Browsing by Author "Krumm, Stefanie A."

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Protein-protein and protein-small-molecule inhibitor interactions in the measles virus replication complex
(2013) Krumm, Stefanie A.; Wolf, Dieter (Prof. Dr.)
The disease measles is caused by the highly contagious measles virus (MeV). MeV belongs to the paramyxovirus family together with respiratory syncytial virus, human parainfluenza viruses and metapneumovirus. Paramyxoviruses are responsible for major pediatric morbidity and mortality. Despite the availability of an effective MeV vaccine, measles case numbers increased alarmingly in the past few years especially in Europe. The return of endemic measles in the European population can directly be linked to the decrease in acceptance/use of the measles-mumps-rubella (MMR) vaccine. The Measles Initiative has set a goal to eliminate measles by 2015. The addition of an effective antiviral to quickly treat sporadic outbreaks and the surrounding communities would greatly aid in the measles eradication efforts. Fundamental understanding of the viral replication mechanism at the molecular level will be critical for the successful development of antivirals. Therefore the following dissertation examined the protein-protein interactions in the measles virus polymerase complex to understand the events taking place at the molecular level. Additionally, it engaged in protein-small-molecule interactions to identify small-molecule inhibitors of viral replication and their targets. The first part of the thesis focused on molecular interactions in the viral replication complex. The viral replication complex is an attractive target for antiviral therapy since it possesses unique features and is expressed and functions in a sub cellular compartment distinct from host cell polymerases. The polymerase complex consists of the phosphoprotein (P) and the polymerase (L) protein. The P-L complex only interacts with nucleoprotein (N) encapsidated RNA (RNP) for transcription and replication. MeV N contains a core domain involved in RNA encapsidation and a 125-residue carboxy (C)-terminal tail (Ntail) considered to mediate P-L binding to RNP for polymerization. Ntail of MeV is largely unstructured, but a terminal microdomain is implicated in P binding. C-terminal tail truncated N mutant proteins progressively eliminating this microdomain and upstream tail sections demonstrated that the interaction of the Ntail microdomain with a C-terminal domain in P is not required for polymerase recruitment and initial binding of L to its template. Additional investigations showed that disrupting the domain organization by insertion of an epitope tag in the Ntail did not affect polymerase activity, but rather affected particle assembly. Cell free virions contained reduced levels of envelope proteins which did not affect cell-to-cell fusion kinetics. However, the N-mutant virus was observed to have a kinetic delay of viral mRNA and genome production. Studies to identify and characterize small-molecule antiviral compounds and their targets were conducted in the second part of this thesis. Non-nucleoside small-molecules are suitable antiviral therapeutics. There are two main approaches in identifying antivirals. First, compounds that target the virus, for example the RNA replication machinery, can be assayed for. Alternatively, compounds that target a host factor that the virus requires can also be a viable strategy. Cellular factors may also be necessary for the entire family of viruses and therefore compounds aiming for host factors may be more likely to be broadly active inhibitors. A potent pathogen-directed small-molecule compound class had been identified in a high-throughput screen. Hit-to-lead chemistry yielded a highly potent and water soluble compound ERDRP-0519. It targets the L subunit of the morbillivirus polymerase complex directly, since resistance-mediating mutations were exclusively located in the L protein. Unparalleled efficacy of this orally available small-molecule inhibitor was demonstrated and pioneered a path towards an effective morbillivirus therapy that can support measles eradication efforts. Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. JMN3-003 is a potent broadly active inhibitor of viral RdRp activity with a host factor mediated profile. It inhibited a wide range of different viral targets. Its antiviral activity was host cell species dependent and induced a temporary cell cycle arrest. While the compound inhibited viral mRNA and genome production, it left host cell mRNA and protein production unaffected. Taken together, this PhD studies changed the prevailing paradigm in polymerase recruitment and provided strong proof of concept for the potential of the development of pathogen- and host-directed antiviral therapy. These studies demonstrated how basic molecular research of protein-protein interactions critical for virus replication can complement a translational approach to identify, characterize, and improve novel antiviral candidates.