Neuraminidase treatment of respiratory syncytial virus-infected cells or virions, but not target cells, enhances cell–cell fusion and infection

Cell-cell fusion between eukaryotic cells is a general process involved in many physiological and pathological conditions, including infections by bacteria, parasites, and viruses. As obligate intracellular pathogens, viruses use intracellular machineries and pathways for efficient replication in their host target cells. Interestingly, certain viruses, and, more especially, enveloped viruses belonging to different viral families and including human pathogens, can mediate cell-cell fusion between infected cells and neighboring non-infected cells. Depending of the cellular environment and tissue organization, this virus-mediated cell-cell fusion leads to the merge of membrane and cytoplasm contents and formation of multinucleated cells, also called syncytia, that can express high amount of viral antigens in tissues and organs of infected hosts. This ability of some viruses to trigger cell-cell fusion between infected cells as virus-donor cells and surrounding non-infected target cells is mainly related to virus-encoded fusion proteins, known as viral fusogens displaying high fusogenic properties, and expressed at the cell surface of the virus-donor cells. Virus-induced cell-cell fusion is then mediated by interactions of these viral fusion proteins with surface molecules or receptors involved in virus entry and expressed on neighboring non-infected cells. Thus, the goal of this review is to give an overview of the different animal virus families, with a more special focus on human pathogens, that can trigger cell-cell fusion.

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Alternative Mechanisms of Respiratory Syncytial Virus Clearance in Perforin Knockout Mice Lead to Enhanced Disease

Journal of Virology ◽

10.1128/jvi.75.20.9918-9924.2001 ◽

2001 ◽

Vol 75 (20) ◽

pp. 9918-9924 ◽

Cited By ~ 52

Author(s):

Sandra Aung ◽

John A. Rutigliano ◽

Barney S. Graham

Keyword(s):

Respiratory Syncytial Virus ◽

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Target Cell ◽

Cell Lysis ◽

Target Cells ◽

Alternative Mechanism ◽

Wild Type ◽

Infected Cells ◽

Syncytial Virus

ABSTRACT Virus-specific cytotoxic T lymphocytes are key effectors for the clearance of virus-infected cells and are required for the normal clearance of respiratory syncytial virus (RSV) in mice. Although perforin/granzyme-mediated lysis of infected cells is thought to be the major molecular mechanism used by CD8+ cytotoxic T lymphocytes for elimination of virus, its role in RSV has not been reported. Here, we show that viral clearance in perforin knockout (PKO) mice is slightly delayed but that both PKO and wild-type mice clear virus by day 10, suggesting an alternative mechanism of RSV clearance. Effector T cells from the lungs of both groups of mice were shown to lyse Fas (CD95)-overexpressing target cells in greater numbers than target cells expressing low levels of Fas, suggesting that Fas ligand (CD95L)-mediated target cell lysis was occurring in vivo. This cell lysis was associated with a delay in RSV-induced disease in PKO mice compared to the time of disease onset for wild-type controls, which correlated with increased and prolonged production of gamma interferon and tumor necrosis factor alpha levels in PKO mice. We conclude that while perforin is not necessary for the clearance of primary RSV infection, the use of alternative CTL target cell killing mechanisms is less efficient and can lead to enhanced disease.

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Functional Analysis of Recombinant Respiratory Syncytial Virus Deletion Mutants Lacking the Small Hydrophobic and/or Attachment Glycoprotein Gene

Journal of Virology ◽

10.1128/jvi.75.15.6825-6834.2001 ◽

2001 ◽

Vol 75 (15) ◽

pp. 6825-6834 ◽

Cited By ~ 171

Author(s):

Sunee Techaarpornkul ◽

Naina Barretto ◽

Mark E. Peeples

Keyword(s):

Cell Culture ◽

Respiratory Syncytial Virus ◽

G Protein ◽

Viral Entry ◽

Target Cells ◽

Virion Assembly ◽

F Protein ◽

Infected Cells ◽

Syncytial Virus ◽

Small Hydrophobic

ABSTRACT Respiratory syncytial virus (RSV) produces three envelope glycoproteins, the attachment glycoprotein (G), the fusion (F) protein, and the small hydrophobic (SH) protein. It had been assumed, by analogy with other paramyxoviruses, that the G and F proteins would be required for the first two steps of viral entry, attachment and fusion. However, following repeated passage in cell culture, a viable mutant RSV that lacked both the G and SH genes was isolated (R. A. Karron, D. A. Buonagurio, A. F. Georgiu, S. S. Whitehead, J. E. Adamus, M. L. Clements-Mann, D. O. Harris, V. B. Randolph, S. A. Udem, B. R. Murphy, and M. S. Sidhu, Proc. Natl. Acad. Sci. USA 94:13961–13966, 1997). To explore the roles of the G, F, and SH proteins in virion assembly, function, and cytopathology, we have modified the full-length RSV cDNA and used it to rescue infectious RSV lacking the G and/or SH genes. The three resulting viruses and the parental virus all contain the green fluorescent protein (GFP) gene that serves to identify infected cells. We have used purified, radiolabeled virions to examine virus production and function, in conjunction with GFP to quantify infected cells. We found that the G protein enhances virion binding to target cells but plays no role in penetration after attachment. The G protein also enhances cell-to-cell fusion, presumably via cell-to-cell binding, and enhances virion assembly or release. The presence or absence of the G protein in virions has no obvious effect on the content of F protein or host cell proteins in the virion. In growth curve experiments, the viruses lacking the G protein produced viral titers that were at least 10-fold lower than titers of viruses containing the G protein. This reduction is due in large part to the less efficient release of virions and the lower infectivity of the released virions. In the absence of the G protein, virus expressing both the F and SH proteins displayed somewhat smaller plaques, lower fusion activity, and slower viral entry than the virus expressing the F protein alone, suggesting that the SH protein has a negative effect on virus fusion in cell culture.

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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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RhoA Signaling Is Required for Respiratory Syncytial Virus-Induced Syncytium Formation and Filamentous Virion Morphology

Journal of Virology ◽

10.1128/jvi.79.9.5326-5336.2005 ◽

2005 ◽

Vol 79 (9) ◽

pp. 5326-5336 ◽

Cited By ~ 70

Author(s):

Tara L. Gower ◽

Manoj K. Pastey ◽

Mark E. Peeples ◽

Peter L. Collins ◽

Lewis H. McCurdy ◽

...

Keyword(s):

Electron Microscopy ◽

Respiratory Syncytial Virus ◽

Cell Fusion ◽

Infectious Virus ◽

Syncytium Formation ◽

Velocity Sedimentation ◽

Rsv Infection ◽

Infected Cells ◽

Syncytial Virus ◽

Rhoa Signaling

ABSTRACT Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants, the elderly, and immunocompromised adults. RSV infection of HEp-2 cells induces the activation of RhoA, a small GTPase. We therefore asked whether RhoA signaling is important for RSV replication or syncytium formation. The treatment of HEp-2 cells with Clostridium botulinum C3, an enzyme that ADP-ribosylates and specifically inactivates RhoA, inhibited RSV-induced syncytium formation and cell-to-cell fusion, although similar levels of PFU were released into the medium and viral protein expression levels were equivalent. Treatment with another inhibitor of RhoA signaling, the Rho kinase inhibitor Y-27632, yielded similar results. Scanning electron microscopy of C3-treated infected cells showed reduced numbers of single blunted filaments, in contrast to the large clumps of long filaments in untreated infected cells. These data suggest that RhoA signaling is associated with filamentous virus morphology, cell-to-cell fusion, and syncytium formation but is dispensable for the efficient infection and production of infectious virus in vitro. Next, we developed a semiquantitative method to measure spherical and filamentous virus particles by using sucrose gradient velocity sedimentation. Fluorescence and transmission electron microscopy confirmed the separation of spherical and filamentous forms of infectious virus into two identifiable peaks. The C3 treatment of RSV-infected cells resulted in a shift to relatively more spherical virions than those from untreated cells. These data suggest that viral filamentous protuberances characteristic of RSV infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play a role in initiating cell-to-cell fusion.

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Iduronic Acid-Containing Glycosaminoglycans on Target Cells Are Required for Efficient Respiratory Syncytial Virus Infection

Virology ◽

10.1006/viro.2000.0293 ◽

2000 ◽

Vol 271 (2) ◽

pp. 264-275 ◽

Cited By ~ 154

Author(s):

Louay K. Hallak ◽

Peter L. Collins ◽

Warren Knudson ◽

Mark E. Peeples

Keyword(s):

Respiratory Syncytial Virus ◽

Virus Infection ◽

Respiratory Syncytial Virus Infection ◽

Target Cells ◽

Iduronic Acid ◽

Syncytial Virus

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Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

Frontiers in Microbiology ◽

10.3389/fmicb.2015.01552 ◽

2016 ◽

Vol 6 ◽

Cited By ~ 1

Author(s):

Mai Izumida ◽

Haruka Kamiyama ◽

Takashi Suematsu ◽

Eri Honda ◽

Yosuke Koizumi ◽

...

Keyword(s):

Cell Fusion ◽

Envelope Protein ◽

Target Cells ◽

Cell Cell

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Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans

Journal of Virology ◽

10.1128/jvi.00986-09 ◽

2009 ◽

Vol 83 (20) ◽

pp. 10710-10718 ◽

Cited By ~ 55

Author(s):

Steven Kwilas ◽

Rachael M. Liesman ◽

Liqun Zhang ◽

Edward Walsh ◽

Raymond J. Pickles ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

G Protein ◽

Cell Cultures ◽

Vero Cells ◽

Target Cells ◽

Apical Surface ◽

Virus Attachment ◽

C Terminus ◽

Syncytial Virus

ABSTRACT Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.

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The Avian Retrovirus Avian Sarcoma/Leukosis Virus Subtype A Reaches the Lipid Mixing Stage of Fusion at Neutral pH

Journal of Virology ◽

10.1128/jvi.77.5.3058-3066.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3058-3066 ◽

Cited By ~ 52

Author(s):

Laurie J. Earp ◽

Sue E. Delos ◽

Robert C. Netter ◽

Paul Bates ◽

Judith M. White

Keyword(s):

Cell Fusion ◽

Receptor Binding ◽

Low Ph ◽

Target Cells ◽

Dependent Manner ◽

Neutral Ph ◽

Subtype A ◽

Avian Sarcoma ◽

Virus Subtype ◽

Cell Cell

ABSTRACT We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at ≥22°C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and ≥22°C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37°C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.

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