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Protein interactions involved in viral replication characterized by T7 phage display
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein interactions are essential for many cellular processes. By utilizing protein-protein interactions, viruses can infect their host cells and replicate. Phage display can be used for identification and characterization of protein-protein interactions. This technique includes screening of a bacteriophage library that display peptides/protein fragments on the surface in fusion with a coat protein. Depending on the characteristics of the interaction of interest, size and copy number of the displayed insert can be varied for optimal selection. This thesis describes attempts to use different formats of the T7 phage display system in identification and characterization of interactions with proteins involved in viral replication.

Large T-antigen (LT) is essential for initiation of DNA-synthesis in polyomavirus. A peptide library made in phage T7 was screened against three LT-specific monoclonal antibodies: LT1, F4, and F5. Whereas a consensus sequence corresponding to a linear epitope in LT was displayed in phage clones selected with F5, mimetic peptides were displayed in clones selected with LT1 and F4. Synthetic peptides containing the consensus sequences could be used as specific competitors in affinity purification of LT.

The cellular protein La interacts with the untranslated regions of several RNA viruses, including hepatitis C virus (HCV), and has been hypothesized to participate in viral replication. To investigate whether other viral gene products may be involved in this interaction, we constructed HCV cDNA libraries in phage T7. Affinity selection of peptide and cDNA libraries against La resulted in selection of phage clones displaying the N-terminal region of HCV NS5A and a basic consensus motif found in HCV NS5B. These interactions with La were confirmed with NS5A and NS5B translated in vitro.

To investigate a highly conserved segment in the DnaJ-domain of mouse polyomavirus T-antigens, 13LLELLKL19, mutant LTs with substitutions of amino acid residues 13, 14, and 15 were affinity purified and characterized. While mutant L13V was defective in viral DNA replication, it was active in all replication-related reactions, i.e. binding, unwinding and melting viral DNA and interaction with DNA polymerase alpha p48 subunit and c-Jun. Considering all the results, we suggest that the defect is a result of impaired assembly of replication complexes, possibly due to underphosphorylation of the mutant protein.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2001. , 60 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 995
Keyword [en]
Biochemistry, protein-protein interaktion, fagdisplayteknik, HCV, polyomavirus
Keyword [sv]
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Virology
URN: urn:nbn:se:uu:diva-585ISBN: 91-554-4927-1OAI: oai:DiVA.org:uu-585DiVA: diva2:166619
Public defence
2001-02-22, C8:305, BMC, Husargatan 3, 751 23 Uppsala, Uppsala, 09:15
Available from: 2001-02-01 Created: 2001-02-01Bibliographically approved

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