Identifizierung und Charakterisierung des HCMV Portal Proteins pUL104

Die HCMV DNA-Verpackung beruht auf der Spaltung konkatemerer DNA durch die virale Terminase, deren Interaktion mit einem im Kapsid vorliegenden Portalprotein und der anschließenden Translokation der DNA in das Kapsid. In der vorliegenden Arbeit konnte pUL104 als das HCMV Portalprotein identifiziert...

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Bibliographic Details
Main Author: Dittmer, Alexandra
Contributors: Radsak, Klaus (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2006
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HCMV DNA-packaging is based on cleavage of concatenated DNA by the viral terminase, its interaction with a capsid associated portal protein und the subsequent DNA translocation into the capsid. In this dissertation pUL104 was identified as the HCMV portal protein. Using indirect immunofluorescence pUL104 could be shown to have a non-linear NLS and to localize to the cell nucleus during solitary expression. In infected cells pUL104 is recruited into replication centers and late after infection appears in the cytoplasm as part of the virion. After purification of a monospecific polyclonal antibody pUL104 was identified in infected cells by immunoprecipitation as a protein of 75 kDa with a dimer-form of 150 kDa. Under native conditions both protein forms are part of the same complex. Cell fractionation also showed both a nuclear and a cytoplasmic distribution. Furthermore, pUL104 could be shown to be a structural component of extracellular virions. Performing immuno-gold labelling of ultra-thin sections of infected cells pUL104 was observed at single capsid vertices. Analyses of pUL104 structure showed recombinant rpUL104 to sediment as a high-molecular weight complex of at least 670 kDa both on a sucrose gradient and in a gelpermeation-chromatography. Oligomerisation also could be observed in cross-linking and single-particle analyses, whereas the latter showed a ring structure with a barrel shaped side view. An interaction of pUL104 with pUL56, the large terminase subunit, could be demonstrated both in an in vitro binding assay and by co-immunoprecipitation in vivo, and could be inhibited with the HCMV replication inhibitor Cl4RB, a benzimidazole-D-ribonucleoside. Binding of pUL104 to dsDNA could also be shown. With these results pUL104 fulfils both the structural and the functional requirements for a portal protein. Using UL104 specific siRNA both pUL104 expression and HCMV replication were repressed. EM-analysis of ultra-thin sections of infected cells showed an accumulation of empty capsids in the cell nucleus as well as a lack of infectious virions in the cytoplasm. This demonstrates that pUL104 is necessary for DNA packaging. In summary, pUL104 is a capsid-associated oligomer, which interacts with the viral terminase and is essentiell for DNA packaging.