Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium.

Talita B Gagliardi,Monty E Goldstein,Daniel Song,Maxinne A Ignacio,Jae W Jung,Kelsey M Gray,Gregg A Duncan,Margaret A Scull,Kimberly M Stroka,Shin-Ru Shih

doi:10.1371/journal.ppat.1010159

Talita B Gagliardi, Monty E Goldstein + Show 8 more

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https://doi.org/10.1371/journal.ppat.1010159

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Abstract

The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality–aspects of infection that may contribute to pathogenesis in vivo.

Highlights

Rhinoviruses (RVs) are responsible for over 40% of respiratory virus infections in the human population [1,2,3,4]
We performed a detailed analysis of RV-C15 replication in a physiologically-relevant model of human airway epithelium
Our single-cell, microscopy-based approach revealed that–unlike other RVs–the endoplasmic reticulum is the primary site for RV-C15 replication

Summary

Introduction

Rhinoviruses (RVs) are responsible for over 40% of respiratory virus infections in the human population [1,2,3,4]. No vaccine or direct-acting antiviral is currently available due in part to the diversity of RVs in circulation, with over 160 genotypes identified [9,10]. These genotypes comprise three species (RV-A, RV-B, and RV-C) where RV-A and RV-C are the most prevalent and RV-C is associated with more severe clinical disease, especially in children [4,11,12]. The restricted expression of CDHR3 to ciliated cells in the upper and lower airway epithelium [20,21,22,23] limits the cellular tropism of RV-C, compared to other RVs that utilize low-density lipoprotein receptor (LDLR) or intercellular adhesion molecule (ICAM)-1 as receptors [18]. Mechanisms of infection and replication are not always conserved between rhinovirus species

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