The Mechanism of Enterovirus A71 Induced Heat Shock Protein 27 Response to Promote Viral Infection


Student thesis: Doctoral Thesis

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Award date25 Jun 2019


Enterovirus A71 (EV-A71) is a human pathogen that causes hand, foot and mouth disease (HFMD) and fatal neurological diseases without effective treatment. Identification and characterization of key host factors are important for understanding the disease pathogenesis. In this study, we found that both protein and mRNA levels of Hsp27 were increased at 6-9 hours post EV-A71 infection. However, UV-inactivated EV-A71 could not induce the expression of Hsp27. More ever, EV-A71 infection induced the translocation of Hsp27 from the cytosol to the nucleus. siRNAs of Hsp27 were designed to investigate the role of Hsp27 in EV-A71 infection. Knockdown of Hsp27 decreased the levels of viral RNA, VP1 protein, and viral titers. And overexpression of Hsp27 enhanced EV-A71 replication.

To further confirm the role of Hsp27, Hsp27 knockout (Hsp27-KO) cell lines were created by CRISPR/Cas9-mediated genome editing. As expected, knockout of Hsp27 markedly suppressed the replication of EV-A71. After re-introducing Hsp27 expression into the Hsp27-KO cells, the reduced replication of EV-A71 was successfully restored. Further studies showed that knockout of Hsp27 decreased viral internal ribosome entry site (IRES)-mediated translation, while ectopic expressed Hsp27 increased the IRES-mediated translation. The 2A protease (2Apro) of EV-A71 has been demonstrated to play a positive role in regulating viral IRES activity. I found that knockout of Hsp27 could also inhibit 2Apro-induced viral IRES activity and overexpression of Hsp27 promoted 2Apro-mediated viral IRES activity in a dose-dependent manner. It was also demonstrated that hsp27 did not affect the expression of eIF4G. However, eIF4G cleavage induced by 2Apro was obviously inhibited in Hsp27-KO cells as compared with the wild type RD cells. At the same time, ectopic expression of Hsp27 promoted the cleavage of eIF4G. More importantly, knockout of Hsp27 could also inhibit 2Apro-induced viral protein production upon EV-A71 infection.

IRES trans-acting factors (ITAFs) are recruited to the IRES and helps to initiate and enhance viral IRES-dependent translation. Knockout of Hsp27 could regulate the mRNA and protein level of ITAFs, including FBP1, hnRNP A1 and HuR. Moreover, EV-A71 infection induced translocation of hnRNP A1 from the nucleus to the cytoplasm and this process was almost completely blocked after knockout of Hsp27.

The activation of the p38 MAPK signaling pathway is involved in EV-A71 infection. In this study, we found that p38 MAPK-induced EV-A71 replication was dramatically suppressed in Hsp27-KO cells compared with the RD wild type cells. Hsp27 also acts as a target and could be phosphorylated by p38 MAPK at serine residues 15, 78, and 82. I constructed the dephosphorylation mutation (Hsp27-3A) and mimic-phosphorylation mutation (Hsp27-3D) plasmids. Compared with the wild type Hsp27, Hsp27-3A mutation inhibited EV-A71 replication, while Hsp27-3D mutation promoted virus infection. An inhibitor of Hsp27, TDP, significantly reduced EV-A71 infection-induced cytopathic effects (CPE). Viral reproduction and viral IRES activity were both inhibited in a dose-dependent manner after TDP treatment. The above data indicated that Hsp27 may act as a potential new target to develop drugs to against EV-A71 and possibly other viral infections.