Functions of the respiratory syncytial virus SH and P proteins.

摘要

Respiratory syncytial virus (RSV) is the leading cause of pediatric hospitalizations due to lower respiratory tract infections. Immunocompromised and elderly patients can also develop severe respiratory illness. Immunoprophylaxis with monoclonal antibodies against the RSV fusion protein is the only currently available treatment option. Although research is ongoing to develop a safe and effective vaccine against RSV, no vaccine is available yet. Understanding the way RSV uses the cell for survival and proliferation is an important step towards finding effective infection prevention methods.;Some members of the Paramyxoviridae family, which includes RSV, encode for a small hydrophobic (SH) protein of unknown function. Previous studies by our lab show that the SH protein of parainfluenza virus 5 (PIV5) inhibits apoptosis through the TNF-alpha pathway. The mumps virus SH protein was found to be a functional counterpart of the PIV5 SH despite the lack of sequence homology between these proteins. To understand the function of the RSV SH protein, a recombinant PIV5 containing the RSV SH protein in place of the PIV5 SH protein (PIV5DeltaSH - RSV SH) was generated. Analysis showed that the RSV SH could functionally replace the PIV5 SH protein since infection of cells with this virus did not induce apoptosis. Expression of the RSV SH protein in the absence of all other viral proteins was sufficient to inhibit the TNF-alpha pathway that leads to NF-kappaB activation. An RSVDeltaSH mutant was generated in a different approach at understanding the role RSV SH protein during infection. The RSVDeltaSH virus induced apoptosis in A549 cells, a human lung epithelial cell line. Interestingly the levels of TNF-alpha in the media of RSVDeltaSH infected A549 cells were not sufficient to induce apoptosis in this cell line, suggesting that the RSV SH has a different mechanism of inhibiting apoptosis in A549 cells.;The RSV SH is an accessory protein involved in RSV pathogenesis but not necessary for virus growth. In contrast, the RSV phosphoprotein (P) is necessary for viral RNA synthesis and a P deletion mutant is not viable. The P protein is an integral part of the polymerase complex involved in transcription and replication and it is the major phosphorylated species of the virus. The role of P protein phosphorylation for the proteins function is not completely understood. Recent studies in our lab suggest that the cellular kinase Akt is important for the RNA synthesis of non-segmented negative-sense RNA viruses. To study the role of Akt in RSV RNA synthesis, Akt activity was inhibited with small molecule inhibitors, siRNA and dominant negative constructs. Inhibition of Akt reduced RSV RNA synthesis, protein production and RSV titers. In addition, a small molecule inhibitor of Akt activation also reduced P protein phosphorylation, suggesting that P is a target of Akt. Serine 86 of P was identified as the Akt phosphorylation site in vitro. Mutation of that serine to an alanine significantly reduced P protein phosphorylation by Akt in an in vitro kinase assay. Phosphorylation at serine 86 was also detected in P protein from RSV infected cells. These results suggest that Akt inhibitors could be developed for RSV therapeutics. Identification of the site of Akt phosphorylation in P during RSV infection could lead to a new strategy in the development of an RSV vaccine.