Nucleocytoplasmic large DNA viruses (NCLDVs) have genome sizes that range from around 100 kilobases (kb) to up to 2.5 megabases, and virion sizes that can reach up to 1.5 μm. Their large size in both of these contexts is atypical and defies the traditional view that viruses are streamlined, "filterable infectious agents". NCLDVs include many diverse groups, including Poxviruses, Asfarviruses, Iridoviruses, Mimiviruses, and Marseilleviruses. Poxviruses are perhaps the most well-studied; these viruses have 135-360 kbp genomes with about half of the genes encoding essential replication genes and the other half encoding genes related to host-virus interactions. Many of the genes involved in host-virus interactions are involved in immunomodulatory processes and have homology to proteins encoded by the host. These viral genes, often referred to as "mimics", are therefore believed to be the result of host-to-virus gene transfer. In this study I sought to examine if common poxvirus immunomodulatory genes were found in other NCLDV lineages, and if so, to analyze the evolutionary history of these genes. I identified 5 protein families of immunomodulatory genes that were found in both poxviruses and other NCLDV lineages, and I used phylogenetic tools to compare viral immunomodulatory genes of NCLDVs to their eukaryotic orthologs to evaluate the number of times different NCLDV lineages have acquired these genes. Our phylogenetic analyses showed that several viral immunomodulatory genes were acquired multiple times by different NCLDV lineages, while others appear to have been transferred between viral groups. Interestingly, some NCLDV genes clustered together with homologs from the unrelated Herpesviridae family, suggesting that inter-viral gene exchange can traverse vast evolutionary distances. The vast diversity of hosts infected by different NCLDV lineages suggests that these immunomodulatory genes play key roles that are useful to viruses in a variety of contexts. This research provides insight into how giant viruses acquire host genes, which contribute to their large genome size, and how those genes evolved to subvert antiviral defenses. / Master of Science / Giant viruses are a relatively recent discovery, from the beginning of this century. Nucleocytoplasmic large DNA viruses (NCLDVs) are a classification of multiple giant virus families. These viruses have large genomes from around 100 kilobases to 2.5 megabases of DNA. For reference, the genome size of the flu virus is approximately 13 kilobases. Most viruses cannot be seen by the human eye, even with microscopes, but giant viruses can get as big as bacteria, which can be seen with microscopes. It is unknown how or why these viruses get so large. One explanation is that they steal genes from their host and those genes evolve to work against the host. In this thesis, I explored some of the genes that these viruses have picked up. I curated a set of 49 previously characterized viral genes to analyze in this context. These genes have to do with modulating the host immune system and are known as "immunomodulatory genes". Viral immunomodulatory genes are often mimics of the host genes which function to help the immune system. However, a virus evolves faster than a host and the virus mimic gene can evolve to work against the immune system. This change can be visualized using phylogenetic tools; the viral genes will be more similar to each other than to the host genes and cluster separately on a phylogenetic tree. About half of the genes of Poxviruses, a giant virus family that has viruses that infect humans, are related to virus-host interactions, and include viral mimic genes. Poxviruses have been far better studied than other NCLDV families because of their public health importance. Variola virus, the virus that causes smallpox, is a poxvirus. Other NCLDV infect animals, algae, and amoeba. Though their hosts are different, their genomes have similar features. I set out to discover whether some of these previously characterized viral immunomodulatory genes that exist in poxviruses also exist in other NCLDV families. I utilized phylogenetic tools and a database of giant virus sequences to figure out which genes are being picked up by which family of NCLDV. I also sought to determine whether the individual NCLDV families have their own acquired immunomodulatory gene or have a gene very similar to all other families, suggesting an ancient acquisition. If the gene is very similar, it suggests that an ancestor of the NCLDV acquired the gene and it has stuck around as the group diverged into families. It is also interesting if different families stole the same type of gene multiple times because that indicates the importance of that gene in subverting the antiviral immune system for viral replication. This work provides insight into how giant viruses acquire host genes, which contribute to their large genome size, and how they evolved those genes to subvert antiviral defenses.

Les virus géants sont un groupe de virus ADN double brin caractérisés par une taille géante du virion et du génome, et un répertoire de gènes qui comprend environ 450 à 2500 gènes prédits. Une proportion importante de ces gènes (jusqu'à 93%) sont des 'ORFans', ou codent pour des protéines de fonction inconnue. Acanthamoeba polyphaga mimivirus est le premier virus géant découvert, il y a une décennie, par co-culture sur Acanthamoeba spp. Il est le membre prototype de la famille Mimiviridae. Le génome de Mimivirus code pour environ 1000 protéines, parmi lesquelles ~50% n'ont pas d'homologue connu dans les banques de séquences publiques. La capside de Mimivirus a un diamètre d'environ 500 nm et est couverte par une couche dense de fibres, à l'exception de l'un de ses sommets. Ces fibres sont d'environ 130 nm de longueur et se composent d'une tige souple et d'une tête de forme globulaire.Dans ce travail de thèse, nous avons cherché à étudier les gènes impliqués dans la formation des fibres de Mimivirus. Dans ce but, nous avons notamment exprimé des gènes candidats dans E. coli, et nous avons mis au point une stratégie qui a utilisé l'interférence ARN afin d'étudier la fonction et la structure des protéines de Mimivirus. Nous avons annoté quatre protéines associées aux fibres. La stratégie utilisant les petits ARN interférant appliquée ici est originale et a été utilisée pour la première fois pour les virus géants qui infectent les amibes. Elle pourrait permettre de décrypter la fonction des gènes des mimivirus et d'annoter potentiellement des centaines de protéines présentes dans les bases de données publiques, et de différencier l'ADN poubelle des gènes réellement utilisés. / Giant viruses are a group of double stranded DNA viruses that are characterized by a giant virion and genome size, and gene repertoires encompassing approximately 450 to 2500 predicted genes. A substantial proportion of these genes (up to 93%) consists in ORFans, or encodes proteins with unknown functions. Acanthamoeba polyphaga mimivirus is the first giant virus that was discovered, a decade ago, after co-culturing on Acanthamoeba spp. It is the prototype member of the family Mimiviridae. Mimivirus encodes about 1000 proteins, among which ~50% have no known homolog in public sequence databases. The Mimivirus capsid is about 500 nm in diameter and is covered by a dense layer of fibers, except at one of its vertices. These fibers are about 130 nm in length and consist of a soft shaft and a globular shaped head.In this thesis work, we aimed to study the genes involved in the formation of the Mimivirus fibers. For this purpose, we have expressed candidate genes in E. coli, and implemented a strategy that used RNA interference to study the function and structure of Mimivirus proteins. We then succeeded in annotating four proteins as fiber associated proteins. The short interfering RNA strategy that we applied here is original and has been used for the first time in giant viruses that infect amoeba. It could allow deciphering the function of the mimivirus gene repertoires and help annotating hundreds of proteins without known function found in public databases and differentiate between junk DNA and truly used genes.

No description available.

No description available.

No description available.

Zipper-Interacting Protein Kinase (ZIPK) is a known modulator of actin-myosin contractility in vertebrate species. Interestingly, rodent and mouse ZIPK has undergone a divergence in regulation in comparison to other vertebrate orthologs including human. Whereas the human ortholog of ZIPK requires phosphorylation of residues TT299/300 for nuclear exit, rodents and mouse require interaction with another protein termed PAR-4. In this project we completed several experiments to examine zebrafish ZIPK in development and its effect on acto-myosin contractility. It was found that zebrafish ZIPK was expressed ubiquitously in maternal stages. In zygotic stages, ZIPK expression dropped dramatically and localized to the anterior portions of the embryo. Zebrafish and human ZIPK, but not rodent ZIPK were able to increase stress fiber formation and myosin light chain-2 (MLC-2) phosphorylation in vitro. Human and zebrafish ZIPK underwent nucleocytoplasmic shuttling without PAR-4 interaction, unlike rodent ZIPK, which required PAR-4 for nuclear exit. Unlike human ZIPK, zebrafish ZIPK TT299/300AA mutants were able to undergo shuttling. Similar to human ZIPK, catalytic mutations to zebrafish ZIPK abolished or dramatically reduced activity. Through these experiments we were able to show human and zebrafish ZIPK homologs function and are regulated similarly, while the rodent ZIPK was much more unique. Although the exhibited phenotypes were similar between human and zebrafish ZIPK orthologs, the mechanism of regulation is not completely conserved.

No description available.

No / Ran is a small ras-related GTPase that controls the nucleocytoplasmic exchange of macromolecules across the nuclear envelope. It binds to chromatin early during nuclear formation and has important roles during the eukaryotic cell cycle, where it regulates mitotic spindle assembly, nuclear envelope formation and cell cycle checkpoint control. Like other GTPases, Ran relies on the cycling between GTP-bound and GDP-bound conformations to interact with effector proteins and regulate these processes. In nucleocytoplasmic transport, Ran shuttles across the nuclear envelope through nuclear pores. It is concentrated in the nucleus by an active import mechanism where it generates a high concentration of RanGTP by nucleotide exchange. It controls the assembly and disassembly of a range of complexes that are formed between Ran-binding proteins and cellular cargo to maintain rapid nuclear transport. Ran also has been identified as an essential protein in nuclear envelope formation in eukaryotes. This mechanism is dependent on importin-β, which regulates the assembly of further complexes important in this process, such as Nup107–Nup160. A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer. Ran is overexpressed in a range of tumors, such as breast and renal, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival. Furthermore, tumors with oncogenic KRAS or PIK3CA mutations are addicted to Ran expression, which yields exciting future therapeutic opportunities.

Der "klassische" Importweg von Proteinen wie Transkriptionsfaktoren, Kernrezeptoren oder viralen Proteinen in den Zellkern erfolgt in Abhängigkeit der Importine alpha und beta. Während nur ein Importin beta existiert, waren zu Beginn der Arbeiten zwei humane alpha-Importine bekannt. In der vorliegenden Arbeit wird die Identifikation, Klonierung und funktionelle Charakterisierung von vier neuen humanen alpha-Importinen beschrieben. Anhand ihrer Primärstruktur wurden die sechs alpha-Importine in drei Subfamilien unterteilt. Um die Hypothese zu testen, dass die verschiedenen Importin alpha Isoformen spezifische Funktionen ausüben und sich nicht vollständig gegenseitig ersetzen können, wurde zunächst ihre Expression auf RNA- und Proteinebene analysiert. Hier ließen sich differentielle Expressionsmuster in verschiedenen humanen Zellen und Geweben nachweisen. In vitro Analysen mit rekombinant exprimierten und aufgereinigten Proteinen deuteten daraufhin, dass die neu identifizierten Isoformen tatsächliche Importfunktion besitzen, dass sich jedoch die verschiedenen alpha-Importine in ihren Substratspezifitäten unterscheiden. Verschiedene neue Substrate der alpha-Importine wurden identifiziert und deren Importwege im Detail analysiert. Unterschiede in der Regulation der Expression der alpha-Importine in Abhängigkeit von Zellproliferation, Zelldifferenzierung bzw. in unterschiedlichen Diabetesmodellen der Ratte deuteten ebenfalls auf spezifische Funktionen der verschiedenen Isoformen hin. Die spezifische Inhibition der Importin alpha Expression in kultivierten HeLa-Zellen mittels RNA-Interferenz führte bei den meisten Isoformen zu einer ausgeprägten Inhibition der Zellproliferation, wodurch erstmals der Nachweis essentieller Funktionen verschiedener alpha-Importine in lebenden humanen Zellen erbracht wurde. In weiterführenden Experimenten sollen die Ursachen für die Inhibition der Zellproliferation bei Importin alpha-Mangel geklärt und die Bedeutung der unterschiedlichen alpha-Importine in vivo weiter analysiert werden. / The "classical" import of proteins like transcription factors, nuclear receptors or viral proteins into the nucleus depends on importins alpha and beta. While only one importin beta is known, two human alpha-importins had been described. In this study the identification, cloning and functional characterisation of four novel human alpha-importins is reported. Based on their primary structures the human alpha-importins can be grouped into three distinct subfamilies. To test the hypothesis that the various alpha-Importins differ in their specific functions and cannot substitute for each other first their expression at the RNA- and protein levels were analyzed. Differential expression patterns in various human cells and tissues could be demonstrated. In vitro analyses using recombinantly expressed and purified proteins indicated, that the newly identified isoforms posses import functions in deed. However, there was evidence for differences in their substrate specific import efficacies. New substrates of the alpha-importins were identified and their import pathways analyzed in detail. Differences in the expression regulation of the alpha-importins depending on cellular proliferation and differentiation as well as in different rat models of diabetes further pointed towards specific functions of the various alpha-importins. Specific expression inhibition of several isoforms of the importin alpha protein family in cultured HeLa-cells using RNA-interference technology caused a strong inhibition of cellular proliferation. This is the first proof for essential functions of different alpha-importins in living human cells. Future experiments shall identify the mechanisms involved in the cellular proliferation inhibition due to importin a deficiency and further analyze the role of the different alpha-importins in vivo.

Positive-stranded (+)RNA viruses replicate inside cells and depend on many cellular factors to complete their infection cycle. In the meanwhile, (+)RNA viruses face the host innate immunity, such as cell-intrinsic restriction factors that could block virus replication. Firstly, I have established that the plant DDX17-like RH30 DEAD-box helicase conducts strong inhibitory function on tombusvirus replication when expressed in plants and yeast surrogate host. This study demonstrates that RH30 blocks the assembly of viral replicase complex, the activation of RNA-dependent RNA polymerase function of p92pol and viral RNA template recruitment. In addition, the features rendering the abundant plant DEAD-box helicases either antiviral or pro-viral functions in tombusvirus replication are intriguing. I found the reversion of the antiviral function of DDX17-like RH30 DEAD-box helicase and the coopted pro-viral DDX3-like RH20 helicase due to deletion of unique N-terminal domains. The discovery of the sequence plasticity of DEAD-box helicases that can alter recognition of different cis-acting elements in the viral genome illustrates the evolutionary potential of RNA helicases in the arms race between viruses and their hosts. Moreover, I discovered that Xpo1 possesses an anti-viral function and exports previously characterized cell-intrinsic restriction factors (CIRFs) from the nucleus to the replication compartment of tombusviruses. Altogether, in my PhD studies, I found plant RH30 DEAD-box helicase is a potent host restriction factor inhibiting multiple steps of the tombusvirus replication. In addition, I provided the evidence supporting that the Nterminal domain determines the functions of antiviral DDX17-like RH30 DEAD-box helicase and pro-viral DDX3-like RH20 DEAD-box helicase in tombusvirus replication. Moreover, I discovered the emerging significance of the Xpo1-dependent nuclear export pathway in tombusvirus replication.