《【病毒外文文獻(xiàn)】2008 Inhibition of human coronavirus 229E infection in human epithelial lung cells (L132) by chloroquine_ Involvement of》由會(huì)員分享，可在線閱讀，更多相關(guān)《【病毒外文文獻(xiàn)】2008 Inhibition of human coronavirus 229E infection in human epithelial lung cells (L132) by chloroquine_ Involvement of（3頁珍藏版）》請?jiān)谘b配圖網(wǎng)上搜索。

Available online at Antiviral Research 77 2008 150 152 Short communication Inhibition of human coronavirus 229E infection in human epithelial lung cells Involvement of p38 T Univer accepted Abstract HCoV CQ than p38 gulated inhibits findings the Keywords Coronavirus 229E Chloroquine p38 MAPK ERK Chloroquine CQ a diprotic weak base that increases the pH malaria tis human Ho the poorly endoc as et infection kinase including al 1995 play a crucial role in infection of coronaviruses such 0166 3542 doi of acidic vesicles has been used for the treatment of and inflammatory diseases such as rheumatoid arthri CQ has antimicrobial effects even against viruses such as immunodeficiency virus type 1 HIV 1 and SARS CoV Sperber et al 1993 Savarino et al 2003 Vincent et al 2005 wever the inhibitory effect on SARS CoV is inactive in vivo Barnard et al 2006 and the detailed mechanisms underlying antimicrobial and anti inflammatory effects of CQ remain understood Coronavirus an enveloped virus enters the cytoplasm by ytosis and matures in the membrane transport system such the trans Golgi network TGN Nauwynck et al 1999 Ng al 2003 ER stress caused by Japanese encephalitis virus induces the activation of p38 mitogen activated protein MAPK and host cell apoptosis Su et al 2002 MAPKs ERK JNK and p38 are involved in cell death Xia et Corresponding author E mail address sirasawa faculty chiba u jp H Shirasawa 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 produc tion 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 replica tion virus in the supernatants was quantified by measuring the HCoV 229E RNA by reverse transcription RT real time 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 prime CAGTCAAATGGGCTGATGCA 3 prime reverse 5 prime AAAGGGCTATAAAGAGAATAAGGTATTCT 3 prime and a probe 5 prime CCCTGACGACCACGTTGTGGTTCA 3 prime van Elden et see front matter 2007 Elsevier B V All rights reserved 10 1016 j antiviral 2007 10 011 Masakazu Kono a b Koichiro Kengo Saito a Takayuki Kuriyama a Department of Molecular Virology Chiba Chiba 260 8670 b Department of Chest Medicine Chiba Univer Chiba 260 8670 Received 13 July 2007 The antiviral effects of chloroquine CQ on human coronavirus 229E significantly decreased the viral replication at concentrations lower mitogen activated protein kinase MAPK and extracellular signal re CPE induced by HCoV 229E infection and viral replication Our replication of HCoV 229E 2007 Elsevier B V All rights reserved L132 by chloroquine MAPK and ERK atsumi b Alberto M Imai a b Hiroshi Shirasawa a sity School of Medicine 1 8 1 Inohana Japan sity School of Medicine 1 8 1 Inohana Japan 17 October 2007 229E infection of human fetal lung cell line L132 are reported in clinical usage We demonstrated that CQ affects the activation of kinase ERK Furthermore p38 MAPK inhibitor SB203580 suggest that CQ affects the activation of MAPKs involved in M Kono et al Antiviral Research 77 2008 150 152 151 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 al 2004 CQ 10 and 25H9262M inhibited the release of HCoV 229E into supernatants significantly in a dose dependent manner Fig 1a Since CQ has both prophylactic and therapeutic antivi ral 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 Fig 2 specific a viability at e w 2 Involvement of p38 and ERK in the infection of HCoV 229E a Effect of CQ h with or without CQ and infected with HCoV 229E at an MOI of 3 Cytoplasmic proteins antibodies against indicated MAPKs b Effect of p38 inhibitor SB203580 on specific p38 MAPK inhibitor subsequently adsorbed with HCoV 229E at an MOI was measured The absorbance of CQ untreated cells was calculated as 100 0 4 10 and 25H9262M of p38 inhibitor L132 cells were pretreated with indicated concentrations xtracted from culture supernatants were used for reverse transcription real time PCR as calculated as 100 Data represent mean S E M of triplicates on the phosphorylation of p38 MAPK and ERK L132 cells were pretreated for extracted at 90 min post infection were immunoblotted and reacted with the CPE L132 cells were treated with indicated concentrations of SB203580 of 3 At 72 h post infection the cells were stained with crystal violet and cell c Effect of p38 inhibitor SB203580 on the viral RNA load in supernatant of SB203580 and adsorbed with HCoV 229E at an MOI of 3 RNA at 72 h post infection The amount of viral RNA load in CQ untreated cells 152 M Kono et al Antiviral Research 77 2008 150 152 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 internaliza tion 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 exam ined 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 20H9262g was separated on 10 SDS polyacrylamide gels and then transferred to a polyvinylidene difluoride mem brane Millipore The membranes were blocked with skim milk and incubated with primary antibodies to phosphory lated p38 MAPK phosphorylated ERK p38 MAPK Cell Signaling Technology and ERK BD Biosciences Pharmin gen overnight at 4 C The membranes were washed and then incubated with 1 2000 dilution of peroxidase conjugated anti mouse 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 25H9262M CQ Representative results ERK HCoV w p i HCoV replication SB203580 data inhibition v if the supernatants titers in We demonstrated that CQ inhibits HCoV 229E replication Additionally our results revealed that CQ inhibits the activa tion of p38 MAPK in HCoV 229E infected cells and evokes the activation of ERK independently of infection Our study demon strated 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 References Banerjee S et al 2002 Murine coronavirus replication induced p38 mitogen activated protein kinase activation promotes interleukin 6 production and virus replication in cultured cells J Virol 76 5937 5948 Barnard D L et al 2006 Evaluation of immunomodulators interferons and known in vitro SARS CoV inhibitors for inhibition of SARS CoV replica tion in BALB c mice Antivir Chem Chemother 17 275 284 Kopecky Bromberg et al 2006 7a Protein of severe acute respiratory syndrome coronavirus inhibits cellular protein synthesis and activates p38 mitogen activated protein kinase J Virol 80 785 793 Nauwynck H J et al 1999 Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor mediated endocytosis J Gen Virol 80 297 305 Ng M L et al 2003 Proliferative growth of SARS coronavirus in Vero E6 cells J Gen Virol 84 3291 3303 Pleschka Sa Sperber Su v V W Xia Y are shown in Fig 2a However the phosphorylation of was enhanced 1 4 and 2 4 fold by CQ with mock and 229E infection respectively while the status of ERK as not significantly affected by HCoV 229E infection at 90 min These data demonstrate that CQ can neutralize the effect of 229E infection on p38 MAPK To confirm the role of p38 MAPK in the HCoV 229E viral p38 MAPK activity was blocked with its inhibitor 10 and 25H9262M which had no effect on cell viability not shown but significantly mediated a dose dependent of CPE Fig 2b This indicates that p38 MAPK acti ation is required for CPE induced by HCoV 229E To examine p38 MAPK is involved in the viral replication of HCoV 229E effect of SB203580 on the HCoV 229E released in culture was tested SB203580 significantly reduced viral in a dose dependent manner Fig 2c demonstrating the volvement of p38 MAPK in HCoV 229E viral replication S et al 2001 Influenza virus propagation is impaired by inhibition of the Raf MEK ERK signalling cascade Nat Cell Biol 3 301 305 varino A et al 2003 Effects of chloroquine on viral infections an old drug against today s diseases Lancet Infect Dis 3 722 727 K et al 1993 Inhibition of human immunodeficiency virus type 1 replication by hydroxychloroquine in T cells and monocytes AIDS Res Hum Retrov 9 9 13 H L et al 2002 Japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response J Virol 76 4162 4171 an Elden L J et al 2004 Frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real time reverse transcriptase polymerase chain reaction J Infect Dis 189 652 657 incent M J et al 2005 Chloroquine is a potent inhibitor of SARS coronavirus infection and spread Virol J 2 69 eber S M et al 2002 Inhibition of mitogen activated protein kinase signal ing by chloroquine J Immunol 168 5303 5309 Z et al 1995 Opposing effects of ERK and JNK p38 MAP kinases on apoptosis Science 24 1326 1331 i A K Krieg A M 1998 Rapid induction of mitogen activated protein kinases by immune stimulatory CpG DNA J Immunol 161 4493 4497