We have previously shown that RhoA is activated during RSV infection (16
). We now report data that suggest that RhoA-mediated signaling and actin polymerization are associated with the filamentous virion morphology and the syncytium-inducing phenotype. RhoA is an essential host cell protein with GTPase activity and is known to influence a variety of signaling pathways and basic cell functions (18
). The role of RhoA in virus-induced fusion and subsequent signaling events may have significance in virus infection to ensure the coordinated control of cellular activities required for virus replication, such as the stage of the cell cycle and reorganization of the actin cytoskeleton. There are many examples of viruses using the host cell machinery to complete their life cycles, so it is not surprising that RSV has adapted to utilize an essential GTPase to modify its morphological properties.
In C3-treated RSV-infected cells, there was no formation of syncytia (Fig. ). Interestingly, blocking RhoA signaling with C3 or Y-27632 did not affect the efficiency with which RSV initiated infection or the production of infectious virions in cell culture (Fig. and ). Therefore, we have shown a distinction in the requirement for RhoA signaling events between the entry of cell-free virions (independent of RhoA signaling) and cell-to-cell fusion and syncytium formation (dependent on RhoA signaling). This distinction is not related to changes in the overall expression of RSV F, since Western blotting and flow cytometric analysis of C3-treated and untreated RSV-infected cells showed equivalent levels of F expression (Fig. ).
The fusion assays whose results are shown in Fig. showed that there were distinct requirements for cell-to-cell fusion for the effector cells, which expressed the RSV envelope glycoproteins, and for the target cells, which did not express RSV envelope glycoproteins. This suggests that RhoA signaling and actin polymerization are required for an infected cell to fuse with an uninfected target cell. On the other hand, the data suggest that RhoA signaling through Rho kinase and actin polymerization are not required in uninfected cells for fusion to occur.
In Fig. , we show that C3 partially inhibited cell-to-cell fusion in treated targets. A recent study showed that a C3 treatment does not remove RhoA from the membrane but may cause it to be redistributed into detergent-soluble regions of the membrane (29
), thereby reducing its density within cholesterol-rich membrane microdomains (lipid rafts). Specific proteins, including RhoA and CD44, localize to these membrane microdomains (29
). It has been shown by our group and others that RSV proteins colocalize with cellular proteins associated with lipid microdomains, including caveolin-1 (5
) and CD44, as well as with RhoA (27
). The ADP-ribosylation of RhoA by C3 inactivates RhoA signaling, results in an overall decrease in both the number and length of viral filaments, and shifts the localization of F to nonlipid microdomain regions of the membrane (27
). This suggests that the selective incorporation of RSV proteins into lipid microdomains may result in virus assembly in microvilli providing the filamentous viral morphology associated with syncytium formation. Viruses such as HIV-1, influenza virus, and simian virus 40 have also been shown to enter cells or to bud from lipid rafts (1
). The redistribution of RhoA into non-lipid-raft membrane domains of uninfected target cells by C3 does not influence replication or infection with individual virions, but it may diminish the ability of RSV-induced filaments to mediate cell-to-cell fusion. It is possible that there is a greater requirement for membrane order and the coalescence of factors within lipid rafts to support cell-to-cell fusion than for virus-to-cell membrane fusion. This may be related either to a required mixture of proteins and heparin binding domains which are needed to mediate cell-to-cell fusion or to the need for a threshold concentration.
RSV-infected cells form long filamentous protrusions expressing the F glycoprotein within 24 h after infection (16
). Several viruses, including influenza virus and Ebola viruses, can also form long filamentous particles during infection, but it is unclear what role these filaments play in the spread of virus infection and in syncytium formation (11
). We were able to visualize these filamentous structures in RSV-infected cells by TEM (Fig. ), SEM (Fig. ), and F-specific indirect immunofluorescence (16
) (Fig. ). The treatment of cells with C3 or Y-27632 beginning 24 h before virus infection caused an altered pattern of F staining in infected cells by immunofluorescence (16
). C3- and Y-27632-treated cells had diffuse staining for F throughout the cell, and there were no distinct filamentous structures present on or around the infected cells (16
). By using SEM, we observed that viral filaments can bridge from the infected cell to a neighboring cell (Fig. ). The viral filaments were blunted and more diffusely expressed on the surfaces of HEp-2 cells treated with C3 (Fig. ), suggesting that filaments are needed to bridge cell junctions in order to initiate the cell-to-cell fusion process.
Interestingly, the RhoA-induced phosphorylation of moesin via Rho kinase can cause the production of microvilli which colocalize with CD44, a lipid raft protein, and resemble the RSV filaments shown by immunofluorescence (16
) and SEM (Fig. ) (34
). These microvilli are important for cell-to-cell adhesion in epithelial cells (48
). The inhibition of RhoA signaling by C3 inhibits microvillus formation (Fig. ) (44
), suggesting that RhoA-induced microvilli may play an important role in the normal assembly of virus particles and the formation of filamentous virus structures. These data indicate a strong association between the ability of RSV to form filaments and its ability to undergo cell-to-cell fusion, further suggesting that viral filaments may be important for cell-to-cell fusion and syncytium formation. Immuno-TEM of microvilli and filamentous virions showed that the microvilli are coated with the RSV F glycoprotein (Fig. ). These data suggest that the microvilli are sites of viral assembly and represent budding viral particles.
In order to confirm that C3 affects filamentous virus production, we developed a sucrose gradient velocity sedimentation technique to separate spherical and filamentous viruses. There were two peaks of RSV PFU in both C3-treated and untreated RSV-infected groups (Fig. ). The relative separation of spherical and filamentous morphological forms into two identifiable peaks was further supported by immunofluorescence staining of virus particles with an anti-F monoclonal antibody and TEM (Fig. ). The results showed that the C3 treatment shifted the relative production of viral morphologies to more spherical particles than those obtained with untreated RSV (Fig. ). A previous report has shown that cytochalasin D treatment enhances spherical influenza virion release and reduces the formation of filamentous influenza virus particles (39
). These data are consistent with our findings and suggest that the assembly of viral filaments requires an intact actin microfilament network.
It has been reported that filamentous structures similar to those described for wild-type RSV formed at the cell surface, even when all three envelope glycoproteins were replaced by a single foreign viral glycoprotein (vesicular stomatitis virus G protein) carrying the RSV F cytoplasmic domain (33
). The engineered recombinant virus induces filaments at the cell surface and causes cell-cell membrane fusion at pH 5.0 but not at pH 7.0. The requirement of viral genes for particle morphology differs among several viruses. For efficient particle assembly, vesicular stomatitis virus and rabies virus require the M protein (26
), influenza virus requires the M1 and M2 proteins (38
), and simian virus 5 (another paramyxovirus) requires the coexpression of the N protein, the M protein, and one of the homologous transmembrane glycoproteins (42
). For RSV, the interactions of the F protein cytoplasmic domain and the M protein with cellular proteins, RhoA, and the actin cytoskeleton may play a role in filamentous virus formation.
Our findings indicate that RhoA signaling is associated with RSV-induced filament formation and that the production of infectious virions in vitro does not require a syncytium-inducing phenotype. In addition, we report an association between RhoA-induced viral filaments and RSV-induced syncytium formation. A temperature-sensitive (ts
) strain of RSV with a non-syncytium-inducing phenotype in cell culture has been reported (25
). This virus produces equivalent numbers of progeny viruses as normal syncytium-inducing strains of RSV, but the virus is severely attenuated in vivo (25
). In addition, the non-syncytium-inducing phenotype correlates with the lack of viral filaments seen by SEM (25
). Thus, RSV infection in the presence of C3 and Y-27632 treatment may be analogous to ts
RSV strains, which replicate with equal efficiencies in vitro as their counterpart syncytium-inducing strains, but without significant syncytium formation (25
). However, the ts
strains are attenuated in vivo, suggesting that viral filament formation and cell-to-cell fusion may represent virulence determinants. Learning how to produce non-syncytium-inducing viruses would be valuable for the development of live attenuated vaccines for two reasons. First, the virus may be less virulent, and second, if the virion morphology were more homogeneous, then the virus might be easier to purify and concentrate. More work is needed to define the precise steps at which RhoA activation is required for syncytium formation and to determine whether RhoA-induced signaling events are involved in RSV pathogenesis.