Reprogramming Emesvirus zinderi

The genomes of all of today’s organisms are encoded in deoxyribonucleic acid (DNA). However, at the early stages of the emergence of life, ribonucleic acids (RNA) might have played a comparable role. Potential descendants of these ancient RNAs can be found in the form of RNA viruses, especially small single stranded RNA viruses. A well-studied example for these viruses is Emesvirus zinderi, more commonly known as bacteriophage MS2. In this study, the genome of MS2 served as a starting point for the design of an RNA based replisome, encoding for genetic information beyond the replicase gene. Furthermore, harnessing the infection mechanism of MS2, based on the MS2 maturation protein, it was tried in parallel to establish a new RNA delivery system. While these approaches are of interest for synthetic biology and biochemical technologies, they also might shed light on the role of RNA at the beginnings of life. Using classical biochemical and microbiological methods in combination with modern synthetic biology, a variety of tools could be established and repurposed for this endeavour. Deploying an in vitro translational system, used in synthetic biology, led to the identification of amino acids in the maturation protein crucial for viral infection, while classical methods for protein purification and detection of this infection enabled the in vitro reconstitution of minimal infectious units of MS2. Using the same translational system, sequential reduction/simplification of it led to the identification of the minimal complex for RNA replication and regulating co-factors. In addition, the characterisation of the replication machinery led to the discovery of novel RNA replicators, providing a platform for the design of RNA replisomes. These replisomes, similar in function to plasmid-based DNA replisomes, could be shown to be capable of in vitro replication and translation. Finally, during this thesis additional methods were established that exhibited potential for future studies of RNA replisomes, but also provide a strategy for replisome generation based on alternative viral systems. In conclusion, the goal of this study, the design of an RNA based replisome was achieved and this discovery opens new possible applications in the research fields of synthetic biology and the emergence of life.Die Genome aller heutigen Organismen sind in Desoxyribonukleinsäure (DNA) kodiert. In den frühen Stadien der Entstehung des Lebens könnten jedoch Ribonukleinsäuren (RNA) eine vergleichbare Rolle gespielt haben. Mögliche Nachfolger dieser ursprünglichen RNAs finden sich in Form von RNA-Viren, insbesondere kleinen einzelsträngigen RNA-Viren. Ein gut untersuchtes Beispiel für diese Viren ist Emesvirus zinderi, besser bekannt als Bakteriophage MS2. In dieser Studie diente das Genom von MS2 als Ausgangspunkt für den Entwurf eines RNA-basierten Replisoms, das für genetische Informationen über das Replikase-Gen hinaus kodiert. Ausserdem wurde unter Ausnutzung des Infektionsmechanismus von MS2, der auf dem MS2-Reifungsprotein basiert, parallel dazu versucht ein neues RNA-Transportsystem zu entwickeln. Diese Ansätze sind nicht nur für die synthetische Biologie und biochemische Technologien von Interesse, sondern könnten auch Aufschluss über die Rolle von RNA am Anfang des Lebens geben. Durch den Einsatz klassischer biochemischer und mikrobiologischer Methoden in Kombination mit der modernen synthetischen Biologie konnte eine Vielzahl von Werkzeugen für dieses Vorhaben etabliert und umgenutzt werden. Der Einsatz eines in der synthetischen Biologie verwendeten In-vitro-Translationssystems führte zur Identifizierung von Aminosäuren im Reifungsprotein, die für die virale Infektion entscheidend sind, während klassische Methoden zur Proteinaufreinigung und zum Nachweis dieser Infektion die In-vitro-Rekonstitution von minimalen infektiösen Einheiten von MS2 ermöglichten. Unter Verwendung desselben Translationssystems führte die sequenzielle Reduktion/Vereinfachung desselben zur Identifizierung der essenziellen Proteinfaktoren für die RNA-Replikation und zusätzlicher regulierender Co-Faktoren. Darüber hinaus führte die Charakterisierung der Replikationsmaschinerie zur Entdeckung neuartiger RNA-Replikatoren, die eine Plattform für den Entwurf von selbstreplizierenden RNA-Vektoren bildeten. Diese Replisomen, die in ihrer Funktion den plasmidbasierten DNA-Replisomen ähneln, waren nachweislich in der Lage in vitro zu replizieren und translatiert zu werden. Schließlich wurden im Rahmen dieser Arbeit zusätzliche Methoden etabliert, die Potenzial für künftige Untersuchungen von RNA Replisomen aufweisen, aber auch eine Strategie für die Erzeugung von Replisomen auf Basis anderer viraler Systeme bieten. Zusammenfassend lässt sich sagen, dass das Ziel dieser Studie, die Entwicklung eines RNAbasierten Replisoms, erreicht wurde und diese Entdeckung neue Anwendungsmöglichkeiten in den Forschungsbereichen der synthetischen Biologie und der Entstehung des Lebens eröffnet

Relation between genomic and capsid structures in RNA viruses.

We described a new computer program for calculation of RNA secondary structure. Calculation of 20 viral RNAs with this program showed that genomes of the icosahedral capsid viruses had higher folding probabilities than those of the helical capsid viruses. As this explains virus assembly quite well, the information of capsid structure must be imprinted not only in the capsid protein structures but also in the base sequence of the whole genome. We compared folding probability of the original sequence with that of the random sequence in which base composition was the same as the original. All the actual genomes of RNA viruses were more folded than the corresponding random sequences, even though most transcripts of chromosomal genes tended to be less folded. The data can be related to encapsidation of viral genomes. It was thus suggested that there exists a relation between actual sequences and random sequences with the same base ratios, and that the base ratio itself has some evolutional meaning

Strain variation of respiratory syncytial virus in Qatar and its relationship to B-cell epitopes from the attachment (G) protein of RSV (B) strain

Respiratory Syncytial Virus (RSV) is the major cause of acute lower tract infection in early childhood. Annual epidemics occur which are well documented in developed countries during winter months, placing considerable pressure on the provision of health care. Little is known about the epidemiology of RSV infection in the Middle East and other desert climate regions of the world. The aim of this project was to study the specificity of the immune response to RSV B in children, and to relate this to the infecting RSV strain, with particular emphasis on antibody response to RSV attachment (G) protein. RSV is an important cause of hospital admission in children (54%) during winter months in Qatar. 63% of these infections are due to RSV A. A case study involving analysis of RSV strains from hospitalized children was carried out in Qatar over 2 winter seasons (1999-2000 and 2000-2(01). RSV incidence was found to correlate with high relative humidity and low temperature. A comparison of enzyme immuno assay (EIA) and polymerase chain reaction (peR) for the detection of RSV in clinical samples demonstrated that peR was more sensitive than EIA. All positive RSV samples obtained during the study period were classified as belonging to RSV A and B subtypes using Multiplex peR. In this project, primer sets were designed and optimized to amplify the whole of the G gene of RSV A and B strains. Derived sequence analysis allowed deduction of the molecular epidemiology of RSV G gene for RSV A and B strains in Qatar and elsewhere. Sequence data of the G gene from Qatar RSV A&B isolates confirmed the variability of this protein and showed that variability occurs among group B RSV viruses isolated in Qatar (0.8%), although to a lesser extent than among the group A viruses (5%) from same location. However, the group B viruses isolated in Qatar were highly variable in G gene sequences compared to the prototype strain RSVB N2 (13%) and to strain world-wide (10%) at the nucleotide level. In order to produce an epitope map of the RSVB G protein, synthetic peptides representing linear B-cell epitopes of a representative Qatar RSV B isolate (B/Q/28/00) were used. The reactivity of human sera with the synthetic peptides was studied using sera from young children from whom RSV had been isolated. The sera from these children had variable peptide binding responses against different regions of the G protein of RSV B and the responses were focused on the conserved region. The results indicated that peptide 14 (a. a 150-165) of the G protein, in the conserved region of the protein, is a major antigenic site. This peptide sequence was recognized by a majority of the tested sera (93% sera). To examine the relationship between neutralization antibody titre and reactivity to the synthetic peptides in children's sera, a modified micro-neutralization method was used. There was no significant correlation between the peptide binding activity in the sera and the neutralization titre of these sera. Sera from children infected with RSV A bound to peptides from the G protein of RSV B strain. However, children infected with RSV B had greater neutralizing antibody titre to RSV B strain than to the two RSV A strains. There was no difference in the neutralization antibody titre in sera to RSV A when assessed with prototype strains isolated many years ago (RSV A2), and with the recently-isolated strain AlQ/IO/OO