Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells.
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.
Lennemann, NJ; Evans, AS; Coyne, CB
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