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

Abstract

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.

p38 and OGT Sequestration into Viral Inclusion Bodies in Cells Infected with Human Respiratory Syncytial Virus Suppresses MK2 Activities and Stress Granule Assembly

“…Previous evidence indicated roles for p38 MAPK and ERK, in particular, in RSV infection (11,12,34). Our study demonstrated that RSV interferes with the p38 signaling pathway by spatially sequestering p38-P into viral IBs.…”

“…Previous work implicated the mitogen-activated protein kinases (MAPKs), in particular the extracellular signal-regulated kinase (ERK) and p38 MAPK, in the tropism as well as entry of RSV (10)(11)(12). The p38 MAPK is a central mediator involved in regulating cellular inflammatory and stress responses, as well as cellular protein synthesis (13,14).…”

Respiratory Syncytial Virus: Infection, Detection, and New Options for Prevention and Treatment

“…There have been many candidate cellular receptors described for RSV entry, including annexin II (70), CX3 chemokine receptor 1 (CX3CR1) (71,72), epidermal growth factor (EGF) receptor (73), calcium-dependent lectins (70), Toll-like receptor 4 (TLR4) (74,75), intercellular adhesion molecule 1 (ICAM-1) (76), nucleolin (77,78), and heparan sulfate proteoglycans (HSPGs) (79). Some receptors like EGF are purportedly used by only certain strains of RSV (73).…”

Respiratory syncytial virus therapy and prophylaxis: have we finally turned the corner?

“…For RSV, our group has taken the approach of targeting specific aspects of host cell machinery that function as chokepoints of viral replication. Specifically, p38 mitogen-activated protein (MAP) kinase is a cell-signalling enzyme used by RSV during its replicative lifecycle [12], and we showed that treatment of cultured cells with a p38 MAP kinase inhibitor dramatically inhibited replication of several respiratory viruses (including RSV), without the drug causing overt cellular toxicity [13]. While these findings are intriguing, further studies are needed to extend our in vitro observations to the in vivo state.…”