The Mechanism of Hsp27 Phosphorylation Facilitates Enterovirus A71 Infection

Hsp27蛋白磷酸化促進腸病毒A組71型複製的機制研究

Student thesis: Doctoral Thesis

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Award date2 Oct 2024

Abstract

Enterovirus A71 (EV-A71) is a common neurotropic virus that can cause hand, foot, and mouth disease (HFMD), severe neural complications, and even death. To date, China is the only country that has licensed EV-A71 vaccines for genotype C, and there is no effective approved drug for EV-A71 treatment. As a positive-sense (+) single-stranded RNA (ssRNA) virus, EV-A71 depends on IRES-dependent viral polypeptide translation to initiate virus replication after entry with the help of host proteins. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has been identified to bind to the viral internal ribosome entry site (IRES) and initiate viral protein translation. Our previous study showed that EV-A71 hijacks heat shock protein 27 (Hsp27) to induce the cytosol redistribution of hnRNP A1 initiating viral protein translation. However, the underlying mechanism of this process is still elusive.

Hsp27 undergoes phosphorylation in response to various stimuli, including heat shock and viral infection. In this study, we reported that inhibition of Hsp27 phosphorylation using the p38 kinase inhibitor SB203580 abolished its nuclear translocation. Phosphorylation-deficient Hsp27 mutants, Hsp27-3A (Hsp27S15/78/82A) and Hsp27S78A, failed to translocate into the nucleus and induce the cytosol redistribution of hnRNP A1 in Hsp27 knockout cells. In contrast, Hsp27S15A and Hsp27S82A mutants displayed similar effects to wildtype Hsp27 for nuclear translocation and hnRNP A1 cytosol re-localization. Furthermore, we demonstrated that the viral 2A protease (2Apro) activity was a key factor in regulating Hsp27 nuclear translocation and hnRNP A1 cytosol re-localization. Moreover, Hsp27S78A almost eliminated the effects of Hsp27 on the IRES activity and viral replication, which were partially reduced by Hsp27S82A, while Hsp27S15A displayed the same activity as wild-type Hsp27. We further developed a peptide S78, which dramatically suppressed EV-A71 protein translation and propagation through the blockage of EV-A71-induced Hsp27 phosphorylation and redistribution of Hsp27 and hnRNP A1. The competitive inhibition of S78 on Hsp27 function can be impaired by a single-site mutation (S78A), indicating the crucial role of Serine 78 phosphorylation of Hsp27 for EV-A71 infection.

Hsp27 has three functional domains: the WDPF domain, the ACD (α-crystallin) domain, and a flexible C-terminal domain. The WDPF domain locates in the crucial core of Hsp27 oligomers. The ACD domain is essential for the formation of dimers. The C-terminal flexible domain is associated with the protein interaction, which is important for chaperone function of Hsp27. We verified that the deletion of WDPF domain disturbed the subcellular localization of Hsp27, resulting in partial nuclear translocation. Deletion of ACD domain and WDPF domain blocked the hnRNP A1 translocation upon EV-A71 infection, and IRES-dependent translation. Interestingly, WDPF peptide was constructed and dramatically inhibited the Serine 78 phosphorylation of Hsp27. Consequently, the hnRNP A1 translocation, viral IRES activity, and viral protein translation and propagation were all suppressed by WDPF peptide, but not by the control peptide ΔWDPF with a shorter region of WDPF domain. More strikingly, the WDPF peptide dramatically decreased the RNA level of Human Coronavirus OC43 (HCoV-OC43), the DNA level of Herpes Simplex Virus 1 (HSV-1), and viral titer of HCoV-OC43 and HSV-1. An additional inhibitory effect on HCoV-OC43 and HSV-1 infection was achieved by the combined treatment of the WDPF peptide and a multiple kinase inhibitor Sorafenib, a commonly used anticancer drug.

Taken together, these findings uncovered the crucial role of Serine 78 phosphorylation of Hsp27 and WDPF domain in EV-A71-induced hnRNP A1 nuclear translocation and IRES-dependent viral protein translation and propagation. Additionally, the WDPF peptide effectively inhibited the replication and reproduction of HCoV-OC43 and HSV-1, which was further suppressed by the combined treatment of peptide WDPF and Sorafenib. These results suggest a new path for target-based, pan-antiviral combined strategy with high efficiency and selectivity.

    Research areas

  • Hsp27, hnRNP A1, EV-A71, Peptides