Inhibition of PKC-alpha activity blocks respiratory syncytial virus infection*1

Abstract Rationale RSV infection activates several signaling pathways leading to the production of inflammatory mediators. However, the majority of studies have utilized A549 epithelial carcinoma cells and it remains unknown whether these pathways are operational in normal human bronchial epithelial (NHBE) cells. The role of specific PKC isozymes in RSV infection of NHBE cells was examined in this study. Methods Cultured NHBE cells were exposed to RSV at an MOI of 20 for different time periods. The PKC isoforms and their activation status were determined by Western blotting and immunocytofluorescence by confocal microscopy. Results Western blot analysis revealed that NHBE cells express PKC-alpha, beta-2, gamma, delta, epsilon, theta, and iota. Furthermore, immunocytofluorescence studies indicate that PKC-alpha tranlocates to the plasma membrane and colocalizes with RSV as early as 10 min after infection. PKC-alpha translocation is paralleled by an increase in its phosphorylation suggesting that it is activated. Moreover, inhibition of PKC activity using Calphostin C and pseudosubstrate peptide impairs RSV infection showing that PKC-alpha is required for successful RSV infection. Finally, analysis of RSV fusion using R18-labeled virus shows that inhibition of PKC-alpha blocks viral fusion. Conclusions These results demonstrate that PKC-alpha activation, because of its role in membrane fusion, is critical for RSV infection. Thus, PKC-alpha may be potential target for inhibiting RSV infection and consequently reducing RSV-associated respiratory diseases and exacerbation of asthma.