Inhibition of human coronavirus 229E infection in human epithelial lung cells (L132) by chloroquine: involvement of p38 MAPK and ERK

Abstract

The antiviral effects of chloroquine (CQ) on human coronavirus 229E (HCoV-229E) infection of human fetal lung cell line, L132 are reported. CQ significantly decreased the viral replication at concentrations lower than in clinical usage. We demonstrated that CQ affects the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK). Furthermore, p38 MAPK inhibitor, SB203580, inhibits CPE induced by HCoV-229E infection and viral replication. Our findings suggest that CQ affects the activation of MAPKs, involved in the replication of HCoV-229E.

Fig. 1

Inhibitory effects of CQ on virus replication and infectivity of HCoV-229E. (a) Effect of CQ on the released virus in supernatants. L132 cells were treated with indicated concentrations of CQ and adsorbed with HCoV-229E at an MOI of 3. RNA was extracted from culture supernatants and used for reverse transcriptional real-time PCR at 3 h post infection. The amount of viral RNA in CQ-untreated cells was calculated as 100%. (b) Effect of CQ on the cytoplasmic viral RNA. L132 cells were pretreated with indicated concentrations of CQ, and infected by HCoV-229E at an MOI of 3. At 3 h post infection, cellular RNA was extracted and used for reverse transcriptional real-time PCR. Data represent mean ± S.E. in triplicate.

Fig. 2

Involvement of p38 and ERK in the infection of HCoV-229E. (a) Effect of CQ on the phosphorylation of p38 MAPK and ERK. L132 cells were pretreated for 2 h with or without CQ and infected with HCoV-229E at an MOI of 3. Cytoplasmic proteins extracted at 90 min post infection were immunoblotted and reacted with specific antibodies against indicated MAPKs. (b) Effect of p38 inhibitor SB203580 on the CPE. L132 cells were treated with indicated concentrations of SB203580, a specific p38 MAPK inhibitor, subsequently adsorbed with HCoV-229E at an MOI of 3. At 72 h post infection, the cells were stained with crystal violet and cell viability was measured. The absorbance of CQ-untreated cells was calculated as 100%. (c) Effect of p38 inhibitor SB203580 on the viral RNA load in supernatant at 0, 4, 10, and 25 μM of p38 inhibitor. L132 cells were pretreated with indicated concentrations of SB203580 and adsorbed with HCoV-229E at an MOI of 3. RNA extracted from culture supernatants were used for reverse transcription real-time PCR at 72 h post infection. The amount of viral RNA load in CQ-untreated cells was calculated as 100%. Data represent mean ± S.E.M. of triplicates.