Periodic Reporting for period 1 - VIRLUMINOUS (Illuminating the enteroviral life cycle)
Periodo di rendicontazione: 2023-04-01 al 2025-09-30
Enteroviruses are highly prevalent pathogens that include poliovirus, coxsackievirus, enterovirus-A71, enterovirus-D68, and rhinovirus. There is an urgent need for technologies that allow sensitive real-time observation of the dynamics and localisation of viral RNA, viral proteins, and host factors at the single-cell level to answer important questions about the enterovirus life cycle. The EU-funded VIRLUMINOUS project aims to develop recombinant reporter viruses for real-time imaging technology to visualise and dissect the spatial and temporal regulation of different phases of the virus life cycle. The technology will be used to study viral RNA replication, virus assembly and release in living cells. The project's insights into the molecular interplay between enteroviruses and their hosts will be crucial for developing antiviral therapies.
A toolbox to visualize vRNA, viral proteins, and host factors.
We set out to develop a toolbox to visualize vRNA, viral proteins, host factors and their interactions. Previously, we produced viruses containing SunTag epitopes, allowing us to visualize translation of the viral genomic RNA (described in detail in Nature Protocols, 2025, in press). In the current project, we set out to produce viruses containing an RNA aptamer, a structured RNA element that upon binding of an otherwise non-fluorescent dye becomes fluorescent. Our first system allowed us to visualize viral RNA in living cells in the mid- and late-phases of infection, but appeared too insensitive to visualize the incoming viral RNA. To also detect the incoming viral genomic RNA, we are now employing a second RNA aptamer system. This system appears more sensitive and allows us to detect the incoming viral RNA at the single molecule level. Moreover, we set out to develop systems to visualize viral proteins during infection in living cells. For this, we use a split GFP system in which a small part of the GFP molecule is genetically fused to the viral protein of interest. Using cells expressing the remainder of the GFP molecule, we managed to construct a virus that allows us to visualize the synthesis, localization, and dynamics of the viral 2C protein in living cells. In addition, we managed to construct a two-color split-GFP virus allowing us to study two different fluorescent viral proteins at the same time.
Visualize and study viral RNA translation, replication and assembly.
To visualize and study where viral RNA replication and assembly takes place and where the viral RNA, viral proteins and host factors localize and interact in living cells, we have constructed viruses containing (combinations of) SunTag, RNA aptamers, and split-GFP viral proteins. In parallel, we implemented a novel fixed cell imaging method (4i) in our lab to dissect the composition of the sites of vRNA translation, replication and encapsidation in unprecedented detail. To study the molecular composition and organization of the viral replication complexes (i.e. the ensemble of the viral proteins that replicate the viral RNA) and the virus-induced replication organelles with which these replication complexes are associated, we have set up a cryo-electron tomography (cryo-ET) pipeline. We are using split-GFP labeled viral replication proteins and correlative light and EM microscopy (CLEM) in combination with cryo-ET to identify the viral replication and assembly complexes.
We set out to develop a toolbox to visualize vRNA, viral proteins, host factors and their interactions. Previously, we produced viruses containing SunTag epitopes, allowing us to visualize translation of the viral genomic RNA (described in detail in Nature Protocols, 2025, in press). In the current project, we set out to produce viruses containing an RNA aptamer, a structured RNA element that upon binding of an otherwise non-fluorescent dye becomes fluorescent. Our first system allowed us to visualize viral RNA in living cells in the mid- and late-phases of infection, but appeared too insensitive to visualize the incoming viral RNA. To also detect the incoming viral genomic RNA, we are now employing a second RNA aptamer system. This system appears more sensitive and allows us to detect the incoming viral RNA at the single molecule level. Moreover, we set out to develop systems to visualize viral proteins during infection in living cells. For this, we use a split GFP system in which a small part of the GFP molecule is genetically fused to the viral protein of interest. Using cells expressing the remainder of the GFP molecule, we managed to construct a virus that allows us to visualize the synthesis, localization, and dynamics of the viral 2C protein in living cells. In addition, we managed to construct a two-color split-GFP virus allowing us to study two different fluorescent viral proteins at the same time.
Visualize and study viral RNA translation, replication and assembly.
To visualize and study where viral RNA replication and assembly takes place and where the viral RNA, viral proteins and host factors localize and interact in living cells, we have constructed viruses containing (combinations of) SunTag, RNA aptamers, and split-GFP viral proteins. In parallel, we implemented a novel fixed cell imaging method (4i) in our lab to dissect the composition of the sites of vRNA translation, replication and encapsidation in unprecedented detail. To study the molecular composition and organization of the viral replication complexes (i.e. the ensemble of the viral proteins that replicate the viral RNA) and the virus-induced replication organelles with which these replication complexes are associated, we have set up a cryo-electron tomography (cryo-ET) pipeline. We are using split-GFP labeled viral replication proteins and correlative light and EM microscopy (CLEM) in combination with cryo-ET to identify the viral replication and assembly complexes.
Visualization of viral RNA at the single-molecule level in live cells.
We have successfully inserted RNA aptamers in the viral RNA, allowing us for the first time to visualize the incoming viral genome RNA, viral RNA translation (using SunTag) and replication (using aptamers and split-GFP viral replication proteins), viral RNA assembly and the release of virus particles in living cells. These novel technologies will be applied to address unresolved questions in enterovirus biology, thereby providing breakthrough insights.
Dissection of the role of the viral 2A protease in translation and replication using SunTag technology.
Recently, we constructed an enterovirus with a proteolytically inactive 2A protease. This novel mutant virus allowed us to dissect the role of 2A in enhancing viral RNA translation and replication using SunTag technology (i.e. by introducing SunTag epitopes in the 2A dead virus and characterizing these viruses). Using this 2A dead virus, we furthermore revealed that 2A protease activity is essential for suppressing host antiviral responses (by disturbing nucleocytoplasmic transport of cellular signaling proteins and cellular mRNAs, as well as by suppressing the formation of antiviral stress granules and the production of type I interferon) (Schipper et al, 2025, bioRxiv 2025.02.20.639337).
Identification of novel restriction factors that affect viral RNA translation and/or replication.
As part of our ongoing efforts to identify proviral and antiviral host factors for enteroviruses, we use CRISPR-cas9 screening methodologies. In a screen with the 2A dead virus, we identified several novel antiviral host factors that restricted replication of this 2A dead virus but not of the wild-type virus. These cellular proteins were not identified before with wild-type enteroviruses as they are countered by proteolytic activity of 2A, underscoring the value of our recombinant virus approach. We will investigate the underlying mechanism using SunTag, PP7 and split-GFP technologies described above. Insights into how antiviral factors curtail enterovirus infection are essential for understanding basic aspects of viral translation, replication, assembly and release.
Development of a sensor to detect and visualize accessible viral dsRNA in infected cells in real time.
Double-stranded (ds)RNA formed during viral replication can potently activate an antiviral response that can restrict virus replication and spreading. Viruses are thought to evade this response by shielding dsRNA from host sensors. However, it is poorly understood how dsRNA shielding is achieved, and where and when during infection dsRNA is sensed to activate the antiviral response. We developed a sensitive, live-cell reporter to selectively visualize cytosol-accessible dsRNA and dsRNA sensing in single living cells. We observed that during picornavirus infection most dsRNA is efficiently shielded from cytosolic sensors, and we identified roles for viral proteins and replication organelles in dsRNA shielding. Importantly, kinetic analysis revealed that small amounts of dsRNA often escape shielding during late-stage infection, and this population of cytosol-accessible dsRNA is critical for antiviral response activation (manuscript submitted).
We have successfully inserted RNA aptamers in the viral RNA, allowing us for the first time to visualize the incoming viral genome RNA, viral RNA translation (using SunTag) and replication (using aptamers and split-GFP viral replication proteins), viral RNA assembly and the release of virus particles in living cells. These novel technologies will be applied to address unresolved questions in enterovirus biology, thereby providing breakthrough insights.
Dissection of the role of the viral 2A protease in translation and replication using SunTag technology.
Recently, we constructed an enterovirus with a proteolytically inactive 2A protease. This novel mutant virus allowed us to dissect the role of 2A in enhancing viral RNA translation and replication using SunTag technology (i.e. by introducing SunTag epitopes in the 2A dead virus and characterizing these viruses). Using this 2A dead virus, we furthermore revealed that 2A protease activity is essential for suppressing host antiviral responses (by disturbing nucleocytoplasmic transport of cellular signaling proteins and cellular mRNAs, as well as by suppressing the formation of antiviral stress granules and the production of type I interferon) (Schipper et al, 2025, bioRxiv 2025.02.20.639337).
Identification of novel restriction factors that affect viral RNA translation and/or replication.
As part of our ongoing efforts to identify proviral and antiviral host factors for enteroviruses, we use CRISPR-cas9 screening methodologies. In a screen with the 2A dead virus, we identified several novel antiviral host factors that restricted replication of this 2A dead virus but not of the wild-type virus. These cellular proteins were not identified before with wild-type enteroviruses as they are countered by proteolytic activity of 2A, underscoring the value of our recombinant virus approach. We will investigate the underlying mechanism using SunTag, PP7 and split-GFP technologies described above. Insights into how antiviral factors curtail enterovirus infection are essential for understanding basic aspects of viral translation, replication, assembly and release.
Development of a sensor to detect and visualize accessible viral dsRNA in infected cells in real time.
Double-stranded (ds)RNA formed during viral replication can potently activate an antiviral response that can restrict virus replication and spreading. Viruses are thought to evade this response by shielding dsRNA from host sensors. However, it is poorly understood how dsRNA shielding is achieved, and where and when during infection dsRNA is sensed to activate the antiviral response. We developed a sensitive, live-cell reporter to selectively visualize cytosol-accessible dsRNA and dsRNA sensing in single living cells. We observed that during picornavirus infection most dsRNA is efficiently shielded from cytosolic sensors, and we identified roles for viral proteins and replication organelles in dsRNA shielding. Importantly, kinetic analysis revealed that small amounts of dsRNA often escape shielding during late-stage infection, and this population of cytosol-accessible dsRNA is critical for antiviral response activation (manuscript submitted).