Article

RhoA signaling is required for respiratory syncytial virus-induced syncytium formation and filamentous virion morphology.

Vaccine Research Center, Building 40, Room 2502, NIAID, NIH, 40 Convent Dr., MSC 3017, Bethesda, MD 20892-3017, USA.
Journal of Virology (Impact Factor: 4.65). 06/2005; 79(9):5326-36. DOI: 10.1128/JVI.79.9.5326-5336.2005
Source: PubMed

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.

Download full-text

Full-text

Available from: Teresa R. Johnson, Jun 26, 2015
0 Followers
 · 
100 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Respiratory syncytial virus (RSV) primarily causes bronchiolitis and pneumonia in infants. In spite of intense research, no safe and effective vaccine has been developed yet. For understanding its pathogenesis and development of anti-RSV drugs/therapeutics, it is indispensable to study the RSV-host interaction. Although, there are limited studies using electron microscopy to elucidate the infection process of RSV, to our knowledge, no study has reported the morphological impact of RSV infection using atomic force microscopy. We report the cytoplasmic and nuclear changes in human epidermoid cell line type 2 using atomic force microscopy. Human epidermoid cell line type 2 cells, grown on cover slips, were infected with RSV and fixed after various time periods, processed and observed for morphological changes using atomic force microscopy. RSV infected cells showed loss of membrane integrity, with degeneration in the cellular content and cytoskeleton. Nuclear membrane was disintegrated and nuclear volume was decreased. The chromatin of the RSV infected cells was condensed, progressing towards degeneration via pyknosis and apoptosis. Membrane protrusions of ∼150-200 nm diameter were observed on RSV infected cells after 6 h, suggestive of prospective RSV budding sites. To our knowledge, this is the first study of RSV infection process using atomic force microscopy. Such morphological studies could help explore viral infection process aiding the development of anti-RSV therapies.
    Journal of Microscopy 11/2013; DOI:10.1111/jmi.12095 · 2.15 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Respiratory syncytial virus (RSV) is a major cause of pneumonia and wheezing in infants and the elderly, but to date there is no licensed vaccine. We developed a gold nanorod construct that displayed the major protective antigen of the virus, the fusion protein (F). Nanorods conjugated to RSV F were formulated as a candidate vaccine preparation by covalent attachment of viral protein using a layer-by-layer approach. In vitro studies using ELISA, electron microscopy and circular dichroism revealed that conformation-dependent epitopes were maintained during conjugation, and transmission electron microscopy studies showed that a dispersed population of particles could be achieved. Human dendritic cells treated with the vaccine induced immune responses in primary human T cells. These results suggest that this vaccine approach may be a potent method for immunizing against viruses such as RSV with surface glycoproteins that are targets for the human immune response.
    Nanotechnology 06/2013; 24(29):295102. DOI:10.1088/0957-4484/24/29/295102 · 3.67 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cholesterol and sphingolipid enriched lipid raft micro-domains in the plasma membrane play an important role in the life-cycle of numerous enveloped viruses. Although human respiratory syncytial virus (RSV) proteins associate with the raft domains of infected cells and rafts are incorporated in RSV virion particles, the functional role of raft during RSV infection was unknown. In the current study we have identified rafts as an essential component of host cell that is required for RSV infection. Treatment of human lung epithelial cells with raft disrupting agent methyl-beta-cyclodextrin (MBCD) led to drastic loss of RSV infectivity due to diminished release of infectious progeny RSV virion particles from raft disrupted cells. RSV infection of raft deficient Niemann-Pick syndrome type C human fibroblasts and normal human embryonic lung fibroblasts revealed that during productive RSV infection, raft is required for release of infectious RSV particles.
    Virology 11/2011; 422(2):205-13. DOI:10.1016/j.virol.2011.10.029 · 3.28 Impact Factor