Charakterisierung der Proteaseaktivität und Spezifität der OTU-Domäne (ovarian tumour domain) des L-Proteins des Krim-Kongo-Fieber-Virus
Final Report Abstract
The original study to experimentally verify the ubiquitin protease activity of the Crimean-Congo hemorrhagic fever virus large protein (CCHFV-L) and to determine function and target specificity was published by a large collaborative study of several research institutes in the USA and Europe. In this publication, CCHFV-L protein was shown to hydrolyze ubiquitin and ISG15 from cellular target proteins and biological significance of this activity was evidenced by their capacity to inhibit NF-κB dependent signalling and to antagonize the antiviral effects of ISG15 during viral infection in vivo. During my work from January 2008 to March 2008 that was carried out in Benedikt Kessler's lab in the scope of a DAAD short-term fellowship, we could observe changes in host cell DUB levels during the Paramyxovirus infection cycle. Based on these experiments, we therefore decided to apply the techniques described to specify CCHFV-L protein protease activity to further characterize observed changes in cellular deubiquitinating enzyme (DUB) activity during Paramyxovirus infection. Both human respiratory syncytial virus (RSV) and human parainfluenza virus 3 (PIV3) as representatives of the Pneumoviridae (Pneumoviruses) and Paramyxovirinae (Respiroviruses) subfamilies, respectively, were analysed for their potential to regulate deubiquitinating activity in infected cells using small active site modifiers that are specific for DUBs. Changes in DUB activity levels during PIV3 infection were further characterised by reverse phase liquid chromatography (LC) and tandem mass spectrometry (MS/MS). The nucleoprotein of PIV3 was found to coimmunoprecipitate with inhibitor bound DUB molecules. Ongoing work concentrates on identification of the interaction partner of the PIV3-N protein to obtain further insight into the mechanisms involved in regulation of DUB activity during PIV3 infection. Additionally, a large quantitative comparison study was carried out to compare the proteome of RSV infected cells versus uninfected cells in order to determine novel pathways regulated during RSV infection. RSV infected and uninfected HEp2 cells were lysed and proteins were separated by isoelectric focussing using OFFGEL fractionation. Following tryptic digestion, purified peptides were characterised by label-free quantitative expression profiling, using nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry (UPLC-MSE). Data sets were analysed using the Waters expression profiling system. Selected proteins that were uniquely identified in infected cells were screened by quantitative real-time PCR for regulation on the transcriptional level. 5'-3'-exoribonuclease 2 (XRN2) and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) were found to be upregulated on both protein and mRNA levels upon RSV infection. Analysing the function of those proteins in the context of viral infection will shed further light on mechanisms of RNA virus replication and the host cell defence machinery.
Publications
- (2007). Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps. Virology Journal, 4:51
Ternette N, Stefanou D, Kuate S, Überla K and Grunwald T
- (2007). Immunogenicity and efficacy of codonoptimized DNA vaccines encoding the F-protein of respiratory syncytial virus. Vaccine 25:7271–7279
Ternette N, Tippler B, Überla K and Grunwald T
- (2009). Protective efficacy and immunogenicity of an adenoviral vector vaccine encoding the codon-optimized F protein of respiratory syncytial virus. J Virol. 83:23
Kohlmann R, Schwannecke S, Tippler B, Ternette N, Temchura V, Tenbusch M, Überla K and Grunwald T
