Toll-Like Receptor 4-Mediated Activation of p38 Mitogen-Activated Protein Kinase Is a Determinant of Respiratory Virus Entry and Tropism

Respiratory viruses exert a heavy toll of morbidity and mortality worldwide. Despite this burden there are few specific treatments available for respiratory virus infections. Since many viruses utilize host cell enzymatic machinery such as protein kinases for replication, we determined whether pharmacological inhibition of kinases could, in principle, be used as a broad antiviral strategy for common human respiratory virus infections. A panel of green fluorescent protein (GFP)-expressing recombinant respiratory viruses, including an isolate of H1N1 influenza virus (H1N1/Weiss/43), was used to represent a broad range of virus families responsible for common respiratory infections (Adenoviridae, Paramyxoviridae, Picornaviridae, and Orthomyxoviridae). Kinase inhibitors were screened in a high-throughput assay that detected virus infection in human airway epithelial cells (1HAEo-) using a fluorescent plate reader. Inhibition of p38 mitogen-activated protein kinase (MAPK) signaling was able to significantly inhibit replication by all viruses tested. Therefore, the pathways involved in virus-mediated p38 and extracellular signal-regulated kinase (ERK) MAPK activation were investigated using bronchial epithelial cells and primary fibroblasts derived from MyD88 knockout mouse lungs. Influenza virus, which activated p38 MAPK to approximately 10-fold-greater levels than did respiratory syncytial virus (RSV) in 1HAEo- cells, was internalized about 8-fold faster and more completely than RSV. We show for the first time that p38 MAPK is a determinant of virus infection that is dependent upon MyD88 expression and Toll-like receptor 4 (TLR4) ligation. Imaging of virus-TLR4 interactions showed significant clustering of TLR4 at the site of virus-cell interaction, triggering phosphorylation of downstream targets of p38 MAPK, suggesting the need for a signaling receptor to activate virus internalization.Respiratory virus infections cause considerable morbidity and mortality worldwide; it was recently reported that hospitalizations due to respiratory syncytial virus (RSV) exceed 2 million per year in the Unites States alone (16). An H1N1 swine influenza pandemic took place during the 2009-2010 winter season (14), and there is the lingering threat of an H5N1 avian influenza pandemic, with mortality due to direct bird-to-human H5N1 infection in hospitalized patients between 30 and 100% (3). The severe acute respiratory syndrome (SARS)-associated coronavirus, isolated in 2003, resulted in devastating respiratory tract infections with few treatment options (40). For most common respiratory viruses, treatment is symptomatic, and for pathogens such as influenza viruses for which specific treatments are available, oseltamivir (Tamiflu)- and amantidine-resistant strains are emerging and being transmitted globally (33).All functions within a cell are triggered and regulated by cell signaling cues. Since viruses are obligate intracellular parasites, they rely upon cell signaling to regulate all processes within the cell that drive virus replication. In this study we investigated the effects of kinase inhibitors as a therapeutic strategy and to investigate the roles played by some kinases during virus replication. The extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPKs) have been shown by us and others to play important roles during virus replication in vitro (19, 20, 26, 30, 42), and we have recently reported that inhibition of p38 MAPK activation is an effective and novel antiviral strategy in vivo (29). The significance of p38 MAPK activity in vivo is such that inadvertent and coincident activation of this kinase by some pharmaceutical agents enhances virus replication (29). Antiviral strategies may exist whereby inhibition of host cell kinases may stem the spread and replication of numerous different viral species. Such broad antiviral strategies would permit administration of kinase inhibitors to patients suspected of having respiratory viral infection, and to health care workers or inhabitants within the locale of a viral outbreak, prior to the availability of results from laboratory diagnostic testing.The activation of p38 MAPK by pattern recognition receptors (PRRs) has been studied in the context of the antiviral immune response (reviewed in reference 22). We report here that viruses usurp these responses for the benefit of virus replication through activation of p38 MAPK, mediated by a PRR (Toll-like receptor 4 [TLR4]) and MyD88, providing the basis for a broad-spectrum antiviral.