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The HCMV Encoded Mrna-Export Factor Pul69 – Functional Conservation Within the Betaherpesvirinae and Identification of Mrna-Targets During Infection

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The HCMV Encoded Mrna-Export Factor Pul69 – Functional Conservation Within the Betaherpesvirinae and Identification of Mrna-Targets During Infection The HCMV encoded mRNA-export factor pUL69 – functional conservation within the Betaherpesvirinae and identification of mRNA-targets during infection Der HCMV kodierte mRNA-Exportfaktor pUL69 – funktionelle Konservierung innerhalb der Betaherpesvirinae und Identifikation von gebundenen mRNAs während der Infektion Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Barbara Zielke aus Nürnberg Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 3. Mai 2012 Vorsitzender der Promotionskommission: Prof. Dr. R. Fink Erstberichterstatter: Prof. Dr. T. Stamminger Zweitberichterstatter: Prof. Dr. A. Burkovski The most reliable way to forecast the future is to try to understand the present. John Naisbitt Table of contents Table of contents A Summary ............................................................................................................ 1 A Zusammenfassung............................................................................................ 2 B Introduction ....................................................................................................... 3 C Objectives ........................................................................................................ 14 D Materials and Methods.................................................................................... 15 1. Biological Materials..................................................................................................15 1.1. Bacteria ...................................................................................................................15 1.2. Eukaroytic cell cultures............................................................................................15 1.3. Virus strains.............................................................................................................15 1.4. Antibodies................................................................................................................16 1.4.1. Monoclonal antibodies....................................................................................16 1.4.2. Polyclonal antibodies......................................................................................16 1.4.3. Secondary antibodies.....................................................................................16 2. Nucleic acids.............................................................................................................17 2.1. Oligonucleotides......................................................................................................17 2.2. Vectors and plasmids ..............................................................................................20 2.2.1. Vectors and vector systems ...........................................................................20 2.2.2. Ready-to-use DNA constructs........................................................................20 2.2.3. Newly generated plasmids and BACmids ......................................................22 2.3. Additional nucleic acids ...........................................................................................24 3. Enzymes, chemicals and media..............................................................................24 3.1. Enzymes..................................................................................................................24 3.2. Media.......................................................................................................................24 3.2.1. Bacterial media...............................................................................................24 3.2.2. Mammalian cell culture media........................................................................24 3.3. Chemicals................................................................................................................25 3.4. Standard buffers......................................................................................................25 4. Methods.....................................................................................................................26 4.1. Standard molecular biology techniques...................................................................26 4.2. In vitro mutagenesis ................................................................................................26 4.3. Eukaryotic cell culture techniques ...........................................................................26 4.3.1. Maintenance of eukaryotic cell cultures..........................................................26 4.3.2. Transfection of mammalian cells....................................................................27 4.3.3. Infection of human foreskin fibroblasts (HFFs)...............................................27 Table of contents 4.4. Indirect immunofluorescence analysis.....................................................................27 4.5. Heterokaryon analysis .............................................................................................28 4.6. Nuclear mRNA-export assay ...................................................................................28 4.7. Coimmunoprecipitation............................................................................................29 4.8. RNA techniques.......................................................................................................29 4.8.1. Total cellular RNA preparation from infected cell culture cells .......................29 4.8.2. Cytoplasmic RNA preparation from infected cell culture cells........................29 4.8.3. RNA-immunoprecipitation...............................................................................30 4.8.4. Reverse transcription polymerase chain reaction (RT-PCR)..........................31 4.8.5. Quantitative SYBR-Green PCR (qPCR).........................................................31 4.9. Generation and characterization of recombinant viruses ........................................32 4.9.1. Generation of recombinant HCMVs using the BACmid technology ...............32 4.9.2. Homologous recombination using linear DNA fragments...............................32 4.9.3. Preparation, restriction enzyme digestion and PCR analysis of BACmid DNA..............................................................................................33 4.9.4. Reconstitution of recombinant viruses ...........................................................33 4.9.5. Virus titration based on IE1-gene expression.................................................34 4.9.6. Absolute quantification of virus genome copy numbers using Taqman probes ..............................................................................................34 4.9.7. Characterization of recombinant viruses by multistep growth curve analysis.................................................................................................34 4.10. Generation and characterization of a cDNA-library after RNA-immuno- precipitation of pUL69 from HCMV-infected HFFs ................................................35 E Results ............................................................................................................. 36 1. Functional characterization of the betaherpesviral pUL69-protein family..........36 1.1. Expression analyses of HCMV pUL69 and homologous proteins of the Betaherpesvirinae ...................................................................................................36 1.2. Nuclear localization of pUL69 and homologous betaherpesviral proteins...............37 1.3. Homo- and heterodimerization of betaherpesviral pUL69-homologs ......................38 1.4. Analysis of the nuclear mRNA-export activity of pUL69 and betaherpesviral homologs.................................................................................................................42 1.4.1. Analysis of nuclear mRNA-export by transient transfection experiments.......42 1.4.2. Stimulation of nuclear export of unspliced RNAs is restricted to the cytomegaloviral proteins pUL69, pC69 and pRh69........................................43 1.5. Nucleocytoplasmic shuttling of HCMV pUL69 and its betaherpesviral homologs ...45 1.6. Interaction with UAP56/URH49 is a prerequisite for stimulation of mRNA-export by pUL69, pC69 and pRh69 .............................................................47 1.7. Interaction of pUL69 with UAP56/URH49 but not RNA-binding is essential for efficient replication of human cytomegalovirus...................................................51 Table of contents 1.8. Characterization and functional analyses of chimeric fusion proteins between HCMV pUL69 and MCMV pM69 .............................................................................53 1.8.1. Verification of protein expression and analysis of subcellular localization of chimeric proteins pCh1 to pCh4 .................................................................55 1.8.2. pM69 acquires mRNA-export activity by fusion of the UAP56-interaction motif of HCMV pUL69 to its N-terminus .........................................................57 1.8.3. Transfer of the UAP56-interaction motif of pUL69 to pM69 enables the chimeric protein to substitute for pUL69 during HCMV-infection...................59 2. Identification of viral and cellular mRNAs that are targeted by the HCMV encoded mRNA-export
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