Rhinovirus 3C Protease Facilitates Specific Nucleoporin Cleavage and Mislocalisation of Nuclear Proteins in Infected Host Cells

Philip G Bardin,Belinda J Thomas,Erin J Walker,Parisa Younessi,Alex J Fulcher,Robert Mccuaig,David A Jans,Reena Ghildyal,Matthew Bogyo

doi:10.1371/journal.pone.0071316

Philip G Bardin, Belinda J Thomas + Show 7 more

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https://doi.org/10.1371/journal.pone.0071316

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Abstract

Human Rhinovirus (HRV) infection results in shut down of essential cellular processes, in part through disruption of nucleocytoplasmic transport by cleavage of the nucleoporin proteins (Nups) that make up the host cell nuclear pore. Although the HRV genome encodes two proteases (2A and 3C) able to cleave host proteins such as Nup62, little is known regarding the specific contribution of each. Here we use transfected as well as HRV-infected cells to establish for the first time that 3C protease is most likely the mediator of cleavage of Nup153 during HRV infection, while Nup62 and Nup98 are likely to be targets of HRV2A protease. HRV16 3C protease was also able to elicit changes in the appearance and distribution of the nuclear speckle protein SC35 in transfected cells, implicating it as a key mediator of the mislocalisation of SC35 in HRV16-infected cells. In addition, 3C protease activity led to the redistribution of the nucleolin protein out of the nucleolus, but did not affect nuclear localisation of hnRNP proteins, implying that complete disruption of nucleocytoplasmic transport leading to relocalisation of hnRNP proteins from the nucleus to the cytoplasm in HRV-infected cells almost certainly requires 2A in addition to 3C protease. Thus, a specific role for HRV 3C protease in cleavage and mislocalisation of host cell nuclear proteins, in concert with 2A, is implicated for the first time in HRV pathogenesis.

Highlights

Human Rhinoviruses (HRVs) belong to the picornavirus family of small positive strand RNA viruses
Recent work has shown that HRV infection results in the cleavage of translation and transcription factors, as well as the nucleoporin (Nup) proteins that make up the nuclear pore, the only avenue for transport into and out of the nucleus [19,20,21]; cleavage of nucleoporin proteins (Nups) such as Nup153 and Nup62, can result in inhibited nuclear import [20,21,22] leading to the mislocalization of host cell proteins
Cell lysates were analysed for 3C protease, Nup62, Nup98, Nup153, Nup93, Nup133, heterogeneous nuclear ribonucleoprotein particle A1 and C1/C2 proteins and nucleolin, with tubulin as a loading control. 3C protease and its precursor 3CD were clearly evident in cell lysates from 6 h p.i., as was 3CD’, the product of a 2A cleavage within 3CD [19]

Summary

Introduction

Human Rhinoviruses (HRVs) belong to the picornavirus family of small positive strand RNA viruses. As in the case of poliovirus, HRV infection results in the shut-down of key host cell processes [3], where recruitment of several host proteins by the picornavirus internal ribosome entry site (IRES) element results in their non-availability for cellular functions, enabling optimal viral polyprotein translation [3,4,5,6]. Recent work has shown that HRV infection results in the cleavage of translation and transcription factors, as well as the nucleoporin (Nup) proteins that make up the nuclear pore, the only avenue for transport into and out of the nucleus [19,20,21]; cleavage of Nups such as Nup153 and Nup, can result in inhibited nuclear import [20,21,22] leading to the mislocalization of host cell proteins. HRV 2A protease was previously implicated in the cleavage of Nup62 [22] in HRV-infected cells, but the protease responsible for cleavage of the other Nups that are degraded in HRV infection, and the molecular changes that result in the mislocalisation of nuclear proteins in HRV infected cells have not yet been elucidated

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