Funktion von ISG15 bei viralen Herzmuskelerkrankungen
Final Report Abstract
Our group identified the pathological significance of the type I Interferon-induced ubiquitin-like modifier, the Interferon Stimulated Gene of 15 kDa (ISG15), in a mouse model of CoxsackievirusB3 (CV)-induced heart muscle infection. We found that the protective function of the ISG15 system involves a modification of protein substrates with this molecule, yet the cellular targets of ISGylation were unknown. ISGylation of protein substrates occurs in a posttranslational manner. Since the vast majority of de novo protein synthesis in infected cells is attributed to viral proteins, we questioned whether the ISGylation machinery might target CV-proteins. We conducted co-expression experiments of ISG15 components together with different CV proteins, which were followed by immunoprecipitation-studies. Substrate identification by this approach did not yield the expected results. Assuming that only a fraction of viral proteins is subjected to ISGylation, we decided to follow an alternative approach. In order to achieve a detection of even minor amounts of ISGylated viral proteins, a Bioluminescence Resonance Energy Transfer (BRET) assay was adopted as a mean of detection and these experiments are currently ongoing. Irrespective of the reported technical issues, we identified a highly interesting modification of the 3A viral protein and the abundance of this specific modification was influenced by the ISG15 system. We conducted cell biology studies to define a function of this specific protein modification. Also, we are on our track to identify the respective modifier and several possibilities could already be excluded. Ongoing mass spectrometry research will hopefully elucidate, which modification is present on 3A during infection. Based on these results, we will continue our investigation on the pathological impact of modified 3A. In addition to resolving putative targets within the CV proteome for the ISGylation system, we took further steps to differentiate the pathological function of ISG15 in the mouse model. Heart tissue damage upon viral infection involves both the direct virusmediated cytotoxicity as well as adverse effects of activated immune cells and their response signals on intact heart muscle cells. We aimed to define whether ISG15 functions as an immune-modulatory molecule e.g. influencing the activity of myeloid immune cells. The alternative scenario was that ISG15 primarily acts upon inhibition of viral replication and thereby diminishes pathology. Our results clearly demonstrate that ISG15 interfered primarily with viral replication in cells targeted by the virus, whereas immune-cell derived ISG15 did not contribute to disease progression. Also, ISGylation in non-hematopoietic cells turned out to be essential to control viral cytotoxicity in the heart muscle. Based on these results, the major effector function of ISG15 was spatially narrowed down to cardiomyocytes. We conclude that interference with the ISGylation system in these cells might present a promising approach to reduce viral cytotoxicity.
Publications
- Ubiquitin-Like Protein ISG15 (Interferon-Stimulated Gene of 15 kDa) in Host Defense Against Heart Failure in a Mouse Model of Virus-Induced Cardiomyopathy. Circulation. 2014;130:1589-600
Rahnefeld A, Klingel K, Schuermann A, Diny NL, Althof N, Lindner A, Bleienheuft P, Savvatis K, Respondek D, Opitz E, Ketscher L, Sauter M, Seifert U, Tschope C, Poller W, Knobeloch KP and Voigt A
(See online at https://doi.org/10.1161/CIRCULATIONAHA.114.009847)
