Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 173-176.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 158-177.

Rift Valley fever virus (RVFV) is a negative-sense RNA virus that is classified as an overlap select agent by the USDA and the HHS. It was first discovered in the Rift Valley of Kenya in the early 1930s. RVFV is an arbovirus that is transmitted by mosquitoes and infects ruminants and humans. RVFV in humans causes an acute self-limiting febrile illness but in a small percentage of cases, a severe version is noted by ocular disease, hepatitis, hemorrhagic fever, and death. In ruminants, the disease is similar with young livestock being the most susceptible. RVFV is also known to cause "abortion storms" where infected pregnant ruminants abort their fetuses with a near 100% fatality rate. Viruses are obligate intracellular parasites utilizing host-factors to replicate. This study identified three host-protein interactors of the viral Gn and L proteins that aid in viral replication. UBR4 was determined to be an interactor of Gn via immunoprecipitation followed by either LC/MS/MS or western blot analysis. Its inhibition via siRNA or CRISPR-Cas9 knockout showed a reduction of viral titers and viral RNA production. It was determined that UBR4 specifically affects viral RNA production and not entry or egress. Conversely, CK1α and PP1α were identified as binding partners of the L protein using similar methods. CK1α, a kinase, and PP1α, a phosphatase, were chosen for further verification due to data demonstrating the L protein is phosphorylated on at least one serine residue, in addition to PP1α already being shown to impact RVFV replication. Inhibition of CK1 and PP1 via small molecule inhibitors, D4476 and 1E7-03, respectively, showed a decrease in viral titers and RNA production. Strand-specific RT-qPCR demonstrates that CK1 and PP1 impact genomic replication. Upon treatment with D4476 a decrease in L protein phosphorylation was observed. Additionally, it has already been shown that treatment with 1E7-03 increases L protein phosphorylation. These data indicate that CK1 and PP1 modulate L protein phosphorylation, contributing to changes in RVFV replication. This study identifies three host-proteins that affect viral replication, which could be used as a foundation for host-based therapeutic and vaccine development. / Doctor of Philosophy / Rift Valley fever virus (RVFV) is a major biological threat due to its ability to infect both livestock and humans and be passed by mosquito bite. RVFV was first discovered in Africa in the early 1930s. To date, there is no approved therapeutic or vaccine. RVFV usually causes very mild disease but in a small percentage of cases, it progresses to include liver disease, vision loss, swelling of the brain, bleeding, and death. A virus itself is not alive; it needs a living host in order to replicate. To do this, it utilizes things naturally occurring inside the host. The purpose of this study is to identify host-factors that the virus uses in order to efficiently make more viruses. The first viral protein of interest is the glycoprotein, Gn, which is important for viral entry and assembly of the viral particles. It was determined that the host-protein UBR4 is an interactor of Gn and that the inhibition of UBR4 decreases the amount of infectious virus being produced. Similarly, the host-proteins, CK1α and PP1α, were found to be interactors of the viral L protein. The L protein is responsible for synthesizing the building blocks of the virus. It was determined that when CK1 and PP1 are inhibited, the L protein is less efficient at making these building blocks. Understanding the host-factors the virus utilizes is important to the basic understanding of how RVFV infects the host and the development of therapeutics to combat an outbreak.

Most existing chemotherapeutics lack adequate specificity for transformed cells and therefore have high rates of collateral damage to normal tissue. Moreover, such therapies often depend on p53 to induce cell death and are ineffective on the large number of human cancers that have lost p53 function. The discovery of novel p53-independent cancer therapies is therefore of significant interest. The Chicken Anemia Virus protein Apoptin selectively induces apoptosis in transformed cells in a p53-independent manner while leaving normal primary cells unaffected. This selectivity is thought to be largely due to cell type-specific localization: in primary cells Apoptin is cytoplasmic, whereas in transformed cells the protein localizes to the nucleus. The basis for this cell type-specific localization remains to be determined. In this study, Apoptin is revealed to be a nucleo-cytoplasmic shuttling protein whose localization is mediated by an N-terminal nuclear export signal (NES) and a C-terminal nuclear localization signal (NLS). Both signals are required for cell type-specific localization, as Apoptin fragments containing either the NES or NLS fail to localize differently between transformed and primary cells. Significantly, cell type-specific localization can be rescued in trans by co-expression of the two separate fragments, which are able to interact through an Apoptin multimerization domain. Interestingly, this multimerization domain overlaps with the NES suggesting that these two activities may be functionally coupled in cytoplasmic retention in primary cell types. Factors present in transformed cells induce localization of Apoptin to the nucleus where a biochemically distinct, more soluble form of the protein exists. Using affinity-purification and mass spectroscopy it was found that, specifically in transformed cells, Apoptin is associated with APC1, a subunit of the anaphase-promoting complex/cyclosome (APC/C). The APC/C is required to establish a mitotic cell-cycle checkpoint, and its inhibition results in G2/M arrest and apoptosis. Expression of wild type Apoptin in transformed cells inhibits APC/C function and induces G2/M arrest and apoptosis, whereas Apoptin mutants that are unable to associate with APC1 have no effect. In p53 null cells, ablation of APC1 by RNA interference induces a G2/M arrest and apoptosis analogous to that observed following Apoptin expression. Furthermore, Apoptin was found to induce the formation of PML bodies and to recruit APC/C subunits to these nuclear structures suggesting a mechanism involving sequestration and subsequent inhibition of the APC/C. Thus, the results of this study clarify Apoptin cell type-specific localization behavior and explain the ability of Apoptin to induce apoptosis in transformed cells in the absence of p53. This study advances a newly emerging field of viral mechanisms of apoptosis involving G2/M arrest and APC/C modulation. The resultant p53-independent apoptosis suggests that the APC/C may be an attractive target for the development of anti-cancer drugs.

E3-Ubiquitin-Ligasen übertragen Ubiquitin auf die von ihnen gebundenen Substratproteine. Durch diese Ubiquitinierung werden Proteine für den kontrollierten Abbau im Ubiquitin-Proteasom-System markiert. Dieser Prozess beeinflusst aber auch die Aktivität verschiedener Signalwege, die Lokalisation von Proteinen oder die strukturelle Integrität zellulärer Komponenten. MuRF1, MuRF2 und MuRF3 sind E3-Ubiquitin-Ligasen, die hauptsächlich in quergestreifter Muskulatur exprimiert werden. Von MuRF1 ist bereits bekannt, dass es u. a. über die Ubiquitinierung von Myosinen und deren anschließender Degradation an der Entwicklung der Herz- und Skelettmuskelatrophie beteiligt ist. Da das Wissen über MuRF2 und MuRF3 in diesem Zusammenhang noch begrenzt ist, sollte die Auswirkung der kombinierten Keimbahndeletion von MuRF2 und MuRF3 in einem Mausmodell untersucht werden. Der Doppelknockout (DKO) von MuRF2 und MuRF3 führte zu Veränderungen der Morphologie und der Funktionsfähigkeit der Skelett- und Herzmuskulatur. In Skelettmuskelfasern kam es zur Ablagerung myosinhaltiger Proteinaggregate, zu einer Zunahme an langsam kontrahierenden Muskelfasern sowie zum Auftreten von Myozyten mit zentral gelegenen Nuclei als Anzeichen von Regenerationsprozessen. Isolierte Skelettmuskeln von DKO-Mäusen entwickelten eine geringere maximale spezifische Kraft als Muskeln aus Kontrolltieren. Ihre Herzen waren morphologisch unauffällig. Dennoch waren die Kontraktion des linken Ventrikels und das Schlagvolumen reduziert. Darüber hinaus zeigten isolierte Kardiomyozyten Beeinträchtigungen der Kontraktionsfähigkeit und der Kalziumströme in vitro. Eine massenspektrometrische Untersuchung ergab, dass in den Muskeln der MuRF2/3-DKO-Mäuse im Vergleich zu den Kontrollmäusen 12 Proteine in erhöhter Menge vorhanden waren. Eine Anreicherung von MAPKAP-K3, einem dieser Proteine, und von MAPKAP-K2 konnte im Western Blot von Proteinlysaten aus Skelettmuskeln und dem Herz der MuRF2/3-DKO-Mäuse detektiert werden. / E3 ubiquitin ligases attach the small modifier ubiquitin to their substrate proteins. This ubiquitin-tag not only marks proteins for the proteasome dependent degradation, but also influences the activity of signalling pathways, the localisation of proteins or the structural integrity of cellular components. MuRF1, MuRF2, and MuRF3 are E3 ubiquitin ligases predominantly expressed in striated muscles. MuRF1 is involved in cardiac and skeletal muscle atrophy by mediating proteasome-dependent degradation of myosins. The knowledge about MuRF2 and MuRF3 in this context is limited. Therefore, a mouse model was used to analyse the impact of the combined deletion of MuRF2 and MuRF3. The double knockout (DKO) of MuRF2 and MuRF3 influenced the structure and function of skeletal and cardiac muscle. Skeletal muscle fibres exhibited myosin-containing protein aggregates, a fibre-type shift towards slow fibres, and myoycytes with central nuclei which is an indication of regeneration. Maximal force development was reduced in isolated hindlimb muscles M. soleus and M. extensor digitorum longus of MuRF2/3-DKO mice. Hearts were morphologically normal. No protein aggregates or signs of fibrosis were detected. However, heart performance was impaired. The contractibility of the left ventricle and the ejection fraction were reduced. Isolated cardiomyocytes showed a diminished contractibility. Furthermore, their speed of contraction and relaxation was reduced and they had impaired calcium transients. Mass spectrometric analysis of muscle lysates identified 12 enriched proteins in MuRF2/3-DKO muscles. Western Blot analysis confirmed that MAPKAP-K3, one of these proteins, and MAPKAP-K2 were enriched in lysates of skeletal muscles and left ventricles of MuRF2/3-DKO mice. Further investigations will show how MAPKAP-K2- and MAPKAP-K3-signalling pathways are involved in the development of the MuRF2/3-DKO-phenotype.