Furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells.
The intracellular cleavage of respiratory syncytial virus (RSV) fusion (F) protein by furin was examined. In RSV-infected LoVo cells, which express an inactive form of furin, and in RSV-infected Vero cells treated with the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk), the F protein was expressed as a non-cleaved 73 kDa species. In both cases the F protein was initially expressed as an endoglycosidase H (Endo H)-sensitive precursor (F0(EHs)) which was modified approximately 40 min post-synthesis by the addition of complex carbohydrates to produce the Endo H-resistant form (F0(EHr)). The size and glycosylation state of F0(EHr) were identical to a transient intermediate form of non-cleaved F protein which was detected in RSV-infected Vero cells in the absence of inhibitor. Cell surface biotinylation and surface immunofluorescence staining showed that F0(EHr) was present on the surface of RSV-infected cells. RSV filaments have been shown to be the predominant form of the budding virus that is detected during virus replication. Analysis of the RSV-infected cells using scanning electron microscopy (SEM) showed that, in the presence of dec-RVKR-cmk, virus budding was impaired, producing fewer and much smaller viral filaments than in untreated cells. A comparison of immunofluorescence and SEM data showed that F0(EHr) was routed to the surface of virus-infected cells but not located in these smaller structures. Our findings suggest that activation of the F protein is required for the efficient formation of RSV filaments.
Sugrue
Richard J
RJ
MRC Virology Unit, Institute of Virology, Church Street, G11 5JR, Glasgow, UK1.
Brown
Craig
C
MRC Virology Unit, Institute of Virology, Church Street, G11 5JR, Glasgow, UK1.
Brown
Gaie
G
MRC Virology Unit, Institute of Virology, Church Street, G11 5JR, Glasgow, UK1.
Aitken
James
J
Division of Virology, University of Glasgow, Institute of Virology, Church Street, G11 5JR, Glasgow, UK2.
McL Rixon
Helen W
HW
MRC Virology Unit, Institute of Virology, Church Street, G11 5JR, Glasgow, UK1.
D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure / Q000821:virology; D002522:Chlorocebus aethiops; D005455:Fluorescent Antibody Technique; D045683:Furin; D006031:Glycosylation; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D012136:Respiratory Syncytial Viruses / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D019065:Virus Assembly / Q000187:drug effects
D015842:Serine Proteinase Inhibitors; D014760:Viral Fusion Proteins; D013381:Subtilisins; D045683:Furin