Characterisation of immediate early 1 protein of primate and rodent cytomegaloviruses

The HCMV protein hIE1 is a multifunctional regulator that can, on one hand, antagonise the immune system in different ways and, on the other hand, promote lytic replication through interaction and stabilisation of the cellular protein hFEN1 or by transactivation of viral promoters. IE1 antagonises PML-NBs, a part of the intrinsic immune system, through interaction with PML followed by it´s deSUMOylation and disruption. Quantitative analysis, using NanoBRET assays, showed that the interaction of PML with IE1 is species-specific. Using further biological methods, it was shown that the deSUMOylation and disruption of PML is also species-specific. The species specificity, similarity of crystal structures and low sequence identity of hIE1 and rIE1 core were used to search for a possible interaction surface of hIE1 with PML. Stably expressed mutants were generated in which helices (H1, H1/2, H4, H5, H8 or H10/11) of hIE1 were exchanged with helices of rIE1. As none of these mutants were able to interact with PML or disperse it, we assume that the interaction surface consists of several protein helices. Furthermore, the functional integrity of the mutants was investigated by analysing additional hIE1 functions. A possible interaction site of hFEN1 was identified in helix 1 of hIE1, while the other helices have no influence on the interaction. Furthermore, since we found that interaction of hIE1 with hFEN1 is not sufficient for protein stabilisation, we assume that recruitment of an additional cellular factor is required for hFEN1 stabilisation. Transactivation of promoters was investigated by Gaussia luciferase assays and showed that only the mutant carrying helix 1 exhibited activity. In summary, these results indicate that the exchange of protein helices affects different functions of hIE1. However, no interaction surface of IE1 and PML could be identified. In addition, various functions of rIE1 were analysed in comparison to hIE1. It could be shown that while hFEN1 interaction and stabilisation is species-specific, the core domain of rIE1 is sufficient to activate viral promoters and antagonise the innate immune system through STAT2 interaction and relocalisation. Furthermore, since rIE1, unlike hIE1, cannot colocalise with mitotic chromatin, RCMV probably encodes a different protein that ensures genome maintenance. These results suggest that specific functions of IE1 have been conserved during evolution, which are probably crucial for maintaining CMV in different species.