Previous studies have shown that at early stages of enterovirus infection, ROs are observed in contact with the endoplasmic reticulum (ER) membrane. Enteroviruses induce extensive remodeling of the host secretory pathway during infection, which results in the accumulation of large clusters of vesicular structures in the cytoplasm that harbor viral replication proteins. Thus, a better understanding of the interactions of these viruses with the host cell can aid in the development of anti-enterovirus small molecule therapeutics.Īll positive-strand RNA viruses manipulate host cell membranes to form membranous structures, termed replication organelles (ROs), that concentrate viral and host factors to allow for efficient genome replication. Currently, there are no antivirals and vaccines are only available for enterovirus 71 and poliovirus. Infection by these viruses can lead to the development of severe disease, including acute flaccid paralysis, meningitis, and encephalitis. Enteroviruses, including coxsackievirus B3 (CVB) and enterovirus 71 (EV71), are small, non-enveloped, positive-strand RNA viruses. Positive-strand RNA viruses represent a large group of viruses that are responsible for the development of severe disease manifestations worldwide. 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. 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. We found that 2APB treatment had no effect on the kinetics of infection or the percentage of infected cells. Lastly, we applied our system to study the effects of a calcium channel inhibitor, 2APB, on virus-induced manipulation of host membranes. 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. This system thus allows for the monitoring of organelle-specific changes induced by infection in real-time. 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. However, this process has not been well-studied in living cells throughout the course of infection. Enteroviruses manipulate host membranes to form replication organelles, which concentrate viral and host factors to allow for efficient replication.
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