A Chimeric Respiratory Syncytial Virus Fusion Protein Functionally Replaces the F and HN Glycoproteins in Recombinant Sendai Virus

ABSTRACT Entry of most paramyxoviruses is accomplished by separate attachment and fusion proteins that function in a cooperative manner. Because of this close interdependence, it was not possible with most paramyxoviruses to replace either of the two protagonists by envelope glycoproteins from related paramyxoviruses. By using reverse genetics of Sendai virus (SeV), we demonstrate that chimeric respiratory syncytial virus (RSV) fusion proteins containing either the cytoplasmic domain of the SeV fusion protein or in addition the transmembrane domain were efficiently incorporated into SeV particles provided the homotypic SeV-F was deleted. In the presence of SeV-F, the chimeric glycoproteins were incorporated with significantly lower efficiency, indicating that determinants in the SeV-F ectodomain exist that contribute to glycoprotein uptake. Recombinant SeV in which the homotypic fusion protein was replaced with chimeric RSV fusion protein replicated in a trypsin-independent manner and was neutralized by antibodies directed to RSV-F. However, replication of this virus also relied on the hemagglutinin-neuraminidase (HN) as pretreatment of cells with neuraminidase significantly reduced the infection rate. Finally, recombinant SeV was generated with chimeric RSV-F as the only envelope glycoprotein. This virus was not neutralized by antibodies to SeV and did not use sialic acids for attachment. It replicated more slowly than hybrid virus containing HN and produced lower virus titers. Thus, on the one hand RSV-F can mediate infection in an autonomous way while on the other hand it accepts support by a heterologous attachment protein.

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Respiratory syncytial virus (RSV) fusion protein expressed by recombinant Sendai virus elicits B-cell and T-cell responses in cotton rats and confers protection against RSV subtypes A and B

Vaccine ◽

10.1016/j.vaccine.2007.10.038 ◽

2007 ◽

Vol 25 (52) ◽

pp. 8782-8793 ◽

Cited By ~ 50

Author(s):

Xiaoyan Zhan ◽

Julia L. Hurwitz ◽

Sateesh Krishnamurthy ◽

Toru Takimoto ◽

Kelli Boyd ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

T Cell ◽

Fusion Protein ◽

B Cell ◽

Sendai Virus ◽

T Cell Responses ◽

Cell Responses ◽

Cotton Rats ◽

Syncytial Virus

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Sendai virus recombinant vaccine expressing a secreted, unconstrained respiratory syncytial virus fusion protein protects against RSV in cotton rats

International Immunology ◽

10.1093/intimm/dxu107 ◽

2014 ◽

Vol 27 (5) ◽

pp. 229-236 ◽

Cited By ~ 9

Author(s):

Xiaoyan Zhan ◽

Karen S. Slobod ◽

Bart G. Jones ◽

Robert E. Sealy ◽

Toru Takimoto ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Fusion Protein ◽

Sendai Virus ◽

Recombinant Vaccine ◽

Virus Fusion ◽

Cotton Rats ◽

Virus Recombinant ◽

Syncytial Virus

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Cleavage at the Furin Consensus Sequence RAR/KR109 and Presence of the Intervening Peptide of the Respiratory Syncytial Virus Fusion Protein Are Dispensable for Virus Replication in Cell Culture

Journal of Virology ◽

10.1128/jvi.76.18.9218-9224.2002 ◽

2002 ◽

Vol 76 (18) ◽

pp. 9218-9224 ◽

Cited By ~ 25

Author(s):

Gert Zimmer ◽

Karl-Klaus Conzelmann ◽

Georg Herrler

Keyword(s):

Cell Culture ◽

Respiratory Syncytial Virus ◽

Fusion Protein ◽

Reverse Genetics ◽

Consensus Sequence ◽

Vero Cells ◽

Proteolytic Processing ◽

Furin Cleavage ◽

F Protein ◽

Syncytial Virus

ABSTRACT Proteolytic processing of the respiratory syncytial virus F (fusion) protein results in the generation of the disulfide-linked subunits F1 and F2 and in the release of pep27, a glycopeptide originally located between the two furin cleavage sites FCS-1 (RKRR136) and FCS-2 (RAR/KR109). We made use of reverse genetics to study the importance of FCS-2 and of pep27 for BRSV replication in cell culture. Replacement of FCS-2 in the F protein of recombinant viruses by either of the sequences NANR109, RANN109 or SANN109, respectively, abolished proteolytic processing at this position, whereas the cleavage of FCS-1 was not affected. All mutants replicated in calf kidney and Vero cells in the absence of exogenous trypsin, although somewhat higher titers of BRSV containing the NANR109 or the RANN109 motif were achieved in the presence of trypsin. The virus mutants showed a reduced cytopathic effect which was lowest in the case of the SANN109 mutant. These findings demonstrate that cleavage at FCS-2 is dispensable for replication of respiratory syncytial virus in cell culture. A deletion mutant containing FCS-1 but lacking FCS-2 and most of pep27 replicated in cell culture as efficiently as the parental virus, indicating that this domain of the F protein is not essential for virus maturation and infectivity.

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Insertion of the Two Cleavage Sites of the Respiratory Syncytial Virus Fusion Protein in Sendai Virus Fusion Protein Leads to Enhanced Cell-Cell Fusion and a Decreased Dependency on the HN Attachment Protein for Activity

Journal of Virology ◽

10.1128/jvi.00078-08 ◽

2008 ◽

Vol 82 (12) ◽

pp. 5986-5998 ◽

Cited By ~ 22

Author(s):

Joanna Rawling ◽

Blanca García-Barreno ◽

José A. Melero

Keyword(s):

Respiratory Syncytial Virus ◽

Fusion Protein ◽

Membrane Fusion ◽

Cell Fusion ◽

Sendai Virus ◽

Cleavage Sites ◽

Attachment Protein ◽

F Protein ◽

Syncytial Virus ◽

Cell Cell

ABSTRACT Cell entry by paramyxoviruses requires fusion of the viral envelope with the target cell membrane. Fusion is mediated by the viral fusion (F) glycoprotein and usually requires the aid of the attachment glycoprotein (G, H or HN, depending on the virus). Human respiratory syncytial virus F protein (FRSV) is able to mediate membrane fusion in the absence of the attachment G protein and is unique in possessing two multibasic furin cleavage sites, separated by a region of 27 amino acids (pep27). Cleavage at both sites is required for cell-cell fusion. We have investigated the significance of the two cleavage sites and pep27 in the context of Sendai virus F protein (FSeV), which possesses a single monobasic cleavage site and requires both coexpression of the HN attachment protein and trypsin in order to fuse cells. Inclusion of both FRSV cleavage sites in FSeV resulted in a dramatic increase in cell-cell fusion activity in the presence of HN. Furthermore, chimeric FSeV mutants containing both FRSV cleavage sites demonstrated cell-cell fusion in the absence of HN. The presence of two multibasic cleavage sites may therefore represent a strategy to regulate activation of a paramyxovirus F protein for cell-cell fusion in the absence of an attachment protein.

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