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

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.

The Human Respiratory Syncytial Virus Matrix Protein Is Required for Maturation of Viral Filaments

“…HRSVinduced surface filaments have been suggested to be the equivalent of virions (27,49,53,60), and the loss of viral filaments typically results in the loss of the majority of viral infectivity (7,42,49,53). An exception to the above-mentioned data was a study in which wt HRSV-infected HEp-2 cells were treated with RhoA inhibitors (22). In those instances, the amount of surface filaments was strongly reduced, but the yield of infectious virus was unchanged relative to the yield from untreated cells.…”

“…Several reports have implicated cytoskeletal elements in the processes that lead to virus egress, in particular microtubules, myosin V, actin, and actin-regulatory proteins such as profilin and RhoA (30,32,59). In addition, a role for an actin/myosinbased motility system in HRSV exit has been proposed (6,53,59), and there is strong evidence for the involvement of lipid rafts (8,9,22,27,31,36,49,50,53).…”

“…Viral filaments range in length from 2 to 8 m; contain high concentrations of N, G, and F proteins; and often appear as aggregates (1,16,31,43,51,53,55). In a previous study where anti-RhoA drugs were used to block the formation of viral filaments, the virion morphology was reported to shift from predominantly filamentous to predominantly round, without affecting the total yield of infectivity (22). This finding suggests that while the filamentous phenotype is perhaps the "preferred" morphology, the virus has significant potential to generate infectious virions of a nonfilamentous morphology.…”

“…HRSV infectivity is associated predominantly with a filamentous form (1,22,41,51,60). These viral filaments are produced at the cell surface late in the infection cycle and remain largely attached to the infected cells, where they can be readily visualized by immunofluorescence (IF) or electron microscopy (EM).…”

Heat shock protein inhibitors increase the efficacy of measles virotherapy

“…The active form is located on the plasma membrane, and the inactive form, in the cytosol. 44 RhoA activation has been shown to affect cell fusion and syncytia formation after infection with paramyxoviurses such as respiratory syncytia virus 45 and Hendra virus. 25 RhoA is regulated by heat shock proteins: HSP90 is required for RhoA activation in thrombin-induced signaling to cytoskeleton, and the activation of RhoA could be specifically inhibited by GA in this reaction.…”