A dual role for RhoA in respiratory syncytial virus replication and syncytia formation

Respiratory Syncytial Virus (RSV) is an important human pathogen which causes severe lower respiratory infections in young children, the institutionalized elderly, and immunocompromised bone marrow and lung transplant recipients. Our laboratory has recently shown that the fusion (F) glycoprotein of RSV binds to small GTPase RhoA during infection. Disrupting this interaction with a peptide derived from amino acids 77--95 of RhoA inhibits RSV-induced membrane fusion and RSV replication in cell culture and in mice.;RhoA controls several cellular processes such as cell cycle progression and cell morphology and migration. In the cell, RhoA activates the formation of actin stress fibers, focal adhesions and microvilli as well as gene transcription. Inactive RhoA is localized to the cytoplasm and bound to RhoGDI and GDP. RhoA is activated by several different G-protein coupled receptor ligands including thrombin, angiotensin II, and lysophosphatydic acid (LPA). RhoA is activated by release of RhoGDI, an exchange of GDP for GTP, and attachment to the inside of the plasma membrane by geranylgeranylation at the carboxy terminus of the protein.;I have shown that RhoA is activated during RSV replication and using several different inhibitors of RhoA activation and signaling, I have also demonstrated that there is a dual role for RhoA in RSV replication. First, geranylgeranylated and membrane bound RhoA is required for RSV replication and RSV-induced membrane fusion. Second, RhoA signaling is required for the production of F-expressing microvilli, filamentous virions, cell-to-cell fusion and syncytia formation. However, RhoA signaling is not required for the production of infectious virions in cell culture and in the absence of RhoA signaling RSV virions are non-filamentous. These data indicate that microvilli may play a role in both the production of filamentous virions and cell-to-cell fusion. The results in my dissertation suggest several new targets for antiviral drug development and demonstrate fundamental mechanisms involved in RSV-induced membrane fusion and syncytia formation.