Identifizierung und Charakterisierung der Interaktion von Marburg-Virus VP30 mit den zellulären Proteinen PIAS1, PIAS2 und SETDB1

Marburg virus (MARV) and Ebola virus (EBOV) belong to the family filoviridae and cause a severe, often fatal disease in humans and non-human primates. The Ebola virus outbreak in West Africa 2014-2016 underscored the need for a deeper understanding of the EBOV life cycle. The aim of this study was to determine cellular proteins that interact with MARV VP30 and play an important role during MARV infection. Yeast two-hybrid analyses identified five different proteins as cellular interaction partners of MARV VP30, namely PIAS1, PIAS2, SETDB1, RBBP6 and HECTD1. PIAS1, PIAS2, and SETDB1 were cloned and the respective proteins were characterized. Coimmunoprecipitation analyses confirmed the interaction of PIAS1, PIAS2 and SETDB1 with VP30 in human cells. For SETDB1 only in Huh7 cells an interaction was shown. The interaction of MARV VP30 with PIAS2β was shown to depend on the N-Terminus of VP30. Colocalization studies of VP30 and PIAS2β led to the assumption that the interaction might take place in the nucleus. Furthermore, it was shown that PIAS1 and PIAS2 also interact with EBOV VP30. Since MARV and EBOV VP30 interact with the same proteins it is likely to assume that a conserved domain may be responsible for this interaction. PIAS proteins were identified as inhibitors of STAT-signaling, are E3-sumo-ligases and regulate several transcription factors. In the present study it was shown, that PIAS1 and PIAS2 downregulate the interferon-stimulated antiviral Protein IFIT2, which is not activated during filovirus infections. MARV-specific minigenome-assays showed that PIAS1 and PIAS2 enhance viral transcription and replication. In PIAS2 knockout cells viral transcription and replication of a MARV-specific minigenome and the MARV genome during infection is diminished. Based on these results we assume that PIAS proteins play a supportive role during Marburg virus infection, by inhibiting interferon signaling and enhancing viral transcription. Future studies need to show, whether a protein complex consisting of several of the identified interaction partner plays a role during infection, since interaction among the found proteins suggests this. This study shows, that the identified and characterized interactors are relevant for MARV and EBOV and for viral transcription. Further studies on the exact underlying mechanisms are therefore of great importance.