationdependent manner by a peptide furin inhibitor, indicating that furin or a furin-like enzyme is responsible for this process. While cell-cell fusion was clearly affected by preventing spike protein cleavage, virus-cell fusion was not, indicating that these events have different requirements.The surface glycoproteins of many enveloped viruses are initially synthesized as inactive precursors, proteolytic cleavage of which is often required for maturation and full functional activity. In several virus families, this processing step is carried out by cellular proprotein convertases (21), most commonly furin, a component of the constitutive secretory pathway of many different types of cells (9,33). Furin is a membranebound, calcium-dependent subtilisin-like protease whose primary site of action is the trans-Golgi network (TGN), although cycling of furin between TGN and plasma membrane through the exocytic and endocytic pathways has also been demonstrated (6, 28). The enzyme is also secreted from cells in an active soluble form, which is produced by self-cleavage in the TGN (43,45).The mouse hepatitis coronavirus (MHV) spike (S) protein is responsible for attachment to the viral receptor, for virus-cell fusion during viral entry, and for cell-cell fusion during infection. It is a class I fusion protein (5) that is cotranslationally glycosylated to a 150-kDa glycoprotein, which is processed to a 180-kDa form during transport from the endoplasmic reticulum through the Golgi complex. As a late event in maturation, the protein is cleaved into two 90-kDa subunits, S1 and S2 (10,31,34). The S proteins of murine coronaviruses are cleaved to different extents, depending on the strain and the cell line used (10). Cleavage of strain MHV-A59 S protein takes place between residues 717 and 718 at the sequence RRAHR2SVS (26). This sequence resembles the furin consensus sequence motif, RXR/KR (1,21,27). We now demonstrate, for the first time, that furin or a furin-like enzyme is indeed the protease responsible for cleavage of the S protein. Moreover, we investigated the consequences of cleavage, or rather of cleavage inhibition, of the S protein on its fusion activity and on the infectivity and cell entry of the virus.The importance of S protein cleavage for cell-cell fusion has been studied by several groups with inconsistent results. Using a vaccinia virus expression system (11) some investigators found the S proteins from MHV-A59 and MHV-JHM not to require cleavage for the induction of cell-cell fusion but syncytium formation to be delayed in the absence of cleavage (3,38). Others observed that a mutant MHV-JHM S protein, which was not proteolytically cleaved, induced syncytium formation to the same extent as the wild-type S protein (35). In contrast, transient expression of the uncleaved MHV-2 S protein apparently did not result in cell-cell fusion while a cleavable form of this protein did (47). Strikingly, the S protein from a dif...

Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Processing by the Endoprotease SKI-1/S1P Is Critical for Virus Infectivity

“…The supernatants of SKI-1/S1P-deficient cells infected with these arenaviruses also contained a low abundance of N-containing noninfectious particles deficient in envelope GPC or mature cleavage products (GP1 and GP2) (12,13). In contrast, it has been demonstrated for numerous other viruses that blockage of furin processing of glycoprotein precursors does not inhibit formation of virus particles (4,5,16,29), suggesting that blockage of virion formation may be limited to glycoproteins cleaved by SKI-1/S1P. While unlikely, we cannot rule out that SKI-1/S1P could impact CCHFV budding in a manner unrelated to its protease activity.…”

“…4. Glycoprotein-deficient viral particles are released in the infected cell supernatant of SKI-1/S1P-deficient cells.…”

“…Analysis of other viral systems has demonstrated that disruption of viral glycoprotein cleavage can lead to the formation of virus particles decorated with uncleaved glycoprotein precursors on their surface (4,5,16,25,29). This situation applies to viral glycoproteins incom- FIG.…”

Key Golgi Factors for Structural and Functional Maturation of Bunyamwera Virus

“…Molecular modifications associated with transport of viral precursors to the trans-Golgi, in particular acquisition of Endo-H resistance in viral glycoproteins, trigger a major structural transformation that generates prefunctional particles, ready to exit the cells and become fully infectious. This is unique compared with the processes operating for most viruses, which usually mature by an irreversible proteolytic processing of polypeptide precursors (10,12,50,57,58).…”

Middle East Respiratory Syndrome Coronavirus Spike Protein Delivered by Modified Vaccinia Virus Ankara Efficiently Induces Virus-Neutralizing Antibodies

“…3). We considered furin cleavage sites because spike proteins of some betacoronaviruses and all gammacoronaviruses are typically activated by intracellular furin-dependent cleavage (22,23; for a review, see references 24 and 25); the ProP1.0 server indicated three possible furin cleavage sites at amino acid positions 751, 887, and 1113 of MERS-CoV S (data not shown). We favor putative cleavage at amino acid 887 since this cleavage would produce a predicted S2 subdomain of 54.5 kDa for the nonglycosylated protein matching our Western blot data (Fig.…”