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Human rhinoviruses (HRVs) are the predominant cause of the common cold by infecting the upper respiratory tract and may lead to exacerbation of asthma and COPD. The human immune system has developed innate antiviral responses, including the cGAS-STING pathway, which recognizes cytosolic double-stranded DNA and leads to the expression of type I interferons. While STING expression appeared to inhibit the replication of a variety of viruses, it was shown to promote HRV replication. The mechanism contributing to the pro-viral role of STING in HRV infection remains unknown. Here we further elucidated the role of STING in HRV replication. We found that STING promoted HRV-A16 replication, but had no role in HRV-A2 and HRV-B14 replication. Furthermore, treatment with the STING antagonist H-151 did not affect HRV-A16 replication, whereas a STING autophagy mutant lowered its replication. We also isolated HRV-A16 strains that evolved to replicate independently of STING. Within these strains, missense mutations were found in the genes encoding the 2A and 2C proteins. Lastly, we aimed to establish fluorescently labelled human STING alongside a split fluorescent system for HRV-A16, to enable live cell imaging of their localization. Altogether, this report identified a crucial role for STING during HRV-A16 replication and a potential therapeutic target during HRV infections.

Enteroviruses extensively manipulate cellular functions during infection. One of the most prominent changes is in membrane morphology. Enteroviral infection causes the formation of Replication Organelles (ROs) that increase viral replication. ROs are rearranged membranes with different properties to any cellular membranes and serve as platforms for assembly of replication complexes and new virions. Many cellular pathways are modulated by viral proteins to create an environment suited for viral replication. These include manipulation of intracellular trafficking and lipid metabolism. Lipid droplets also play a role in effective infection by shuttling lipids towards ROs. Late during infection autophagy plays a role in non-lytic release by wrapping ROs in more membranes and allowing for the egress of vesicles filled with virions. Finally, recent advances in cryo-Electron Tomography (cryo-ET) are discussed that can allow the study of viral replication in situ in previously unachievable detail.

The ongoing pandemic of COVID-19 has been holding the whole world in its grip for almost two years. Soon after the initial reports of an unknown lung disease, the cause was identified to be a novel coronavirus called SARS-CoV-2. The pandemic has influenced billions of lives, causing millions of deaths worldwide and the numbers are still rising. In order to prevent a virus outbreak with such global impact in the future, it is crucial to gain more knowledge about how viruses emerge in the human population. If the factors that influence the introduction of novel viruses to humans are known, we might be able to stop the emergence of novel viruses by intervening with these factors. The aim of this paper is to increase the understanding of the origin of SARS-CoV-2 by providing a thorough, unbiased review of the current literature on this topic. Three aspects of the virus’ origin are discussed: how, where and when this virus was introduced in the human population. Two main routes of emergence of the virus are currently being considered: through nature or in a laboratory. Previous coronavirus outbreaks in human history have been linked to animal-to-human transmission and hence have a natural origin, with bats being the most prevalent animal source. While related viruses have been found in nature, no animal source has yet been identified for SARS-CoV-2. Moreover, the unique features that are seen in the SARS-CoV-2 genome urge scientists to reconsider a laboratory origin of the virus. Several laboratories across the world have been working on coronaviruses for decades and state-of-the-art experimental techniques allow researchers to create viruses without leaving any traces of it being unnatural. Regarding the initial location and timing of the virus’ emergence, the consensus is that the pandemic began in Wuhan, China in December 2019. Outside of China, however, investigations from Europe and America have reported the prevalence of the virus before that period. These reports

SARS-CoV-2 has made a large impact on society in the past years due to its high transmissibility which led to the COVID-19 pandemic. Academia and the drug development industry have made fast advances to provide potent inhibitors against the virus, but the holy grail has not yet been found. The potency of these inhibitors can be tested in several assays. Cell-based assays are favourable as, in contrast to biochemical assays, they can assess the cell-permeability and toxicity of the inhibitors. This project, therefore, aimed to develop multiple cell-based assays for the evaluation of potential inhibitors that each target various aspects of the viral replication cycle to advance the discovery of novel and much-needed SARS-CoV-2 inhibitors. The viral replication can be inhibited by targeting the viral Main Protease (Mpro). This protease has been well studied for various coronaviruses and is a popular drug target. A previous assay has been designed around the Mpro (Van der Linden et al., 2014; manuscript in preparation) but this assay lacked sensitivity and was not able to detect inhibitors with a lower inhibitory efficacy. A new assay was therefore designed to also identify less potent inhibitors. This assay is based on a cyclic luciferase, that can be linearized after Mpro cleavage. Linearization enables luciferase activation and loss of signal can therefore imply successful inhibition. The assay was compared to the previous luciferase assay and showed lower luciferase activity than the previous system. Alterations in transfection time and plasmid DNA concentrations did not improve the activity. Other factors came to light that may influence and improve the assay for future use. Currently, this assay can be easily performed but cannot be used for SARS-CoV-2 inhibitor identification in its current state. To assess cell entry inhibition, a cell-cell fusion assay was established that can be performed in a BSL-1 setting, as opposed to the BSL-2 setting that is being used f

The enterovirus genus comprises many human pathogens such as poliovirus, coxsackievirus and rhinovirus. For efficient replication, enteroviruses induce membrane rearrangements which require the interaction between viral and host factors such as PI4KB, ACBD3 and 3A. Although interactions between these proteins have been intensively studied, their localization at replication organelle (RO) membranes is unknown. To investigate components of ROs in-depth, we established protocols to study coxsackievirus B3 (CVB3) ROs using expansion microscopy (ExM) and super-resolution microscopy (SRM), i.e. Olympus SoRaSpin10 system. Our data is the first evidence of 2A localization. The use of SRM allowed us to visualize the localization of the viral protease 2A at different time points of infection in greater detail. In addition, we could locate 3A at an early time point after infection (i.e. 3 h p.i.), which was hardly attainable with conventional immunofluorescence microscopy. Our data suggest that imaging at earlier time points might be possible. Moreover, both viral proteins were visualized as dotted structures which have not yet been observed. We demonstrate the potential for in-depth visualization of these viral proteins and their colocalization with host factors using super-resolution microscopy. The Olympus SoRaSpin10 system offers the possibility to combine SRM with live-cell imaging. Thus, tracking viral protein localization, RO formation and its dynamics could be further investigated.