pH of acidic vesicles, has been used for the treatment of malaria and inflammatory diseases, such as rheumatoid arthritis. CQ has antimicrobial effects even against viruses, such as human immunodeficiency virus type 1 (HIV-1) and SARS-CoV (Sperber et al., 1993; Savarino et al., 2003; Vincent et al., 2005) . However, the inhibitory effect on SARS-CoV is inactive in vivo (Barnard et al., 2006) , and the detailed mechanisms underlying the antimicrobial and anti-inflammatory effects of CQ remain poorly understood.
Coronavirus, an enveloped virus, enters the cytoplasm by endocytosis and matures in the membrane transport system, such as the trans-Golgi network (TGN) (Nauwynck et al., 1999; Ng et al., 2003) . ER stress caused by Japanese encephalitis virus infection induces the activation of p38 mitogen-activated protein kinase (MAPK) and host cell apoptosis (Su et al., 2002) . MAPKs including ERK, JNK and p38 are involved in cell death (Xia et al., 1995) play a crucial role in infection of coronaviruses, such as mouse hepatitis virus and SARS-CoV (Banerjee et al., 2002; Kopecky-Bromberg et al., 2006) . CQ inhibits the activation of p38 MAPK and cytokine production caused by CpG DNA (Yi and Krieg, 1998) . ERK, another MAPK associated with cell proliferation (Xia et al., 1995) , is also affected by virus infection and CQ (Pleschka et al., 2001; Weber et al., 2002) . In this study, we examined the correlation between CQ and the activation of p38 MAPK and ERK in human coronavirus 229E (HCoV-229E) infection, and demonstrated the involvement of p38 MAPK in the replication of HCoV-229E.
To examine the effect of CQ on the viral replication, virus in the supernatants was quantified by measuring the HCoV-229E RNA by reverse transcription (RT) realtime PCR. RNA from culture supernatants was extracted by using the QIAamp Viral RNA Mini Kit (QIAGEN) according to manufacturer's instructions and used for RT real-time PCR using specific primers for CoV229E N gene (forward: 5 -CAGTCAAATGGGCTGATGCA-3 , reverse: 5 -AAAGGGCTATAAAGAGAATAAGGTATTCT-3 ) and a probe al., 2004). CQ (10 and 25 M) inhibited the release of HCoV-229E into supernatants significantly in a dose-dependent manner (Fig. 1a) . Since CQ has both prophylactic and therapeutic antiviral effects for SARS-CoV (Vincent et al., 2005) , its effect on the internalization of HCoV-229E into cells was evaluated. We quantified the HCoV-229E RNA incorporated into the cells at 3h post infection by real-time RT-PCR. The ratios of viral mRNA to 18s rRNA in the reactive units of quantitative real-time PCR were determined. The amount of viral RNA incorporated into the cells was not significantly influenced by CQ (Fig. 1b) . These data demonstrate that CQ has no influence on the process prior to the internalization of HCoV-229E into L132 cells.
Since CQ was not involved in the process of internalization of HCoV-229E into the cells, we examined its effect on the activation of p38 MAPK and ERK, since they might be involved in the process after internalization. The activation of p38 MAPK and ERK at 90 min post infection (p.i.) was examined by immunoblotting. The HCoV-229E-infected cells were harvested at 90 min p.i. and the lysate protein concentration was determined by the Bradford assay (Bio-Rad). Extracted protein (20 g) was separated on 10% SDS-polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane (Millipore). The membranes were blocked with skim milk and incubated with primary antibodies to phosphorylated p38 MAPK, phosphorylated ERK, p38 MAPK (Cell Signaling Technology), and ERK (BD Biosciences Pharmingen) overnight at 4 • C. The membranes were washed and then incubated with 1:2000 dilution of peroxidase-conjugated antimouse immunoglobulin for 1 h. Proteins were visualized with SuperSignal West Dura substrate reagent (Pierce) in the linear range on X-ray films.
HCoV-229E infection induced the phosphorylation of p38 MAPK which was inhibited by 25 M CQ. Representative results are shown in Fig. 2a . However, the phosphorylation of ERK was enhanced 1.4-and 2.4-fold by CQ with mock and HCoV-229E infection, respectively, while the status of ERK was not significantly affected by HCoV-229E infection at 90 min p.i. These data demonstrate that CQ can neutralize the effect of HCoV-229E-infection on p38 MAPK.
To confirm the role of p38 MAPK in the HCoV-229E viral replication, p38 MAPK activity was blocked with its inhibitor SB203580 (10 and 25 M) which had no effect on cell viability (data not shown) but significantly mediated a dose-dependent inhibition of CPE (Fig. 2b) . This indicates that p38 MAPK activation is required for CPE induced by HCoV-229E. To examine if p38 MAPK is involved in the viral replication of HCoV-229E, the effect of SB203580 on the HCoV-229E released in culture supernatants was tested. SB203580 significantly reduced viral titers in a dose-dependent manner (Fig. 2c) , demonstrating the involvement of p38 MAPK in HCoV-229E viral replication.
We demonstrated that CQ inhibits HCoV-229E replication. Additionally, our results revealed that CQ inhibits the activation of p38 MAPK in HCoV-229E-infected cells and evokes the activation of ERK independently of infection. Our study demonstrated that CQ inhibits the activation of p38 and SB203580 (p38 inhibitor) suppresses viral replication. This suggests that CQ may inhibit the CoV replication by suppressing the p38 activation.