Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating.

Mahmoud Soliman,Sang-Ik Park,Kyoung-Oh Cho,Eun-Hyo Cho,Ji-Yun Kim,Mia Madel Alfajaro,Yeong-Bin Baek,Deok-Song Kim,Jong-Soon Choi,Ja-Young Seo,Jun-Gyu Park,Mun-Il Kang,Joseph Kwon,Carlos F Arias

doi:10.1371/journal.ppat.1006820

Mahmoud Soliman, Sang-Ik Park + Show 12 more

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https://doi.org/10.1371/journal.ppat.1006820

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Abstract

The cellular PI3K/Akt and/or MEK/ERK signaling pathways mediate the entry process or endosomal acidification during infection of many viruses. However, their roles in the early infection events of group A rotaviruses (RVAs) have remained elusive. Here, we show that late-penetration (L-P) human DS-1 and bovine NCDV RVA strains stimulate these signaling pathways very early in the infection. Inhibition of both signaling pathways significantly reduced production of viral progeny due to blockage of virus particles in the late endosome, indicating that neither of the two signaling pathways is involved in virus trafficking. However, immunoprecipitation assays using antibodies specific for pPI3K, pAkt, pERK and the subunit E of the V-ATPase co-immunoprecipitated the V-ATPase in complex with pPI3K, pAkt, and pERK. Moreover, Duolink proximity ligation assay revealed direct association of the subunit E of the V-ATPase with the molecules pPI3K, pAkt, and pERK, indicating that both signaling pathways are involved in V-ATPase-dependent endosomal acidification. Acidic replenishment of the medium restored uncoating of the RVA strains in cells pretreated with inhibitors specific for both signaling pathways, confirming the above results. Isolated components of the outer capsid proteins, expressed as VP4-VP8* and VP4-VP5* domains, and VP7, activated the PI3K/Akt and MEK/ERK pathways. Furthermore, psoralen-UV-inactivated RVA and CsCl-purified RVA triple-layered particles triggered activation of the PI3K/Akt and MEK/ERK pathways, confirming the above results. Our data demonstrate that multistep binding of outer capsid proteins of L-P RVA strains with cell surface receptors phosphorylates PI3K, Akt, and ERK, which in turn directly interact with the subunit E of the V-ATPase to acidify the late endosome for uncoating of RVAs. This study provides a better understanding of the RVA-host interaction during viral uncoating, which is of importance for the development of strategies aiming at controlling or preventing RVA infections.

Highlights

Group A rotavirus (RVA), a species of the Rotavirus genus in the Reoviridae family, is recognized as a major pathogen that causes severe acute dehydrating diarrhea in young children and in a wide variety of young animals [1, 2]
Viral particles must transport their genome into the cytoplasm or the nucleus of host cells to initiate successful infection
We demonstrate that infection with late-penetration RVA strains results in phosphorylation of PI3K, Akt, and ERK signaling molecules at an early stage of infection, a process mediated by the multistep binding of RVAs outer capsid proteins

Summary

Introduction

Group A rotavirus (RVA), a species of the Rotavirus genus in the Reoviridae family, is recognized as a major pathogen that causes severe acute dehydrating diarrhea in young children and in a wide variety of young animals [1, 2]. For RVA uncoating to deliver DLP into cytoplasm, the environment of the RVA-containing endosomes has to change, for instance, by endosomal acidification, a drop in calcium concentration, the exchange of membrane components, the formation of additional intraluminal vesicles, or the acquisition of lysosomal components [4, 13]. Among these mechanisms, endosomal acidification plays a crucial role in RVA uncoating of L-P strains such as the human strain Wa, the porcine strain TFR-1 and the bovine strain UK, but not in uncoating of E-P strains such as RRV [4, 11, 14]

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