Viruses such as Influenza A (IAV) and SARS-CoV-2 remain one of the greatest threats to public health and are associated with enormous economic impacts. Despite decades of work, there are few general approaches to antiviral treatment, leaving the world’s population exposed to pandemic viruses. The recent Sars-CoV2 pandemic is an important reminder. 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 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 fundamental mechanism 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 successfully infect cells 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 (i.e. free) 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 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 branching sites, and in this context serves as a key scaffolding protein to assemble largely unknown cellular factors in the APP. 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 and the essential requirement of long, branched, and C-terminally unanchored Ub chains. Additional recent experiments have shown that this pathway is in fact used by multiple enveloped RNA viruses, such as Zika, and others, so that it represents a universal route of entry for multiple highly pathogenic viruses. Following uncoating of the capsid, the viral ribonucleoproteins (vRNPs) must be debundled, and this step is critically dependent on another cellular protein, transportin 1 (TNPO1), a karyopherin involved in the nuclear transport of multiple RNA-binding proteins.
Our experiments have confirmed that HDAC6 binds the C-terminus of 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 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. Furthermore, the type, length, and branching sites of the specific Ub chains employed for viral entry are unknown, although circumstantial evidence for certain branching types exists. 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 crucial 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. 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, 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 these processes. To achieve this, the preparation of specific ubiquitin chains with defined length and branching is required, both to reconstitute the putative APP and enable structural studies on the large molecule assemblies. A key goal of our project will be establishing methods for both the synthesis of complex Ub chains, and their identification and isolation from virus infected cells.
A central hypothesis of our project is that viral entry -more specifically uncoating- is enabled by specific ubiquitin chains interacting with HDAC6 –allowing formation of a functional transient complex with motor proteins and other as yet unidentified partners. The activation of the aggresome pathway will be comprehensively explored with the tools of molecular biology including CRISPR knockouts, DARPin artificial proteins that block HDAC6 activation, or viral M1 uncoating, 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-viral 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. A deeper understanding of how these pathways are utilized, during viral infection and beyond, will lead to significant progress in other biological paradigms.
