Protein Kinase C-α Activity Is Required for Respiratory Syncytial Virus Fusion to Human Bronchial Epithelial Cells

Abstract

Respiratory syncytial virus (RSV) infection activates protein kinase C (PKC), but the precise PKC isoform(s) involved and its role(s) remain to be elucidated. On the basis of the activation kinetics of different signaling pathways and the effect of various PKC inhibitors, it was reasoned that PKC activation is important in the early stages of RSV infection, especially RSV fusion and/or replication. Herein, the role of PKC-␣ during the early stages of RSV infection in normal human bronchial epithelial cells is determined. The results show that the blocking of PKC-␣ activation by classical inhibitors, pseudosubstrate peptides, or the overexpression of dominant-negative mutants of PKC-␣ in these cells leads to significantly decreased RSV infection. RSV induces phosphorylation, activation, and cytoplasm-to-membrane translocation of PKC-␣. Also, PKC-␣ colocalizes with virus particles and is required for RSV fusion to the cell membrane. Thus, PKC-␣ could provide a new pharmacological target for controlling RSV infection.Respiratory syncytial virus (RSV), a pneumovirus, is an important respiratory pathogen that produces an annual epidemic of respiratory illness which is seen primarily in infants, but also in adults, worldwide (39). Effective vaccines against RSV are not currently available. The development of antivirals requires a comprehensive molecular understanding of the early events of virus-host interaction necessary for virus fusion and entry into cells, an understanding which is lacking. RSV activates multiple signaling pathways (1, 2, 8, 13), including those involving protein kinase C (PKC), which appear to play a role in acute inflammation (1,29). However, the role of PKC in RSV infection is poorly understood.PKC comprises a family of serine/threonine kinases with at least 12 members. In general, PKC has a catalytic domain, which also contains the ATP binding site, and a regulatory domain containing the phospholipid and diacylglycerol (DAG) binding site. On the basis of structure and activation requirements, the PKC family can be divided into the following major subclasses: (i) classical PKCs, comprising the ␣, ␤I, ␤II, and ␥ isozymes that are Ca 2ϩ dependent and DAG sensitive; (ii) novel PKCs, comprising the ␦, ε, , , and isozymes that are Ca 2ϩ independent and DAG sensitive; (iii) atypical PKCs, comprising the and / isozymes that are Ca 2ϩ independent and DAG insensitive; and (iv) the PKC-isozyme that is similar to the atypical isozymes but contains an additional specific signal peptide transmembrane domain (28, 32). PKCs play an important role in infection of mammalian cells by different viruses (9,10,30,31,33,38). PKC inhibitors reduce the entry of intracellular mature vaccinia virus by affecting Rac1 activation and actin assembly (44). PKC is also important in the formation of caveolae, which a number of viruses such as human immunodeficiency virus type 1, filoviruses (Ebola), and simian virus 40 (11) utilize to evade the phagolysosomal pathway, thus allowing them to thrive inside cells by indirectly eva...

Respiratory syncytial virus induces insensitivity to β-adrenergic agonists in mouse lung epithelium in vivo

“…First, blockade of PKC, GRK2, or CXCL8 restores responsiveness to terbutaline comparable to that induced by forskolin, indicating that such blockade effectively recouples ␤ 2 -AR to PKA. Second, RSV is known to trigger phosphorylation, sustained activation, and cytoplasmto-membrane translocation of PKC (30,41), which is consistent with this mechanism. Third, membrane ␤ 2 -AR expression increases at day 2, which is incompatible with ␤-agonist desensitization as a result of either receptor internalization or degradation.…”

“…Thus the ratio of phosphoPKC:PKC in the membrane fraction of alveolar cells from RSV-infected mice increased by ϳ300% at day 2 compared with uninfected mice. Although activation of several PKC isoforms, including PKC, has been demonstrated previously in vitro following RSV infection (1,30,41), this is the first demonstration of PKC activation in RSV-infected mouse lung ex vivo.…”

Mucosal delivery of a double-stapled RSV peptide prevents nasopulmonary infection

“…A549 cells were plated in a 35-mm fluorodish (30,000 cells/dish). RSV-A2 (1.6 × 10 6 pfu/ml, 300 μl) was stained with 0.3 nM R-18 (Molecular Probes) for 1 hour, followed by passage through a Sephadex column as described previously (44,45). FITC-labeled KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKGGSGGNFYDPLVF-PSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKGSSGGAVSKV-LHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLL-PIVNKGGHHHHHH**).…”

Differential regulation ofMoraxella catarrhalis-induced interleukin-8 response by protein kinase C isoforms

“…5g). Therefore, the M. catarrhalis activates PKCa, PKCe and PKCh activation in pulmonary epithelial cells The translocation of kinases from cytosol to the cellular membrane is known to be an indicator for activation of these PKC isoforms [33,34]. In order to confirm activation of the PKC isotypes PKCa, PKCe and PKCh, translocated PKC isotypes were detected in the cell membrane following M. catarrhalis infection by Western blotting.…”