Furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of Mononegavirales. BDV cell entry follows a receptor-mediated endocytosis pathway, which is initiated by the recognition of an as-yet-unidentified receptor at the cell surface by the virus glycoprotein G. BDV G is synthesized as a precursor (GPC) that is cleaved by the cellular protease furin to produce the mature glycoproteins GP1 and GP2, which have been implicated in receptor recognition and pH-dependent fusion events, respectively. BDV is highly neurotropic and its spread in cultured cells proceeds in the absence of detectable extracellular virus or syncytium formation. BDV spread has been proposed to be strictly dependent on the expression and correct processing of BDV G. Here we present evidence that cell-to-cell spread of BDV required neither the expression of cellular receptors involved in virus primary infection, nor the furin-mediated processing of BDV G. We also show that in furin-deficient cells, the release of BDV particles induced by the treatment of BDV-infected cells with hypertonic buffer was not significantly affected, while virion infectivity was dramatically impaired, correlating with the decreased incorporation of BDV G species into viral particles. These findings support the view that the propagation of BDV within the central nervous systems of infected hosts involves both a primary infection that follows a receptor-mediated endocytosis pathway and a subsequent cell-to-cell spread that is independent of the expression of the primary receptor and does not require the processing of BDV G into GP1 and GP2.

Section: Discussionsupporting

confidence: 59%

Cell-to-Cell Spread of Borna Disease Virus Proceeds in the Absence of the Virus Primary Receptor and Furin-Mediated Processing of the Virus Surface Glycoprotein

Clemente

Torre

2007

“…Most paramyxovirus F proteins, including the simian virus 5 (SV5) F protein (20), the measles virus F protein (6), and the respiratory syncytial virus F protein (59), are proteolytically processed by furin. Furin is a secretory pathway protease that belongs to the family of mammalian subtilisin-like proprotein convertases (PCs).…”

mentioning

confidence: 99%

Subcellular Localization and Calcium and pH Requirements for Proteolytic Processing of the Hendra Virus Fusion Protein

Pager

Wurth

Dutch

2004

Proteolytic cleavage of the Hendra virus fusion (F) protein results in the formation of disulfide-linked F 1 and F 2 subunits, with cleavage occurring after residue K 109 in the sequence GDVK2L. This unusual cleavage site and efficient propagation of Hendra virus in a furin-deficient cell line indicate that the Hendra F protein is not cleaved by furin, the protease responsible for proteolytic activation of many viral fusion proteins. To identify the subcellular site of Hendra F processing, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F were metabolically labeled and chased in the absence and presence of inhibitors of exocytosis. The addition of carbonyl-cyanide-3-chlorophenylhydrazone, monensin, brefeldin A, or NaF-AlCl 3 or incubation of cells at 20°C all inhibited processing of the Hendra F protein, suggesting that cleavage of Hendra F occurs either in secretory vesicles budding from the trans-Golgi network or at the cell surface. In contrast to proteolytic cleavage of the simian virus 5 (SV5) F protein by the Ca 2؉ -dependent protease furin, proteolytic cleavage of the Hendra F protein was not significantly inhibited by decreases in Ca 2؉ levels following incubation with EGTA or A23187. However, in the presence of weak amines and H ؉ V-ATPase inhibitors, known to raise intracellular pH, cleavage of Hendra F protein was inhibited while processing of the SV5 F protein was not significantly affected. The subcellular location, sensitivity to pH changes, and decreased Ca 2؉ requirement suggest that the protease responsible for cleavage of Hendra F protein differs from proteases previously shown to be involved in the processing of other viral glycoproteins.

“…By analogy with other enveloped viruses such as influenza A virus (5,32,36,(40)(41)(42)60), Semliki Forest virus (27,71), and Newcastle disease virus (76), gp160 cleavage may induce a shift from a low-energy state to a metastable Env configuration that is capable of fusion. The common requirement for cleavage of an Env precursor in many families of enveloped viruses is an indication of the general importance of this event in virus assembly (8,27,42,70,79,83,89,91).HIV-1 Env is the focus of vaccine design strategies intended to elicit virus-neutralizing antibodies. To neutralize HIV-1, an antibody must be able to bind to the native, trimeric virusassociated Env complex (11,12,63,64).…”

mentioning

confidence: 99%

Enhancing the Proteolytic Maturation of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins

Binley

Sanders

Master

et al. 2002

142

165

In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.The Env glycoprotein complex mediates receptor binding and membrane fusion during human immunodeficiency virus type 1 (HIV-1) infection of susceptible cells (66). It is synthesized as a polypeptide precursor (gp160) that oligomerizes to form a heavily glycosylated trimer (20,24). At a late stage of synthesis, most probably in the trans-Golgi network (TGN), gp160 is cleaved by furin (17,18,(55)(56)(57)(58) or other, related subtilisin-like proteases (17,18,28,38,58,90) into the surface (SU; gp120) and transmembrane (TM; gp41) subunits (34,43,(55)(56)(57)(58)82). Cleavage occurs at a motif at the gp120-gp41 juncture that contains a basic amino acid tetrad, R-X-(R/K)-R (where X is any amino acid). The gp120 and gp41 proteins then remain noncovalently associated, forming the functional, native gp120 3 -gp41 3 complex (20,24,66).During fusion, the gp120 protein interacts with the virus receptor and coreceptor on target cells. This triggers conformational changes that lead to the insertion of a hydrophobic fusion peptide, located at the N terminus of gp41, into the target cell membrane (66). Cleavage of gp160 is essential for fusion, since uncleaved gp160 is fusion incompetent (9,33,39,48). Generally, only cleaved Env is incorporated into virions (22), although uncleaved Env can be virion associated (39,48). By analogy with other enveloped viruses such as influenza A virus (5,...