Day 1 :
University of St Andrews, UK
Keynote: Protein biogenesis in the Picornaviridae : Virus-encoded proteinases, ribosome ‘skipping’, alternative initiation and programmed ribosomal frame-shifting
Time : 09:00-09:40
Martin D Ryan has cloned and sequenced the genome of Enterovirus Type 70 as a part of his Ph.D. research work. He then went on to work on FMDV polyprotein processing at the Pirbright Institute. After moving to University of St. Andrews in 1994 he continued this work, focusing on the mechanism of FMDV 2A-mediated ‘cleavage’ – showing it was not a proteolytic element, but mediated a novel form of translational recoding – ‘ribosome skipping’. Other lines of research led to the development of 2A as a protein co-expression system – now used very widely in biotechnology and biomedicine. In 2017, he was awarded the Unilever Colworth Prize for his work on 2A. More recently, his laboratory has been working on FMDV RNA replication using a replicon system encoding fluorescent proteins such that FMDV RNA replication can be monitored by live-cell imaging.
The genome structure of viruses within the Picornavirus family is similar to cellular mRNAs: +v sense RNA genomes comprising a long 5’ non-coding region (NCR), a single open reading frame (ORF; ~2,300 aa), a short 3’-NCR with a poly(A) tail, although the 5’ cap structure is quite different. When introduced into the cytoplasm, the virus RNA can function directly as a messenger RNA. The challenge faced by these viruses is, however, the need to generate a multiplicity of different proteins (capsid, RNA replication proteins) from this single ORF. All picornaviruses encode a proteinase (3Cpro) which ‘processes’ the polyprotein into mature products by a combination of a single, co-translational, intramolecular cleavage (in cis) and multiple post-translational intermolecular cleavages (in trans). Some picornaviruses have acquired a second proteinase (aphthoviruses – Lpro; enteroviruses – 2Apro) which perform a single co-translational cleavage in cis, but then go onto cleave cellular proteins involved in the cap-dependent translation of host-cell mRNAs and proteins involved in interferon production in response to infection. The 2A region of aphtho- and many other Picornavirus genera (unlike enteroviruses not a proteinase) may either be a short oligopeptide sequence, or, a larger protein that mediates a translational ‘recoding event termed ‘ribosome skipping’ at the C-terminus of 2A such that translation stops (releasing the nascent protein) then recommences translation of the down-stream replication protein sequences: an apparent ‘cleavage’, but in actuality a discontinuity in the polypeptide backbone. It is now known that this form of control over protein biogenesis is used by other types of RNA virus and some cellular genes. Uniquely within the picornaviruses, Theiler's murine encephalomyelitis virus (TMEV; genus Cardiovirus) encodes an overlapping, alternative ORF within the leader, or L protein, alternative initiation producing the L* protein which plays an important role in the establishment of persistent CNS infections by TMEV. Recently it has been shown that some picornaviruses also use programmed ribosomal frame-shifting shortly after the ribosome skipping event to (translationally) down-regulate the production of replication proteins at later stages of the infectious cycle. Furthermore, this frame-shifting does not rely solely on an RNA secondary structure but is directed by protein 2A. In conclusion, the picornaviruses have evolved a range of different mechanisms to control their protein biogenesis: not at the level of RNA transcription, but by co- and post-translational events alone.
- Ryan MD, Flint M (1997) Virus-encoded proteinases of the picornavirus super-group. J. Gen. Virol. 78: 699-723.
- Donnelly MLL, Luke G, Mehrotra A, Li X, Hughes LE, Gani D, Ryan MD (2001) Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J. Gen. Virol. 82:1013-1025.
- Sharma P, Yan F, Doronina V, Escuin-Ordinas H, Ryan MD, Brown J (2012) 2A peptides provide distinct solutions to driving stop-carry on translational recoding. Nuc. Acids Res. 40:1-9.
- Chen HH, Kong WP, Zhang L, Ward PL, Roos RP (1995) A picornaviral protein synthesized out of frame with the polyprotein plays a key role in a virus-induced immune-mediated demyelinating disease. Nat. Med. 1:927-931.
- Napthine S, Ling R, Finch LK, Jones JD, Bell S, Brierley I, Firth AE (2017) Protein-directed ribosomal frameshifting temporally regulates gene expression. Nat. Commun. 8:15582.
University Hospital of Zürich, Switzerland
Time : 09:40-10:20
Maurizio Provenzano graduated as a MD from La Sapienza University of Rome and obtained his specialization in Medical Oncology at the University of Milan and his Ph.D. in Virology at the University of Padua. He is a tenured investigator in the position of the Head of Research at the Department of Urology, University Hospital of Zurich and Lecturer at the Faculty of Medicine, University of Zurich. Since 2007, he has been a member of the Cancer Network of Zurich. He has authored more than 70 peer-reviewed publications including, reviews, book chapters, and conference papers, many of which are in highly qualified scientific periodicals and has been serving as an Editorial Board Member of reputed journals. He has been the recipient of professional awards and prizes for the contribution to the field of Viral Oncology and Onco-Immunology.
In recent years the scientific literature in the field of the prostate carcinoma (PCa) pointed out on the genetic heterogeneity occurring in this tumour, while little attention was given to the causes that threaten the genomic integrity of the prostate. Together with chemical and physical agents, biological agents such as viruses with oncogenic potential, which interfere with the cell cycle, are responsible for gene alterations and might be included in the putative genomic evolution of PCa. Polyomavirus BK (BKPyV) encodes two viral oncogenes, the large T antigen (LTag) and small t antigen (tag). Transformation of animal and human cells by BKPyV is operated by these two viral oncoproteins. LTag binds and abolishes the functions of the tumour suppressor p53 and pRB family proteins, whereas tag interacts with the phosphatase PP2A, which activates the Wnt pathway. Moreover, tag activates the phosphatidylinositol 3-kinase, an enzyme involved in pathways crucial for cell proliferation and transformation. LTag is also clastogenic and mutagenic. These activities are able to hit the cellular genome, which accumulates many gene mutations/chromosome aberrations. Considering that BKPyV infection is ubiquitous in the general population, it is difficult to assess a specific role of this virus in cellular transformation. BKPyV is kidney-tropic and remains latent in many human organs/tissues, mainly the urogenital tract. The lifelong period of BKPyV infection, together with the fact that about 95% of PCa are slow-growing organ-confined indolent tumors, could be responsible of those gene/chromosomal damages, which initiate the development of this malignancy. Footprints of this small DNA tumour virus has been revealed at higher prevalence in early stages PCa than in healthy control tissues, thus providing an indication for an increased risk of PCa development with the presence of BKPyV infection. Loss-of-function mutations in p53 gene at very early stages of PCa are rare. Therefore, the sequestration of wild-type p53 exerted by BKPyV LTag oncoprotein at initial phases is a hallmark for BKPyV involvement and may ensure the genetic heterogeneity of PCa.
Helmholtz Centre for Infection Research, Germany
Time : 11:00-11:40
Joachim Wink has completed his PhD in 1985 from Frankfurt University. He then went to the pharmaceutical industry and started his career at the Hoechst AG, where he was responsible for the strain collection and specialized in the cultivation and taxonomic characterization of Actinobacteria and Myxobacteria. During the years, he was responsible for the strain library within the pharmaceutical research and a number of screening projects with Hoechst Marion Russel, Aventis and Sanofi. In the year 2005, he did his habilitation at the Carolo Wilhelma University of Braunschweig and in 2012 he went to the Helmholtz Centre for Infection Research in Braunschweig, where he founded the working group of the strain collection with its focus on Myxobacteria. Here, he is now working on the isolation and taxonomic characterization of Myxobacteria and Actinobacteria, as well as, the analysis of their secondary metabolites. He has published more than 50 papers on secondary metabolites and the taxonomy of the producing microorganisms in reputed journals, a number of reviews, as well as, book chapters and more than 35 patents. He is the Editorial Board Member of a number of international journals.
Members of the class Actinobacteria plays an important role for humans, on one hand, we have producer of antibiotics, which mainly belong to the so called Sporoactinomycetes like Streptomyces or Amycolatopsis and on the other hand the pathogenic ones like many species of the Mycobacteria and Nocardia. Actinobacteria are typical soil bacteria, but also inhabit many other habitats, so that they could be isolated from different materials. Even their morphology has been used for the characterization for many years; today a number of molecular biological methods are also available for their taxonomic classification. In the 90th of the last century the polyphasic approach for the classification of bacteria was introduced and is also today the way to identify a species of the Actinobacteria. This approach combines the methods of the morphology and physiology, together with the chemotaxonomic methods and the methods of the molecular biological characterization. The wide variety of the morphology of the different Actinobacteria, we find coccoid and rod shaped single cell forming species like in the Micrococcales, as well as, organisms that form mycelia and differentiate with spore chains or sporangia, made it very important for the identification and classification of this bacteria to have a number of methods available. Especially after finding the different morphological features also in different genera and families the chemotaxonomy became very important in the 60th of the last century. The molecular biological characterization methods opened a number of additional tools, but there are many examples that they also cannot stand alone. Aim of the workshop is after a short introduction of the bacterial class to show the different aspects of the polyphasic taxonomy basing on the work of Vandamme et al. with the class Actinobacteria as an example. It will be discussed if the molecular biological characterization will replace morphology and chemotaxonomy.
- Landwehr W, Wolf C, Wink J (2016) Actinobacteria and Myxobacteria – Two of the Most Important Bacterial Resources for Novel Antibiotics. in M. Stadler and P. Dersch (eds.) How to Overcome the Antibiotic Crisis. Springer. Volume 398 of the series Current Topics in Microbiology and Immunology. 273-302.
- Wink J, Schumann P, Klenk HP, Zaburannyi N, Westermann M, Martin K, Glaeser SP, Kämpfer P, Atasayar E (2016) Streptomyces “caelicus" an antibiotic producing species of the genus Streptomyces and Streptomyces canchipurensis Li et al. 2014 are later heterotypic synonym of Streptomyces muensis Ningthoujam et al. 2013. Int J Syst Evol Microbiol. doi: 10.1099/ijsem.0.001612. [Epub ahead of print].
- Mohammadipanah, J Wink (2016) Actinobacteria from Arid and Desert Habitats: Diversity and Biological Activity. Front Microbiol 2015 6:1541. doi: 10.3389/fmicb.2015.01541.
- Wink J, Schumann P, Spöer C, Eisenbarth K, Glaeser SP, Martin K, Kämpfer P (2014) Emended descriptions of Actinoplanes friuliensis and description of Actinoplanes nipponensis sp. nov., two Antibiotic Producing Species of the Genus Actinoplanes. IJSEM 64:599-606.
- Müller R, Wink J (2014) Future potential for anti-infectives from bacteria – How to exploit biodiversity and genomic potential. IJMM 304:3-13.5.