Professional guarantor

prof. Dr. Ing. Michaela Rumlová, UCT

Infectious diseases are characterised by complex and dynamic interactions between pathogen virulence factors and host-cell recognition and response systems. Many relationships between pathogens and their hosts have arisen through long-term co-evolution.

As a result, pathogens have developed elegant and diverse mechanisms to evade host immune responses, whereas host cells use their systems to detect and eliminate pathogens.

Our understanding of the complexity of pathogen-host interactions is limited. For this reason, developing new preventive or therapeutic approaches is essential. Research into pathogen-host cell interactions is evolving in response to the emergence of new infectious organisms (SARS-CoV-2 or drug-resistant bacteria). In addition, oncogenic viruses account for 10% of diagnosed cancers.

Despite the importance of the scale of the adverse health and economic impacts of infectious diseases, many unanswered questions remain about the precise mechanisms by which viruses and bacteria multiply in infected organisms and cause disease symptoms.

Professional goals

Masaryk University

MUNI_01_Plevka: Structural characterisation of enterovirus replication in situ

Enteroviruses such as coxsackieviruses and rhinoviruses cause annually billions of human infections, including upper and lower respiratory tract infections, gastroenteritis, hand-foot-and-mouth-disease, and life-threatening encephalitis. Despite the societal and economic impact of the diseases, our understanding of enterovirus replication cycle is incomplete. Cryo-electron microscopy and tomography, will be used to characterise enterovirus replication in situ. We will study, enterovirus genome delivery into the cell cytoplasm, the organisation of enterovirus “replication factories”, and the principles of enterovirus capsid assembly and genomes packaging. Results will provide an insight into the mechanism of enterovirus replication and may identify new targets for future anti-enterovirus therapeutics. Principal investigator Pavel Plevka is expert in enterovirus biology and determining their structures and replication intermediates by X-ray crystallography and cryo-electron microscopy.

MUNI_01_Plevka: Phage infection of bacterial biofilm

An antibiotic-resistant Staphylococcus aureus, causing a range of illnesses, from skin infections to pneumonia, meningitis, sepsis and implant-related infections, is recognised by WHO as a pathogen for which new therapeutics are urgently needed. Phage therapy is a promising alternative approach to the treatment of S. aureus biofilms. Correlative light and electron microscopy, is proposed to analyse the dynamics of the Herelleviridae phage phi812 propagation in S. aureus biofilm, herd immunity against phage infection, and phage replication in infected cells. The results will improve our understanding of yet uncharacterised interactions of phages and bacteria, and may enable development of more efficient phage therapy for the treatment of infections caused by antibiotic-resistant strains of S. aureus. Principal investigator Pavel Plevka used cryo-electron microscopy to describe the structure and genome delivery mechanisms of phi812 and other phages that infect pathogenic bacteria.

MUNI_02_Vácha: Antimicrobial Peptides at Viruses and Bacteria

Membranes of bacteria and eukaryotic cells have very different lipid compositions, which have been utilized in selective targeting of membrane-disruptive peptides acting as antimicrobial peptides or toxins. However, the sequence motives of peptides responsible for adsorption and disruption of membranes with specific lipid composition are missing. We will use computer simulations and develop new models to determine new membrane selective peptides, which will be experimentally verified for their membrane affinity and selectivity. The obtained insight and sequence motives could be utilized in the development of new peptides with potential application as therapeutics, sensors, or biomarkers for bacteria. Principal investigator Robert Vácha used computer simulations to determine interaction between proteins and membranes with specific lipid composition, including DivIVA protein, numerous antibacterial peptides, and candidalysin toxin.

MUNI_02_Vácha: Molecular Mechanisms of Viral Infections

Molecular details of viral infection remain elusive due to the experimental challenges of capturing transient structures. However, such details are advantageous for the design of novel antiviral compounds. We will develop new biophysical models of the recognition and movement of viruses on host surfaces, virus penetration and fusion of cellular membranes, genome release from virus capsids into the cytoplasm, and utilization of host machinery for virus replication. The models will be based and verified with available experimental data. The obtained results will bring a molecular understanding of the processes involved in virus infection and replication that may enable the development of drug delivery systems and new therapeutics. Principal investigator Robert Vácha has developed a coarse-grained model of viruses to demonstrate how virus can get across cellular membrane and subsequently release their genome.

MUNI_03_Demo: Mechanisms of coupled transcription-translation in bacteria

Coupling of transcription to translation, typical for bacterial cells, is important for toxicity of pathogenic bacteria, such as Escherichia coli or Pseudomonas aeruginosa. However, the mechanisms of the transcription-translation initiation and control remain unclear. The coupled transcription-translation complexes and structures will be characterized and visualized by using cryo-EM. Time-resolved cryo-EM studies will be used to capture transient interaction intermediates of bacterial RNA polymerase and ribosome. The results gained from these analyses will improve the current understanding of gene regulation in bacterial cells and contribute to the design of innovative antimicrobials. Principal investigator Gabriel Demo is an expert in coupled transcription and translation in bacteria and determining the coupled complex intermediates by time resolved cryo-electron microscopy.

MUNI_03_Demo: Mechanisms of coupled transcription-translation in virus infected mammalian cells

The ability of many viruses with single-stranded RNA (e.g. Togaviridae) and some double-stranded DNA (e.g. Poxviridae or Asfarviridae) genomes to replicate in cytoplasm of infected eukaryotic cells suggests that viral transcription are directly coupled to host translation. However, structural evidence is still missing. Cryo-electron microscopy will be used to investigate transcription-translation coupling of viruses in eukaryotic cells and to visualize the in vitro and in situ arrangement of ribosomes in relation to viral RNA polymerases and the cellular architecture of coupled systems in infected cells. Detailed structural information will bring insights into the mechanisms of viral pathogenesis and will have clinical implications for the development of specific antivirals. Principal investigator Gabriel Demo an expert in cryo-electron microscopy and eukaryotic translation will use the knowledge to determine structures of coupled 80S ribosomes and viral RNA polymerase.

MUNI_04_Šmajs: Genetics and physiology of treponemes and pathogenesis of treponemal infections

The genus Treponema comprises several human and animal pathogenic species and subspecies, including the causative agent of syphilis – T. pallidum subspecies pallidum. Although treponemal infections are one of the most widespread infections affecting millions of people worldwide, little is known about these pathogens. Recently introduced in vitro cultivation system opens new possibilities in treponemal research. This project will provide i) insights into the pathogenicity, invasiveness and persistence of treponemal infections, ii) identification of important biological functions of known genetic differences among different strains of treponemal pathogens, and iii) better understanding of evolution and infection strategies of treponemal pathogens. Understanding of treponemal infection might enable the development of new diagnostic tools and help to design a functional vaccine against syphilis. Principal investigator David Šmajs is an expert in treponemal genomics and epidemiology. Recently, he has been using in vitro cultivation system for study of treponemes.

MUNI_04_Šmajs: Role of microbes in human health and pathological conditions

Role of microbes in human health and pathological conditions Escherichia coli is a commensal of the human gastrointestinal tract, but also one of the most important human pathogens. While several highly adapted E. coli strains evolved the ability to cause a broad spectrum of diseases, most of E. coli strains are commensal bacteria with important role in human health. However, involvement in various pathologic conditions limits broader application of beneficial E. coli strains in human medicine. Results obtained in this project will provide insights into the different characteristics of commensal E. coli strains ex vivo, which are generally poorly understood. Furthermore, the corresponding genetic markers for adverse and beneficial activities of E. coli will be identified with potential applications to human medicine. Principal investigator David Šmajs focuses on ecological role of Escherichia coli in pathological conditions and also on therapeutic potential of E. coli in human and veterinary medicine.

MUNI_05_Pantůček: Phage-host interactions

Viruses that infect bacteria (bacteriophages) as major biomediators have a key impact on the life of bacterial pathogens and can both exacerbate and alleviate the severity of the disease. Strictly lytic phages, when used to combat bacterial pathogens, might solve the current antimicrobial resistance crisis, where chemical drugs fail in treatment. The results will provide understanding of what impact have lytic phages on resident siphophages and genomic pathogenicity islands, which often increase virulence and mediate horizontal gene transfer; what synergistic effects with antibiotics they have; what are the interactions with eukaryotic cells; and what consequent safety rules are to be introduced into the European pharmacopoeia for the rational use of bacteriophage applications and drug development. Principal investigator Roman Pantůček has a strong background in molecular biology of staphylococcal bacteriophages, horizontal gene transfer mediated by phages, phage therapy, and genomics and evolutionary biology of staphylococci.

MUNI_06_ Těšina: Translation in the context of human host-pathogen interactions

Proteins are produced by ribosome-catalyzed translation of mRNAs in all domains of life. Translation is also critical in the context of human host-pathogen interaction where the ribosome, as the central molecular machine for genetic information expression, is the subject to numerous regulatory and quality control events and pathological interventions. The strategies adopted by viruses to reprogram translation and co-translational quality control machinery to promote infection are poorly understood. Thus, there is an urgent need for further research in this area to develop effective strategies for combating viral infections. To address this knowledge gap, I propose a research aimed at elucidating the mechanistic details of viral reprogramming of translation and translational control processes using state-of-the-art biochemical and structural techniques. By elucidating the mechanistic details behind viral manipulation of these processes at a molecular level, we can lay the foundation for developing effective strategies against viral infections. The principal investigator Zdeněk Těšina and his team deals with mapping high-fitness-maintaining mutational trajectories of SARS-CoV-2 virus for future variants prediction.

Charles University

CUNI_01_Tachezy: Molecular epidemiology and immunology of viral infections.

The human organism is colonized by a large number of different viruses. However, very little data are available on the role of viral communities in immunosuppressed patients who undergo transplantation of solid organs or hematopoietic stem cells. In this project, we will characterize the virome of these patients who are at high risk of reactivation of many latent infections and at risk of newly acquired viral infections. The project will focus on anelloviruses and their possible role in the prediction of transplant rejection and graft versus host disease. An understanding the variability and interaction of the anelloviruses with the human immune system can help reveal their role in the immune response after transplantation. The principal investigator Ruth Tachezy and her team have experience in the characterization of clinical samples by next-generation sequencing and virome analysis.

CUNI_01_Tachezy: Immunoprofiling of virus-driven tumors as a tool for individualization of cancer immunotherapy

Oncogenic viruses are responsible for about 10% of malignancies worldwide. Progression of these tumours often results in patient death. The development of virus-induced tumours is usually accompanied by stimulation of anti-virus/anti-tumour immunity, but progression of tumours can finally be enabled by immune escape mechanisms. In this study, we will analyse the immune characteristics of human papillomavirus-induced tumours to reveal new therapeutic targets and biomarkers specific for different carcinogenesis pathways (integrated DNA-E6/E7 expression versus episomal DNA-E2/E4/E5 expression). Identification of potential mechanisms of resistance to treatment and predictive biomarkers can contribute to enhancing therapy efficacy. Principal investigator Ruth Tachezy is an expert in the analysis of tumours associated with human papillomaviruses including the detection of viral DNA integration and gene expression.

CUNI_02_Dřevínek: Molecular epidemiology and antimicrobial resistance in healthcare associated pathogens

A rapid spread of multidrug-resistant bacteria leads to difficult-to-treat infections and higher mortality. Thus, to monitor the emergence of new epidemic lineages of nosocomial pathogens and or novel determinants of antimicrobial resistance in healthcare settings is of key importance. Besides bacterial pathogens, viral nosocomial and viral congenital, such as cytomegalovirus or adenovirus, will be studied along with the SARS-CoV-2 surveillance. An understanding of the factors driving the emergence of antimicrobial resistance in these nosocomial pathogens, the dynamics of patient colonization and subsequent infection will permit healthcare professionals to tackle nosocomial pathogens. Comparative studies on the genomic and phenotypic level currently circulating epidemic and non-epidemic lineages enable us to identify markers associated with spread in healthcare environment and/or poor outcome of patients. Principal investigator Pavel Dřevínek is an expert in antimicrobial resistance with a focus on healthcare-associated pathogens and chronic respiratory infections in patients with cystic fibrosis.

CUNI_02_Dřevínek: The virome in human disease and its interaction with other exposome components

The aetiology of many human diseases is multifaceted, with a contribution from microbial exposures, in interaction with other exposome components. The virome comprises not only human and vertebrate viruses, but also a huge mass of bacteriophages whose direct or indirect interactions with the human host are now becoming explored. The project will help elucidate the structure and interactions of the human virome observed longitudinally in the course of several disease states, with special emphasis on diseases of childhood. The virome will be assessed in the context of other exposomic features, and its perturbations will be examined for causality or effects on the disease course. Principal investigator Pavel Dřevínek is an expert in virome, bacteriome and parasitome studies of primarily non-infectious human diseases.

CUNI_03_Šašková: Discovery of new antiviral defense mechanisms

Treatment of chronic hepatitis B with nucleot(s)ide analogues inhibits the formation of new infectious viral particles but does not eliminate stable covalently closed circular DNA (cccDNA) in hepatocytes. Pegylated interferon α (IFN-α) treatment can be considered as an alternative therapy. However, IFN-α monotherapy leads to functional cure in less than 8% of chronically infected people. We will investigate the HBV escape mechanism on the level of microenvironment of HBV-infected hepatocytes, the interplay between promyelocytic leukaemia protein nuclear bodies and HBV in the nucleus, and interactions of HBV core protein with proteins of the host. We propose to study mechanisms of HBV escape from intrinsic and innate immunity for discovery of new anti-viral therapeutic targets. Principal investigator Klára Grantz Šašková focuses on the characterization of viral and cellular protein interaction on structural, biochemical and functional level, with the goal to select new target candidates for therapeutic interventions.

CUNI_04_Huerfano: Study of interactions of cellular and viral components during the nuclear phase of polyomavirus infections

Infections by DNA viruses replicating in nucleus induce enormous changes in the nuclear structures; however, there are many gaps in the understating of virus interactions with the nuclear components. We propose that SMC5/6 complex and PML associated proteins either directly or indirectly modify viral chromatin and thus affect viral genome transcription and/or replication. The interaction of these proteins with virus minichromosomes and their functional significance will be studied on the human BK and murine polyomaviruses. The search for novel cellular partners of the multifunctional viral regulatory protein – LT antigen will be another part of the research. Our study will bring new insights into the comprehension of the nuclear phase of DNA viruses. Principal investigator Sandra Huerfano is an expert in polyomavirus biology with strong background in techniques of molecular biology..

CUNI_05_Pospíšek: Role of A-to-I editing in mutual interactions between the virus and the human host

RNA adenosine deaminase ADAR1 is responsible for most of the A-to-I RNA editing events in human cells. ADAR1 is inducible by virus infection; however, its function is poorly understood and, depending on the virus, can be manifested both as antiviral and/or proviral. We will study functional interactions of ADAR1 with RNA viruses using HCV, TOSV and SARS-CoV-2 models. We will employ our newly developed methods of translatome and A-to-I editome analyses to map and evaluate A-to-I edited sites in the viral and cellular RNAs and to decipher a role, which ADAR1 plays in synthesis of viral and host proteins in the course of viral infection. Principal investigator Martin Pospíšek’s expertise includes analysis of transcriptome, translatome and RNA editome, generation and characterization of ADAR1 knock-out cell lines and analysis of translation initiation in viruses.

CUNI_05_Pospíšek: Investigation into synthesis and translation of poxviral postreplicative mRNAs

Poxviruses are important human and animal pathogens and variola virus is ranked among the highest risk bioterrorism and biological warfare agents. Despite the poxviruses were in the forefront of the biological and medical research for many years, our understanding of their biology is incomplete, and only one antiviral drug has been approved for the treatment of smallpox. We discovered that postreplicative poxviral mRNAs are not capped at their 5′ ends. We will study synthesis and translation of these unusual poxviral mRNAs with the aim to answer the long-lasting question how poxviruses can overcome the host cell antiviral mechanisms and usurp the host protein synthesis apparatus for the production of viral proteins. Principal investigator Martin Pospíšek’s expertise includes a broad range of techniques for analysis of viral and cellular transcriptomes, translatomes and proteomes. His team discovered 5′ uncapped mRNAs in poxviruses.

CUNI_06_Volf: Biology of sand fly-borne viruses, with an emphasis on TOSV and RVFV

Although phleboviruses have a significant impact on human and animal health and cause serious economic problems, information about their biology and transmission is mostly missing. Predicted or confirmed vectors of most phleboviruses are bloodsucking sand flies (Diptera: Psychodidae). Thanks to a unique collection of sand fly colonies, we will study: i) the susceptibility of various sand fly species to phleboviruses, ii) the various routes of their transmission, iii) the development of different phlebovirus mutants in sand flies and iv) the virus interactions with another important sand fly-borne pathogens, particularly Leishmania. The obtained results would help to predict establishment of new disease foci, as well as to monitor and possibly control the phlebovirus transmission. Principle investigator Petr Volf is the leading expert in biology of phlebotomine sand flies and sand fly-borne pathogens, particularly Leishmania and phleboviruses.

CUNI_07_Hrabak: Emerging antibiotic resistance in Gram-negative bacteria – evolution and intervention

The increasing incidence of antibiotic resistance represents a major clinical challenge and public health concern worldwide. Therefore, the understanding of evolution of resistant bacteria on molecular level is desperately needed. Whole-genome sequencing of bacterial genomes can provide a deep insight into the evolution, routes of the spread, identification of reservoirs, and high-risk population groups. The data can be used to propose a strategy for alternative combating antibiotic resistance. Describing common mobile genetic elements spreading the antibiotic resistance genes and studying their complex structure, plasticity, and plasmid-gene-host interactions will also help us to set up preventive measures for the spread of antibiotic resistance. Principal investigator Jaroslav Hrabák works in molecular epidemiology of multidrug-resistant Gram-negative bacteria since 2005. He is also a member of several international boards focused on antibiotic resistance (e.g., ECDC).

CUNI_07_Hrabak: Development of novel assays for rapid and accurate identification of multidrug resistant bacteria

Novel techniques used in microbiological diagnostics, especially mass spectrometry-based and molecular genetic methods revolutionized diagnostics of infectious diseases in the last two decades. Further development of functional assays and proteomic analysis of bacteria, especially their antibiotic resistance, can significantly help in diagnostics of life-threatening diseases to improve patient’s outcome. The use of mass spectrometry in microbiological diagnostics is usually limited to taxonomical identification of microbes and detection of resistance mechanisms. The technology, however, can be used for deep analysis, including precise detection of resistance mechanisms and host/pathogen interaction. During the project, new methods for LC/MS and MALDI-TOF MS will be developed. The methods will further decrease turnaround time required for diagnostics and will positively influence patients’ outcome especially in life-threatening infections. Principal investigator Jaroslav Hrabák is focused on development of rapid methods for microbiological diagnostics, especially using mass spectrometry. He is also an inventor of several patents in the topic.

CUNI_09_Zahradnik: Mapping high-fitness-maintaining mutational trajectories of SARS-CoV-2 virus for future variants prediction

Proteins are produced by ribosome-catalyzed translation of mRNAs in all domains of life. Translation is also critical in the context of human host-pathogen interaction where the ribosome, as the central molecular machine for genetic information expression, is the subject to numerous regulatory and quality control events and pathological interventions. The strategies adopted by viruses to reprogram translation and co-translational quality control machinery to promote infection are poorly understood. Thus, there is an urgent need for further research in this area to develop effective strategies for combating viral infections. To address this knowledge gap, I propose a research aimed at elucidating the mechanistic details of viral reprogramming of translation and translational control processes using state-of-the-art biochemical and structural techniques. By elucidating the mechanistic details behind viral manipulation of these processes at a molecular level, we can lay the foundation for developing effective strategies against viral infections. Theprincipal investigator Zdeněk Těšina and his team deals with mapping high-fitness-maintaining mutational trajectories of SARS-CoV-2 virus for future variants prediction.

University of Chemistry and Technology Prague

UCT_01_Rumlová: Involvement of DEAH-box containing RNA helicase in the replication cycle of betaretroviruses

To replicate successfully, viruses are largely dependent on the host-cell machinery. One class of host-cell proteins often utilized by both RNA and DNA viruses are helicases. We identified an RNA helicase (DHX15) that plays a critical role in the replication of betaretroviruses. The ultimate goal is to understand the role of DHX15 in the betaretroviral replication cycle. The results obtained within the project will explain the mechanism of the functional regulation of both a host-cell RNA helicase DHX15 by retroviral components, and retroviral reverse transcriptase by cellular helicase DHX15. The insight into the structural-functional mechanism of helicase recruitment by viral pathogens could also help to understand the role of RNA helicases in protozoal, bacterial and fungal infections. Principal investigator Michaela Rumlová´s expertise comprises retroviruses, mainly HIV-1 and betaretrovirus, their assembly and interactions with host-cell factors.

UCT_01_Rumlová: Flaviviral C protein

Tick-borne encephalitis virus (TBEV) infection causes serious neurological diseases in humans for which there is no effective treatment. TBEV capsid (C) protein, which is responsible for viral RNA packaging and virus assembly, appears a very promising target for antivirals of a new generation. We will characterize the specificity of TBEV C-mediated RNA packaging and search for the putative specific RNA packaging signal. Using our recently solved NMR structure of the C-protein, we will predict amino acids responsible for C-protein dimerization and RNA binding. Their importance will be verified by analysing RNA content in viruses carrying these mutated amino acids. The data elucidating the mechanism of RNA packaging during TBEV infection may contribute to the development of targeted therapy. Principal investigator Michaela Rumlová has a strong background in using of combination of virologic, molecular-biology, gene-engineering, structural and biochemical methods.

UCT_01_Rumlová: Multidrug-resistant bacteria

Antibiotic resistance has become one of the main problems in today’s healthcare system. Common mechanisms of bacterial resistance are antibiotic efflux; altered membrane permeability reduction of the antibiotic’s uptake; antibiotic inactivation by enzymatic modification, or mutation of the target site. We plan to search for inhibitors of transmembrane efflux pumps, which are the major cause of bacterial resistance. By using qPCR, we will determine the mechanisms of antibiotic resistance in clinical isolates. The search for new efflux pumps inhibitors will be based on, but not limited to, synthetic derivatives of known inhibitors and natural substances isolated from medicinal plants. Our aim is to verify the capability of the identified inhibitors to restore the sensitivity to antibiotics in drug-resistant bacterial strains. Principal investigator Michaela Rumlová is an expert in microbiology focused on the retroviral life cycle, antimicrobial therapy including phototherapy and biofilm inhibition and will be advisor for this project.

Institute of Organic Chemistry and Biochemistry of the CAS

IOCB_01_Pichová: Interaction of HBV and cellular proteins critical for virus replication

Hepatitis B virus (HBV) can induce acute and chronic infection, which potentially leads to cirrhosis and hepatocellular carcinoma (HCC) development. HBV thus represents an important environmental carcinogen for humans. HBV persists in the form of covalently closed circular (ccc) DNA, which is responsible for the establishment of a chronic infection. Knowledge of HBV replication regulation, cccDNA formation and degradation, HCC development is limited. A combination of molecular virology, biochemistry, and structural biology will be used to enhance understanding of the viral protein HBx role in regulation of transcription, and development of chronic hepatitis B. We propose to identify host proteins involved in these processes and validate their function. The results may lead to identification of novel targets for drug intervention. Principal investigator Iva Pichová’s expertise includes functional and structural analysis of pathogenic proteins, analysis of HBV life cycle using molecular biology and virology methods.

IOCB_01_Pichová: Unravelling regulation of purine metabolism for anti-mycobacterial inhibitor development

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is one of the top ten causes of death. An increasing number of extensively drug-resistant Mtb strains and development of persistent infection complicates treatment. Purine nucleotides represent promising targets for development of novel inhibitor types. Transcription regulation of the purine metabolism will be investigated under normal growth conditions, hypoxia and nutrient starvation, possible formation of a complex formed from de novo purine biosynthetic enzymes, which can facilitate metabolic flux in a model Mycobacterium smegmatis spp. Essential enzymes from purine biosynthesis will be identified and validated to test the IOCB compound library, in order to get a lead structure for inhibitor development. The results may lead to the development of novel types of inhibitors. Principal investigator Iva Pichová has a strong background in biochemical characterization of metabolic enzymes from Mycobacterium tuberculosis and Mycobacterium smegmatis. Her expertise includes also molecular biology and microbiology methods.

IOCB_02_Bouřa: Structural and functional characterization of coronaviral RNA processing enzymes

Besides the current COVID-19 pandemic situation, coronaviruses are also estimated to be responsible for about 15% of cases of the common cold. The most lethal coronavirus species demonstrated a mortality rate exceeding 30%, representing a permanent threat of deadly pandemics. The assembly, function and regulation of coronaviral multi-protein complexes with key enzymatic functions remain incompletely understood. We propose to structurally and functionally characterize key coronaviral complexes responsible for processing of the viral RNA. The results obtained within this project will provide insight into the mechanism of action of inhibitors against the RNA processing enzymes, especially the methyltransferases (MTases) and the RNA-dependent ENA-polymerase (RdRp). Our results will provide a structural template for further development of inhibitors using the structure-based lead-to-drug candidate optimization method. Principal investigator Evžen Bouřa is an expert in structure-guided inhibitor design against coronaviral enzymes, their biochemistry and in determining their structures by X-ray crystallography.

IOCB_02_Bouřa: RNA replication of flaviviruses

Flaviviruses are the most prevalent arthropod-borne viruses worldwide, and nearly half of the 70 Flavivirus members identified are human pathogens. Many are mosquito-borne viruses, such as Yellow Fever virus, West Nile fever virus, Dengue virus or Zika virus. Despite the huge clinical impact of flaviviruses, there is no specific human antiviral therapy available to treat infection with any of the flaviviruses. We will focus on the most important and most conserved protein involved in RNA replication – the NS5 protein that harbours both the RdRp activity and the MTase activity. We aim to solve structures of multiple flaviviral NS5 proteins in complex with small molecules to assess the possibility of obtaining a universal inhibitor against all flaviviruses. Principal investigator Evžen Bouřa’s expertise includes enzymatic and biochemical characterization of NS5s. He also solved several X-ray structures of flaviviral enzymes both apo and in a complex with a small molecule inhibitor.

IOCB_05_Weber: Identification and characterization of Hepatitis B core-host interaction networks

Chronic hepatitis is a life-long liver disease caused by infection of Hepatitis B virus (HBV). Despite the existence of preventive vaccine, more than 250 million people worldwide suffer from chronic hepatitis that is associated with severe liver conditions ranging from fibrosis and cirrhosis to hepatocellular carcinoma. We propose to characterize HBV core protein (HBc) interactome network and identify key cellular proteins and pathways that are essential for viral replication and persistence. Our goal is to provide detailed knowledge concerning: i) key regulatory events in HBV replication; ii) important HBc interactors and cellular pathways involved in nucleocapsid formation and intracellular trafficking, pgRNA encapsidation and reverse transcription, cccDNA minichromosome formation and maintenance. The results may lead to development of new anti-HBV therapies. Principal investigator Jan Weber has strong background in molecular biology and virology, expertise from academic and corporate settings and is interested in characterization of new HBV-host cell interaction.

IOCB_05_Weber: Identification and targeting coronavirus attachment and entry

The emergence of third coronavirus causing severe acute respiratory syndrome in less than two decades transformed emerging coronaviruses in a new public health concern. One of the best measures is to block the virus before it establishes new infection in the cells, thus inhibition of viral attachment and entry are clear choices. We plan to characterize and compare proteome of uninfected cells and cells infected with different coronaviruses, including SARS-CoV-2, early in the infection. Identified proteins will be further characterized using overexpression, siRNA-mediated knockdown and confocal microscopy to elucidate their effect on coronavirus entry and replication. The better understanding of coronavirus attachment, entry and characterization of druggable virus-host interactions may lead to better preparedness in the case of future coronavirus epidemics. Principal investigator Jan Weber expanded his interest to coronavirus field with the focus on the elucidating the different mechanisms of SARS-CoV-2 entry to the cells.

Institute of Molecular Genetics of the CAS

IMG_01_Hejnar: Interactions of avian leukosis virus with cellular receptors and creating resistance to viruses by CRISPR/Cas9 editing of receptor genes

ALV is a virus complex diversified through the virus-host coevolution (virus-host arms race) into several subgroups, each of which recognizes different cell surface receptor. This unique example of coevolution provides a chance to study processes of virus adaptation, broadening the host range, and heterotransmission (in general, all zoonotic viruses arose in a process of heterotransmission). This knowledge could help in artificial creation of resistant animals using the CRISPR/Cas9 technology of gene editing in chicken, the natural host of ALV. Receptor alleles bearing simple substitutions of critical amino-acids at the virus binding domains (e.g., the recently prepared resistance to ALV-J subgroup) are a good material for studying the escape mutations of the virus adapted to the new versions of receptors. Principal investigator Jiří Hejnar is respected expert in retroviral receptors and uses a unique technology of gene editing in chicken. He already finished four KO/gene editing projects in domestic chicken.

IMG_01_Hejnar: multi-level regulation of Syncytin-induced cell-to-cell fusion

Cell-to-cell fusion during placenta development is critical for the proper formation of its outer layer called syncytiotrophoblast. Fusion of mononuclear cytotrophoblast and formation of the multinuclear syncytium is largely dependent on the human proteins called Syncytins. Syncytin-1 and Syncytin-2, the retroviral envelope glycoproteins of two distinct human endogenous retroviruses. Using our original heterologous ectopic system of retrovirus-receptor interaction and dual luciferase cell fusion technique, we will analyse the contribution of syncytin-1/2 and receptors hASCT1/2 for trophoblast fusion and identify amino-acids critical for syncytin-receptor binding. CryoEM structural analysis will be performed to support the functional analyses. In parallel, epigenetic repression of syncytins in non-placental tissues will be studied. This project might result in identification of mutations behind the human idiopathic infertility. For this project, the principal investigator Jiri Hejnar´s expertise in molecular modelling of virus-receptor interaction is critical. Recently published experimental systems will be widely employed in this RO.

IMG_01_Hejnar: Evolutionary approaches to virus-host interactions

Antiviral restriction factors and virus dependence factors are the battlefield of the long-term virus-host coevolution and bidirectional evolutionary approach is necessary to characterize viral adaptations to new host species during the process of heterotransmission and zoonotic events as well as the changes in relevant host genes. These will include the seeking for antiviral activities of genes already characterized to be restrictive towards HIV-1 in human. This RO will focus on the effects of chicken tetherin against avian viruses (avian leukosis virus, Marek disease virus, avian influenza, avian coronaviruses, etc). Chicken tetherin will be the primary target to genetic knock out or gene editing in vivo using the CRISPR/Cas9 with the final goal to demonstrate improved resilience to chicken diseases. Principal investigator Jiří Hejnar´s expertise includes retroviruses and antiviral restriction factors, epigenetic silencing of retroviruses by epigenetic mechanisms, and genetic basis of antivirus resilience.

Institute of Microbiology of the CAS

IMIC_02_Janata: Antibiotics: Molecular principles of the resistance, action and (bio)synthesis

According to the recent WHO fact sheet, antibiotic resistance is now one of the greatest threats to global health, food security and development. This project will investigate the poorly characterized mechanism of resistance to ribosome-targeting antibiotics mediated by ABCF proteins conferring resistance to several groups of clinically important antibacterial agents, e.g., macrolides and lincosamides. Project activities will advance knowledge of the primary role of antibiotics and the corresponding resistance determinants in the natural environment, and have the potential to explain the evolutionary mechanisms leading from cell signalling and regulatory molecules to antimicrobial resistance. A better understanding of the principles of antibiotic-resistance (ARE) ABCF function and resistance evolution could provide strategies for developing new compounds and even radically innovative therapeutic approaches to overcome this type of resistance. Principal investigator Jiří Janata is an expert in molecular mechanisms of antibiotic action and resistance. His team significantly contributed to description of molecular mechanism of ARE ABCF proteins.

IMIC_03_Krásný: The bacterial transcription machinery and its resistance to antibiotics

The central enzyme of gene expression is RNA polymerase (RNAP). Bacteria contain one type of this enzyme, which can be inhibited by rifamycins. Resistance to rifamycins arises due to mutations in RNAP or is mediated by other proteins. We recently discovered one such protein, HelD, which binds to RNAP and changes its conformation. This results in dissociation of RNAP from nucleic acids as well as in release of rifampicin, if bound. The proposed research will characterize the mechanistic functioning of HelD and describe regulation of its expression. This research will reveal new information about how bacteria cope with antibiotics and may lead to new compounds targeting the transcription machinery. Principal investigator Libor Krásný is an expert in bacterial physiology, antibiotic resistance, gene expression at the transcription level and its regulation.

IMIC_03_Krásný: Antibacterial toxins utilized in interspecies warfare

Bacteria are surrounded by other microorganisms in their environment. To survive they have to constantly battle for resources. Bacillus subtilis is a model bacterium of industrial importance. It is equipped with an arsenal of weapons, toxins, to keep at bay or eliminate enemies. These toxins are of potential medicinal interest. We recently identified the presence of so far unknown toxins in this bacterium. The aims are to (i) identify the gene(s) that are required for toxin production; (ii) purify the toxin(s); and (iii) define the mechanism(s) of action of this toxin(s) against various bacterial species. In summary, this research objective will identify novel bacteria-encoded toxins suitable for future use as antibacterial agents. Principal investigator Libor Krásný is an expert in bacterial physiology, antibiotic resistance, gene expression at the transcription level and its regulation.

Biology Centre CAS

BC_01_Růžek: Human antibodies and antivirals to prevent and/or treat (re)emerging viral infections

Antibodies can often play a dual role during viral infection (protective versus disease enhancing). Here, we will examine antibody-mediated enhancement of the infection by tick-borne encephalitis virus mediated by single monoclonal antibodies and polyclonal sera from a large cohort of clinical samples. By characterizing the human antibodies induced by the virus during natural infection at the molecular level, we aim to gain novel insights into the disease pathogenesis of flavivirus encephalitis, with important clinical implications. Principal investigator Daniel Růžek is an expert in the research of pathogenesis of viral infections, with a particular focus on arboviral and other emerging viral diseases.

BC_02_Růžek: Advanced studies on the pathogenesis of viral neuro-infections towards novel therapeutic strategies

Viruses belong to the most commonly identified cause of meningitis and encephalitis in humans, leading to substantial long-term morbidity. Nearly all virus groups contain members that can infect human nervous tissues. Despite the medical importance of these diseases, the biology of the neurotropic viruses and the mechanisms of pathogenesis of viral infection in the central nervous system are poorly understood. Our study will be focused on several critical aspects of the biology of selected neurotropic viruses, such as neurotropic flaviviruses and/or other arboviruses, and the pathogenesis of infections caused by these agents. In particular, we aim to identify important determinants of virulence of neurotropic viruses and reveal mechanisms of viral neuroinvasion into the CNS. Principal investigator Daniel Růžek is an expert in the research of pathogenesis of viral infections, with a special focus on arboviral and other emerging viral diseases.

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