Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells.

Nicholas J Lennemann,Carolyn B Coyne,Azia S Evans

doi:10.3390/v12101074

Abstract

Enteroviruses manipulate host membranes to form replication organelles, which concentrate viral and host factors to allow for efficient replication. However, this process has not been well-studied in living cells throughout the course of infection. To define the dynamic process of enterovirus membrane remodeling of major secretory pathway organelles, we have developed plasmid-based reporter systems that utilize viral protease-dependent release of a nuclear-localized fluorescent protein from the endoplasmic reticulum (ER) membrane during infection, while retaining organelle-specific fluorescent protein markers such as the ER and Golgi. This system thus allows for the monitoring of organelle-specific changes induced by infection in real-time. Using long-term time-lapse imaging of living cells infected with coxsackievirus B3 (CVB), we detected reporter translocation to the nucleus beginning ~4 h post-infection, which correlated with a loss of Golgi integrity and a collapse of the peripheral ER. Lastly, we applied our system to study the effects of a calcium channel inhibitor, 2APB, on virus-induced manipulation of host membranes. We found that 2APB treatment had no effect on the kinetics of infection or the percentage of infected cells. However, we observed aberrant ER structures in CVB-infected cells treated with 2APB and a significant decrease in viral-dependent cell lysis, which corresponded with a decrease in extracellular virus titers. Thus, our system provides a tractable platform to monitor the effects of inhibitors, gene silencing, and/or gene editing on viral manipulation of host membranes, which can help determine the mechanism of action for antivirals.

Highlights

Positive-strand RNA viruses represent a large group of viruses that are responsible for the development of severe disease manifestations worldwide
To monitor enterovirus infection in real-time, we adapted cell-based reporter methodologies previously used for flaviviruses and hepatitis C virus that rely on viral protease cleavage-dependent translocation of a membrane-anchored cytoplasmic fluorescent proteins to the nucleus [27,28,29]
As a proof of principle, we developed a dual reporter construct (RepER) that expresses an endoplasmic reticulum (ER) lumen localized mCherry containing an ER retention signal (KDEL) and signal peptidase cleavage site fused to a type I transmembrane domain followed by an enterovirus 3C protease (3Cpro ) target sequence and a cytoplasmic GFP containing a nuclear localization signal (GFP-nuclear localization sequence (NLS)) (Figure 1a)

Summary

Introduction

Positive-strand RNA viruses represent a large group of viruses that are responsible for the development of severe disease manifestations worldwide. (CVB) and enterovirus 71 (EV71), are small, non-enveloped, positive-strand RNA viruses. Infection by these viruses can lead to the development of severe disease, including acute flaccid paralysis, meningitis, and encephalitis [1,2,3,4,5,6]. A better understanding of the interactions of these viruses with the host cell can aid in the development of anti-enterovirus small molecule therapeutics. Enteroviruses induce extensive remodeling of the host secretory pathway

Methods

Results

Conclusion