Periodic Reporting for period 2 - CHUbVi (Ubiquitin Chains in Viral Infections)
Reporting period: 2021-09-01 to 2023-02-28
Viruses such as Influenza A (IAV) and others remain one of the greatest threats to public health and are associated with enormous economic impacts. Despite decades of work, there are still almost no general approaches to antiviral treatment, leaving the world’s population exposed to pandemic viruses. The current and enduring Sars-CoV2 pandemic is an important reminder of this. Most current strategies for treating and preventing viral diseases such as influenza are specific to the virus type – or in the case of vaccination – even the specific strain. Yet, the recent outbreaks of Ebola, Zika, West Nile, Chikungunya or Sars-CoV2 viruses, and the continued dangers posed by common viruses, remind us that novel treatments are still required; broad spectrum antiviral approaches are lacking or limited due to issues such as toxicity.
By detailed studies on the molecular mechanism of viral entry into mammalian cells, our team has uncovered a potentially fundamental strategy by which enveloped viruses infect cells. In short, our working hypothesis is that viruses such as influenza A make use of a cellular pathway to escape the endosomes and successfully infect cells. They do so by co-opting the cellular aggresome processing pathway (APP), which is normally activated when misfolded proteins accumulate. Central components of this pathway are the deacetylase HDAC6, unanchored ubiquitin chains as well as motor proteins. HDAC6 is an atypical, largely cytoplasmic, deacetylase that has tandem catalytic domains and which can be activated by binding to ubiquitin via its C- terminal zinc finger domain. Ubiquitin (Ub) is a small 76-amino acid protein that is utilized for multiple signaling purposes in the cell. It can form chains of different length and linkage and serve as a post- translational modification (PTM) by being covalently attached via its carboxyl terminus to usually Lys residues in target proteins. Ubiquitinated proteins are often targeted for degradation by the proteasome. In addition, ubiquitin can also interact with a variety of proteins through non-covalent hydrophobic interactions. Initial studies by our team have conclusively demonstrated the critical role of the APP for influenza virus capsid uncoating. Additional recent experiments have shown that this pathway is in fact used by multiple enveloped RNA viruses, such as Zika, Dengue, Ebola and others, so that it represents a universal route of entry for multiple highly pathogenic viruses . Following uncoating of the capsid, the viral RNA particles (vRNPs) have to be debundled, and this step is critically dependent on another cellular protein, transportin 1 (TNPO1), a karyopherin involved in the nuclear transport of multiple proteins, that was recently shown to prevent phase separation of the FUS protein.
Our experiments have confirmed that HDAC6 is regulated by unanchored ubiquitin chains and plays a critical role in virus uncoating, but the exact molecules and mechanisms involved are unclear. Both the enzymatic activity of HDAC6 as well as its capacity to recruit additional proteins appear to depend on its interaction with unanchored ubiquitin chains. The observation that enveloped virus package free ubiquitin and ubiquitin chains gives rise to the intriguing, but currently circumstantial, idea that viral-derived Ub is essential for cell infection by inducing the APP, or a highly related pathway. In this context, the substrates of HDAC6 that are controlled by ubiquitin chains remain unknown. Viral infection also induces structures related to stress granules (SGs), which are transient cytoplasmic RNA-proteins granules forming under various kinds of stress; the vRNPs debundling has similarity to SGs and TNPO1 itself is present in SGs. Furthermore, HDAC6 is critical for SG formation and we have recently shown that its role is to control the phase separation properties of intrinsically disordered proteins that are SG components, thereby promoting granules maturation. Thus, the APP and SGs pathways appear to be inter-related in the context of viral infection and may represent an attractive target for therapeutic intervention. In addition, it was recently shown that an APP-related pathway is also important for activation of the inflammasome, a large oligomeric cytoplasmic complex controlling formation of the inflammatory response. In summary, it appears that the main players of the APP (ubiquitin, HDAC6) partake in regulating seemingly unrelated processes such as virus infection or pathways of the cellular stress response.
Taken together, these observations invite a detailed study on the role of ubiquitin and ubiquitin chains in viral infection, the role of HDAC6 and its substrates on the aggresome processing pathway and formation of stress granules, and the identification of the key proteins involved in regulating this process.
This key hypothesis of our project is that viral entry -more specifically uncoating- is enabled by specific ubiquitin chains interacting with HDAC6 and allowing formation of a functional complex with motor proteins and other as yet unidentified partners. Therefore, the activation of the aggresome pathway will be comprehensively explored with the tools of molecular biology including CRISPR knockouts, DARPin proteins that block HDAC6 activation, and interrogation with synthetic molecules. By drawing on strengths in synthetic and medicinal chemistry within the team, modern small molecule interventions including targeted protein degraders for HDAC6 or its downstream targets will be prepared to demonstrate that novel small molecule approaches to broad spectrum anti-virals targeting are valuable and suitable for drug development. In addition, the pathways discussed here are also recognized to be relevant in other pathologic conditions, in particular in neurodegenerative diseases. Therefore, a deeper understanding of how these pathways are wired and utilized, during viral infection and beyond, will likely lead to significant progress in other biological paradigms.
By detailed studies on the molecular mechanism of viral entry into mammalian cells, our team has uncovered a potentially fundamental strategy by which enveloped viruses infect cells. In short, our working hypothesis is that viruses such as influenza A make use of a cellular pathway to escape the endosomes and successfully infect cells. They do so by co-opting the cellular aggresome processing pathway (APP), which is normally activated when misfolded proteins accumulate. Central components of this pathway are the deacetylase HDAC6, unanchored ubiquitin chains as well as motor proteins. HDAC6 is an atypical, largely cytoplasmic, deacetylase that has tandem catalytic domains and which can be activated by binding to ubiquitin via its C- terminal zinc finger domain. Ubiquitin (Ub) is a small 76-amino acid protein that is utilized for multiple signaling purposes in the cell. It can form chains of different length and linkage and serve as a post- translational modification (PTM) by being covalently attached via its carboxyl terminus to usually Lys residues in target proteins. Ubiquitinated proteins are often targeted for degradation by the proteasome. In addition, ubiquitin can also interact with a variety of proteins through non-covalent hydrophobic interactions. Initial studies by our team have conclusively demonstrated the critical role of the APP for influenza virus capsid uncoating. Additional recent experiments have shown that this pathway is in fact used by multiple enveloped RNA viruses, such as Zika, Dengue, Ebola and others, so that it represents a universal route of entry for multiple highly pathogenic viruses . Following uncoating of the capsid, the viral RNA particles (vRNPs) have to be debundled, and this step is critically dependent on another cellular protein, transportin 1 (TNPO1), a karyopherin involved in the nuclear transport of multiple proteins, that was recently shown to prevent phase separation of the FUS protein.
Our experiments have confirmed that HDAC6 is regulated by unanchored ubiquitin chains and plays a critical role in virus uncoating, but the exact molecules and mechanisms involved are unclear. Both the enzymatic activity of HDAC6 as well as its capacity to recruit additional proteins appear to depend on its interaction with unanchored ubiquitin chains. The observation that enveloped virus package free ubiquitin and ubiquitin chains gives rise to the intriguing, but currently circumstantial, idea that viral-derived Ub is essential for cell infection by inducing the APP, or a highly related pathway. In this context, the substrates of HDAC6 that are controlled by ubiquitin chains remain unknown. Viral infection also induces structures related to stress granules (SGs), which are transient cytoplasmic RNA-proteins granules forming under various kinds of stress; the vRNPs debundling has similarity to SGs and TNPO1 itself is present in SGs. Furthermore, HDAC6 is critical for SG formation and we have recently shown that its role is to control the phase separation properties of intrinsically disordered proteins that are SG components, thereby promoting granules maturation. Thus, the APP and SGs pathways appear to be inter-related in the context of viral infection and may represent an attractive target for therapeutic intervention. In addition, it was recently shown that an APP-related pathway is also important for activation of the inflammasome, a large oligomeric cytoplasmic complex controlling formation of the inflammatory response. In summary, it appears that the main players of the APP (ubiquitin, HDAC6) partake in regulating seemingly unrelated processes such as virus infection or pathways of the cellular stress response.
Taken together, these observations invite a detailed study on the role of ubiquitin and ubiquitin chains in viral infection, the role of HDAC6 and its substrates on the aggresome processing pathway and formation of stress granules, and the identification of the key proteins involved in regulating this process.
This key hypothesis of our project is that viral entry -more specifically uncoating- is enabled by specific ubiquitin chains interacting with HDAC6 and allowing formation of a functional complex with motor proteins and other as yet unidentified partners. Therefore, the activation of the aggresome pathway will be comprehensively explored with the tools of molecular biology including CRISPR knockouts, DARPin proteins that block HDAC6 activation, and interrogation with synthetic molecules. By drawing on strengths in synthetic and medicinal chemistry within the team, modern small molecule interventions including targeted protein degraders for HDAC6 or its downstream targets will be prepared to demonstrate that novel small molecule approaches to broad spectrum anti-virals targeting are valuable and suitable for drug development. In addition, the pathways discussed here are also recognized to be relevant in other pathologic conditions, in particular in neurodegenerative diseases. Therefore, a deeper understanding of how these pathways are wired and utilized, during viral infection and beyond, will likely lead to significant progress in other biological paradigms.
Despite the disruption caused by the Covid-19 pandemic, our team has been able to effectively perform the groundwork required and we have made good progress.
Among others, we have:
• prepared cell lines to manipulate ubiquitin expression
• established robust aggresome analysis pipelines
• achieved full-length HDAC6 expression, allowing further biochemical analysis
• established methods and started to perform mass spectrometry ubiquitome analysis
• improved chemical synthesis of ubiquitin chains
• prepared initial PROTACS targeting HDAC6
• performed IAV siRNA screens against E2 enzymes
• prepared a small artificial protein preventing Ub recruitment by HDAC6 and analyzed its impact on virus infection and cellular stress pathways
• established purification of ubiquitin chains and ubiquitinated proteins followed by analysis by mass spectrometry
• demonstrated the presence of biochemical complexes involving HDAC6 and forming only in the presence of ubiquitin chains
Among others, we have:
• prepared cell lines to manipulate ubiquitin expression
• established robust aggresome analysis pipelines
• achieved full-length HDAC6 expression, allowing further biochemical analysis
• established methods and started to perform mass spectrometry ubiquitome analysis
• improved chemical synthesis of ubiquitin chains
• prepared initial PROTACS targeting HDAC6
• performed IAV siRNA screens against E2 enzymes
• prepared a small artificial protein preventing Ub recruitment by HDAC6 and analyzed its impact on virus infection and cellular stress pathways
• established purification of ubiquitin chains and ubiquitinated proteins followed by analysis by mass spectrometry
• demonstrated the presence of biochemical complexes involving HDAC6 and forming only in the presence of ubiquitin chains
We anticipate that getting a detailed mechanistic understanding of how the aggresome-related pathway is used by viruses for infection will give the scientific foundation for the possible development of novel therapeutics. These may be useful in the case of viral infection, as well as possibly in situations like inflammation, neurodegeneration or others.
A main advantage of having novel antiviral therapies targeting cellular proteins of the host response is that they may be of use for a broad range of different viruses, unlike the current therapies that are tailored against specific viruses or strains thereof. Thus, such broad specificity antiviral agents could be combined with virus-specific antiviral agents and increase their efficacy.
Although our main focus is the role of unanchored Ub chains, the role of longer ubiquitin chains attached to cellular proteins is also relevant to this project. To this end, the team at ETHZ has also pursued the development of a chemoenzymatic system for the site-specific attachment of assembled Ub chain to specific lysine residues of target proteins. This work was recently published (Akimoto et al., 2022) and provides an unexpectedly simple tool for manipulating Ub chains prepared by the methods described below
A main advantage of having novel antiviral therapies targeting cellular proteins of the host response is that they may be of use for a broad range of different viruses, unlike the current therapies that are tailored against specific viruses or strains thereof. Thus, such broad specificity antiviral agents could be combined with virus-specific antiviral agents and increase their efficacy.
Although our main focus is the role of unanchored Ub chains, the role of longer ubiquitin chains attached to cellular proteins is also relevant to this project. To this end, the team at ETHZ has also pursued the development of a chemoenzymatic system for the site-specific attachment of assembled Ub chain to specific lysine residues of target proteins. This work was recently published (Akimoto et al., 2022) and provides an unexpectedly simple tool for manipulating Ub chains prepared by the methods described below