NIHRDataInsights/HRCSData huggingface dataset:
Title: Molecular mechanisms of HIV-1 restriction by capsid-sensing host cell proteins
Funder: Wellcome Trust

Abstract: Infections by retroviruses, such as HIV-1, critically depend on the viral capsid. Many host cell defence proteins, including restriction factors Trim5&alpha;, TrimCyp and MxB, target the viral capsid at the early stages of infection and potently inhibit virus replication. These restriction factors appear to function through a remarkable capsid pattern sensing ability that specifically recognizes the assembled capsid, but not the individual capsid protein. Using an integrative and multidisciplinary approach, I aim to determine the molecular interactions between the viral capsid and host restriction factors, TrimCyp and MxB, that underpin their capsid pattern-sensing capability and ability to inhibit HIV-1 replication. Specifically, I will combine cryoEM and cryoET with all-atom molecular-dynamics simulations to obtain high-resolution structures and atomic models of the capsid and host protein complexes (in vitro), together with mutational and functional analysis as well as correlative light and cryoET imaging of viral infection process (in vivo and in situ), to reveal the essential interfaces in their 3D organization for HIV-1 capsid recognition and inhibition of HIV-1 infection. Information derived from our studies will allow to design more robust therapeutic agents to block HIV-1 replication by strengthening the pattern recognition feature.

Lay abstract: During early stages of HIV-1 infection, the viral capsid, a protein shell enclosing HIV-1 genome, intimately interfaces with host cellular proteins. These essential molecular interactions occur during a pivotal period in the infection process when the virus is highly susceptible to disruptive interventions, thus, representing promising targets for the development of new drugs and therapeutic strategies for HIV-1 prevention and treatment. Among these proteins are host cell defence factors that specifically bind the viral capsid by recognizing the capsid surface pattern and disrupt its function, thus potently block HIV-1 replication. How the host cell defence proteins sense the incoming viral capsid, however, is not understood. Using an integrative approach combining structural methods with retrovirology, cell biology and computer simulations, I aim to determine the common mechanism(s) of capsid-pattern sensing by host cell defence proteins. I will also examine the consequences for HIV-1 infection upon alteration of host proteins’ pattern-sensing ability. Such studies will reveal a major host immune defence mechanism targeting the viral capsid and advance our understanding of viral pattern-sensing by the host. Information derived from our studies will generate a framework for the development of new therapeutic interventions of HIV-1 and other pathogenic viruses.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Adaptation of avian influenza virus polymerase to humans during pandemic emergence.
Funder: Wellcome Trust

Abstract: Influenza viruses cause intermittent pandemics and seasonal outbreaks that affect millions of humans every year. My goal is to chart the adaptation of influenza virus to the human host from emergence from birds, often associated with severe infections, to endemicity as a seasonal flu virus. I will focus on the viral polymerase because it is a key determinant of host range, pathogenicity and viral evolution. Using next generation sequencing we will establish accurate quantification of the viral polymerase error rate to test whether polymerase constellation determines pandemic potential. Polymerase interacts with a plethora of host proteins, some permissive and others that restrict it. We recently discovered a key host factor, ANP32A, that differs between birds and mammals and explains the major barrier to avian influenza virus polymerase activity in humans. We will uncover the mechanism by which this host factor supports viral replication using a combination of familiar and novel techniques. We will also discover other species-specific host factors that act on polymerase to support or restrict it. Finally, we will explain why partially adapted avian viruses lead to a devastating cytokine storm in humans they infect, as this may guide therapeutic options and inform pandemic preparedness plans.

Lay abstract: Influenza viruses cause devastating pandemics when they emerge from animals and cross over into humans. In 1918, 50 million people died after being infected by the ‘Spanish’ influenza virus. Other viruses that emerge from animal sources such as SARS, MERS and Ebola virus also cause very severe disease in people. What is especially interesting about influenza is that once the new pandemic virus has infected nearly everyone, it evolves further to become a seasonal influenza virus associated with less severe disease, at least in previously healthy people. We do not currently understand why the viruses that cross over to humans from animals are so lethal or how over time they adapt to become less severe. The explanation must be in the way the virus interacts with the host. In birds, influenza virus infection is silent but when an avian influenza infects humans, the mismatch between virus and host factors leads first to replication block, and later after some adaptive mutations, to severe disease. My vision is to discover the key host factors that underlie these observations. These might be excellent new targets for antiviral strategies, and the new knowledge will also inform pandemic preparedness and therapeutic options for severe influenza.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: A regional cryoEM facility at the University of York
Funder: Wellcome Trust

Abstract: The world-renowned York Structural Biology Laboratory (YSBL) plans to expand its technical capability beyond the established techniques of X-ray crystallography and NMR spectroscopy into cryo-electron microscopy with a regional CryoEM hub, opening up structural biology on large, complex systems to a diverse regional user base. Our bid is aligned with partners in Newcastle and Durham, and will dovetail with complementary regional capabilities in Leeds and Sheffield, and with the national facility, eBIC (see letters of support). Key goals include viral structure determination, dissection of supramolecular assemblies and motors, and exploitation of major health-related protein-protein complexes in mammalian systems and bacteria. CryoEM in York will meet growing demand for which existing regional capacity is insufficient. It will also build upon YSBL’s significant crystallographic methods expertise, providing a focus for pioneering new methods developments in partnership with eBIC. We propose a cost-effective and sustainable 200 kV microscope, which will enable both single-particle work and screening of more challenging tomography samples for subsequent analysis at 300kV facilities. The University of York is supporting the bid by establishing a professorial-level CryoEM post, allocating the time of an Experimental Officer, investing a £1M philanthropic donation and committing £1.7M for the necessary building infrastructure.

Lay abstract: How biological organisms work is intricately linked with their structure: the complex three-dimensional arrangement of molecules that make up proteins, lipids (fats), carbohydrates (sugars) and nucleic acids (such as DNA). Knowledge of those 3D structures is therefore key to understanding biological function. Visualising these structures is beyond the capability of ‘normal’ microscopes, and has traditionally used the techniques of magnetic resonance and crystallography. While these remain successful, they have limitations – magnetic resonance only works well for relatively small molecules, while crystallography requires well-defined crystals (often difficult to obtain). Recently, exciting new developments in instrumentation have brought forward an alternative method for visualising biomolecules – cryo electron microscopy (cryo-EM). Its major advantages are that it can image both non-crystalline substances and large, complex biological systems – precisely those at the forefront of biomedical and biotechnological research. The new possibilities opened up by this technology are legion. This proposal is for the acquisition of a Cryo-EM microscope, based at the University of York, to provide access to this essential new technique for the many scientists working in molecular and structural biology in the North East of England, and to form part of a national infrastructure in cryo-EM.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Systematic discovery and analysis of novel molecular mechanisms in protein synthesis
Funder: Wellcome Trust

Abstract: Protein synthesis is one of the most fundamental processes in all organisms. Due to the unique constraints under which RNA viruses evolve, non-canonical mechanisms are particularly prevalent in RNA virus gene expression. Thus, we are using viruses as a source for the discovery of novel gene expression strategies. We will leverage our expertise in computational biology to mine public transcriptomic datasets for 10000s of novel virus species, and use our expertise in virus comparative genomics and molecular biology to identify and characterize novel translational mechanisms. An important feature of the research strategy is close synergistic interaction between the bioinformaticians and experimentalists in my team, which allows us to efficiently investigate new discoveries. Key goals are to: 1. Search ~120,000 RNA-Seq datasets for RNA virus-derived sequences from diverse host organisms from protists to mammals. 2. Use comparative genomics to predict novel cases of non-canonical gene expression. 3. Experimentally characterize the most interesting novel mechanisms. Characterizing these exceptions to the rules of canonical translation will provide new insights into the mechanisms underlying protein synthesis, reveal new modes of cellular gene expression, and provide new tools for molecular biology research, biotechnology and synthetic biology. Thus the results will have broad applications in fundamental molecular biology.

Lay abstract: In cells, messenger RNAs (mRNAs) are translated by ribosomes into proteins. When viruses infect cells, they "hijack" the host protein synthesis machinery to translate viral mRNAs into viral proteins. Viruses have evolved many non-canonical mechanisms to manipulate the protein synthesis machinery to increase viral protein-coding capacity and/or to out-compete host mRNAs for access to ribosomes. The known repertoire of viral non-canonical translation mechanisms derives from just a tiny fraction of the estimated viral diversity on Earth. We will leverage our expertise in computational biology to mine public transcriptomic datasets for 10000s of novel virus species, and use our expertise in comparative genomics and molecular biology to identify and characterize novel gene expression strategies. This work will reveal new ways in which the protein synthesis machinery can be manipulated, identify possible new modes of cellular gene expression, and underpin development of new tools for molecular biology research, biotechnology and synthetic biology.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Molecular mechanisms mediating immune evasion in African trypanosomes
Funder: Wellcome Trust

Abstract: The African trypanosome Trypanosoma brucei is an extraordinarily effective extracellular pathogen. Key for its survival is a protective Variant Surface Glycoprotein (VSG) coat, which is expressed at an extraordinarily high level from a VSG gene located in an RNA polymerase I transcribed VSG expression site. Although an individual trypanosome has multiple bloodstream form expression sites, stringent mono-allelic exclusion ensures that only one is transcribed at a time. We would like to understand how this mono-allelic exclusion operates. We have generated T. brucei strains with disrupted mono-allelic exclusion, which dynamically switch between two different expression sites. What restriction has disappeared? T. brucei additionally contains metacyclic expression sites, which are mono-allelically expressed in the relevant life-cycle stage. How are these different types of expression sites controlled? We have recently made the striking discovery that blocking trans-splicing leads to an abrupt halt in transcription of the active VSG expression site. How does RNA processing feed back to control this unusual RNA polymerase I transcription unit? Last, we have discovered that VSG synthesis is sensed during the cell cycle, and blocking its synthesis triggers a precise pre-cytokinesis cell cycle arrest. How does this happen, and what stress responses are triggered?

Lay abstract: African trypanosomes are single celled parasites which can infect the human bloodstream. They survive immune attack due to their protective Variant Surface Glycoprotein (VSG) coat. VSG is the most abundant protein of bloodstream form trypanosomes (10% total), and arguably one of the most important proteins. VSG coat synthesis could therefore represent an Achilles heel for tackling this pathogen. An individual trypanosome has thousands of genes encoding different coat types. However, only one VSG gene is transcribed at a time from a single expression site. Trypanosomes have multiple expression sites of different types. How are they all controlled so that only one is on at a time? We have found that blocking synthesis of the protective VSG surface coat prevents trypanosomes from dividing. Understanding this connection between VSG coat production and cell division, as well as how coat synthesis is controlled should allow us to tackle this evasive African parasite.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: The complete interaction between Trypanosoma brucei and mammalian host proteins
Funder: Wellcome Trust

Abstract: African trypanosomes have remarkable cell surfaces which mediate their interactions with the molecules of infected mammals. These surfaces are packed with a dense layer of the variant surface glycoprotein (VSG), allowing a population survival strategy based on antigenic variation. Within thisVSG coat operate receptors for mammalian ligands such as transferrin and haptoglobin-haemoglobin. We have shown how these receptors have evolved to bind ligands while minimising exposing of the trypanosome to the adaptive immune system. We have also shown that trypanosome receptors bind to complement components, identifying and characterising receptors for factor H and complement C3. This latter work validated a bioinformatics screen that identified a further 25 putative receptors. The aim of this proposal is to now identify the complete receptor repertoire used by Trypanosoma bruceito exploit and survive within its host. We will assess whether receptors are virulence factors and whether they represent new biology. We will understand the molecular basis for their action. We will also determine what causes the distribution and dynamics of different receptor types within the cell. This will produce detailed mechanistic insight into the cell and molecular biology of trypanosome receptors, yielding a greater understanding of both the trypanosome and the disease.

Lay abstract: African trypanosomes are single celled parasites which cause disease in humans, domestic livestock and wild animals. They can survive for a long time in mammals by exploiting the host for nutrients and avoiding the host immune defences. To do this, trypanosomes have receptor molecules on their surfaces which stick to molecules found in mammalian blood. I aim to understand how trypanosomes are able to bind to and use host nutrients. I also want to understand how their receptors are adapted to allow them to avoid destruction by the host immune defences. This work will reveal what each of the different receptors on the trypanosome does, how they bind to their mammalian ligands and how they avoid being detected The outcome of this work will be a much better understanding of how trypanosomes are able to survive and proliferate in mammals and how they cause disease.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Structure and mechanism of nucleic acid-processing machines in viral biogenesis
Funder: Wellcome Trust

Abstract: The research will focus on analysing the structure and mechanism of nucleic acid machines involved in viral biogenesis. Although the main emphasis is on using structural approaches such as X-ray crystallography and Cryo-EM; we will also use complementary biophysical techniques including surface plasmon resonance, fluorescence-based activity/binding assays, analytical ultracentrifugation and SEC-MALLS, to define composition and stoichiometry of protein-nucleic acid complexes, in addition to the affinity of the interaction. Key goals: 1. To continue investigation into the mechanism of dsDNA packaging motors present in tailed bacteriophages and the closely related human herpes viruses. To obtain high-resolution asymmetric cryo-EM reconstructions of an active motor assembled from components derived from a themostable bacteriophage. 2. To understand the structure-function relationship in FtsK-like DNA packaging motors present in another large class of dsDNA viruses comprising bacterial tectiviruses and eukaryotic poxviruses. 3. To investigate the mechanism of RNA unwinding by 2C helicases present in enteroviruses (such as poliovirus) and to define the RNA-binding surface of the Zika Virus NS3 helicase. To perform pilot studies on exploiting this information for identifying lead compounds for the development of antivirals.

Lay abstract: Viruses are the most common biological entities on our planet, infecting all living things. In humans, viral infections like glandular fever, Dengue fever, Ebola and influenza cause illnesses and deaths worldwide, particularly in the elderly, babies or people with lowered immune defences. Sometimes viruses cause life-long infections as they are impossible to eradicate using current treatments. This can lead to heart problems or cancer. Others, such as Zika virus, can cause serious developmental damage to unborn babies. Viruses can only replicate in cells and use dedicated capsules to protect their genes while passing to another host. During infection, new copies of the virus’ genes are made and loaded into capsules to make new virus particles that then burst out of the host and spread to other cells. The virus employs highly specialised machines to copy its genes and to load them into the capsules. We will use cryo-Electron Microscopy, X-ray crystallography and approaches that sense single molecules, to image the machines and watch them work. The research will provide detailed information about what the machines look like and how they function. This will inform the development of new treatments that can prevent or reduce virus infections.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Wellcome Centre for Integrative Parasitology
Funder: Wellcome Trust

Abstract: The Wellcome Centre for Integrative Parasitology (WCIP) has evolved through Trust support since 1987 into a world-leading and cutting-edge research hub. We now propose a further transformation to take us and the field forward through the integration of disciplines, locations and scales, to tackle disease problems in disease endemic regions (DER). Our 2014 renewal broadened our portfolio of pathogens and technologies and grew the Centre at every level. Our PI complement increased from 9 to 15, including numerous Fellowship and Investigator Award holders (6F/5M). World-leading recruits from Harvard, Oxford and Edinburgh together with young emerging (female) PIs radically extended the Centre’s activities. We have new programmes on immunology, helminths and their population structures, single cell bioinformatics, and clinical pathology of parasitic diseases. Technologically, we have expanded with dedicated technologists in both imaging and metabolomics, while internationally, fruitful engagement with Malawi has embedded Centre members in key areas such as paediatric malaria, epidemiology and enhanced training opportunities. Our strategic growth reflects the evolution of our vision from the intense, largely laboratory-based investigation of parasite biology into the integrated pursuit of WCIP-generated knowledge-based therapies positioned by informed interactions with Global Health stakeholders in Glasgow and selected partners across Europe and particularly DER.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Evasion of innate immunity by HIV-1 during the early stages of viral replication
Funder: Wellcome Trust

Abstract: The entry process of HIV-1 and the early events of its replication cycle are vulnerable to interferon-induced restriction and pattern recognition. We have shown that a family of interferon-induced membrane proteins (IFITMs) restrict the entry of HIV-1 dependent on their CD4 and coreceptor usages. Importantly, viruses that are transmitted between individuals are able to avoid IFITM-mediated restriction, correlating with their inherent resistance to type-1 interferons. Therefore, IFITMs are an important barrier for person-to-person spread. In this proposal, we will identify the determinants of IFITM resistance/sensitivity in the HIV-1 envelope glycoprotein (env) and use this knowledge to answer outstanding questions about the cell biology of HIV-1 entry and tropism. We will characterize how escape from early antibody responses in patients leads to the emergence of interferon-sensitivity in env, and how a polymorphism in ifitm3 limits host control of HIV-1 replication in acute infection. Lastly, we will build on preliminary data to understand how the virion-associated accessory protein, Vpr, shuts-down pattern recognition receptor signalling immediately after cellular entry, and identify the host protein(s) it targets to achieve this. These studies will yield important insights into the avoidance of innate immunity by HIV-1 of relevance for therapeutic intervention and immunogen design.

Lay abstract: Avoidance human innate immunity by HIV-1 is essential for its successful transmission between individuals, and studying these mechanisms will potentially yield new therapeutics to treat HIV/AIDS. We want to understand two aspects of these immune evasion strategies. How HIV-1 avoids a family of membrane proteins, known as IFITMs, which block the entry of the virus into its target cell; and once inside, how it disables the early warning systems that detect invading viruses. We have found that isolates of HIV-1 that represent those transmitted between people are inherently resistant to the activities IFITM proteins, and that this is a major contributor to their overall avoidance innate immunity. We propose to understand how these transmitted viruses IFITM-mediated inhibition. We will determine how IFITMs affect the entry process of the virus itself, the cell types it can replicate in, and how antibody responses that arise in the first few months after infection lead to IFITM sensitivity. We will also study the role of a small viral protein, Vpr, that is associated with the HIV-1 particle. Our preliminary evidence indicates that Vpr shuts-down the cell’s ability to detect virus infection, and we will determine the mechanism by which it does so.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION