Abstract
Enterovirus A71 (EV-A71), a primary causative agent of hand, foot, and mouth disease (HFMD), poses significant public health challenges, particularly among young children. Despite its clinical impact, the molecular mechanisms underlying EV-A71 replication remain incompletely understood.Enterovirus A71 (EV-A71) replication critically depends on translating its single- stranded positive-sense RNA into a single polyprotein, which is subsequently processed by viral proteases. Mass spectrometry analysis performed in our laboratory using purified viral protease 2Apro has revealed potential interactions between 2Apro and heterogeneous nuclear ribonucleoprotein L (hnRNP L). However, the specific functional role of hnRNP L in EV-A71 replication remains unclear and warrants further investigation. In this study, we first found that hnRNP L, the important RNA binding protein (RBP) involved in many human cancers, facilitates EV-A71 infection; although EV-A71 infection does not affect hnRNP L expression. However, the loss- and gain-of-function experiments have proved that hnRNP L played an important role in facilitating EV-A71 reproduction. We further revealed that hnRNP L enhanced viral internal ribosome entry site (IRES) activity upon EV-A71 infection. Initial investigations demonstrated that hnRNP L facilitates viral IRES activity in vitro. This mediation was further corroborated through loss- and gain-of- function experiments in vivo, a process dependent on the viral protease 2Apro, which has been previously reported to enhance IRES activity. Subsequent validation confirmed the interaction between hnRNP L and 2Apro, with the RRM4 domain of hnRNP L identified as the critical interaction region. Intriguingly, while 2Apro does not directly bind the viral IRES, hnRNP L exhibites binding specificity to the IRES region spanning nucleotides 557-741. Functional assays revealed that truncated forms of hnRNP L fail to support full EV-A71 reproduction, potentially due to conformational alterations. Consistent with prior findings on RNA-binding proteins (RBPs) such as hnRNP A1 and hnRNP A2/B1, which translocate from the nucleus to the cytoplasm to promote viral infection, we observed that 2Apro of EV-A71 induced the redistribution of hnRNP L from the nucleus to the cytoplasm. This relocalization likely facilitated hnRNP L's functional role in viral IRES activity.
Previous work from our laboratory identified heat shock proteins, including heat shock cognate protein 70 (Hsc70), as regulators of EV-A71 infection via modulation of viral IRES activity. In this study, mass spectrometry analysis, initially aimed at identifying hnRNP L acetylation sites, unexpectedly detected Hsc70. Further validation confirmed the interaction between hnRNP L and Hsc70, with RRM3 and RRM4 domains of hnRNP L implicated as the interaction regions. In addition, our lab demonstrated the interaction between 2Apro and Hsc70. Through loss- and gain-of-function experiments, as well as rescue assays, we established that hnRNP L and Hsc70 coordinately regulate EV-A71 reproduction by modulating viral IRES activity.
Although mass spectrometry did not identify acetylation sites on hnRNP L, the UniProt database (UniProt ID: P14866) reported an acetylation site at lysine 269 (K269). To assess the functional impact of this modification on EV-A71 infection, we generated lysine point mutants (K269Q, K269R, and K269A), with R354K serving as a control for asymmetric dimethylation. Functional assays revealed that acetylation at K269 did not contribute to EV-A71 reproduction.
In summary, we elucidated the mechanistic role of hnRNP L in facilitating EV-A71 reproduction. The viral protease 2Apro induces the nuclear-to-cytoplasmic redistribution of hnRNP L, thereby enhancing IRES activity in concert with Hsc70 and 2Apro. Our results elucidated a mechanistic link between the roles of host cell RNA-binding proteins (RBPs) and heat shock proteins during viral infection. Collectively, this study establishes hnRNP L as a pivotal factor in the viral replication cycle and highlights its potential as a target for antiviral strategies.
| Date of Award | 3 Dec 2025 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Mingliang HE (Supervisor) & Ruth COLLINS (External Co-Supervisor) |
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