Der Einfluss der Neuwelt-Arenaviren Junín und Tacaribe auf die Apoptose der Wirtszelle

Arenaviruses belong to the segmented negative-strand RNA-viruses and are divided into the two serogroups known as the Old World and New World arenaviruses. In this study the New World arenavirus species Junín (JUNV) and Tacaribe (TCRV), which can be considered prototypes for hemorrhagic fever-causing and non-pathogenic New World arenviruses, respectively, were investigated. JUNV is a human pathogen endemic to Argentina that causes hemorrhagic fever with a high case fatality rate, while its close relative TCRV is not known to be a significant human pathogen. The nucleoprotein NP is the most abundant structural component of the viral particle. It plays an essential role for viral replication/transcription as well as overcoming the interferon response in the host cell. JUNV and TCRV NP show 78% identity between their NP amino acid sequences, nevertheless the characterisation of JUNV and TCRV NP at the beginning of this work revealed significant differences in the expression pattern of JUNV and TCRV NP. In the case of JUNV NP, NP-specific cleavage products with a size of 62, 53, 47, 40 kDa were observed during recombinant expression as well as during JUNV infection. TCRV NP, however, did not show a comparable specific degradation pattern. Examination of the JUNV NP sequence revealed several putative caspase cleavage motifs. Point mutations of these motifs led to alterations in the observed degradation pattern and the loss of certain cleavage events. The DVKD and QEHD motives in JUNV NP were identified as the responsible cleavage sites for the generation of the 47 kDa and 40 kDa cleavage products, respectively. Further it could be shown that the intensity of JUNV NP cleavage directly depended on caspase activity, with increased caspase activity resulting in increased cleavage of NP, while inhibition of caspases decreased cleavage of NP. Based on these data JUNV NP was identified as a substrate of caspases. Interestingly it was further shown that expression of JUNV NP alone, but not of TCRV NP, suppresses the induction of apoptosis in cells treated with an apoptosis activator and that this anti-apoptotic effect also depends on cleavage of JUNV NP. Taken together, these data reveal that JUNV NP, in contrast to TCRV NP, serves as a target for caspase cleavage and is, therefore, able to prevent the induction of apoptosis in JUNV infected cells, possibly by serving as a decoy substrate. To better understand the role of caspase activation in arenavirus infection, the biological relevance of non-cleavage of TCRV NP was also analysed. TCRV, having no anti-apoptotic function mediated by NP, showed significant hallmarks of apoptosis during infection. In the case of TCRV, but not JUNV, a strong cleavage of caspase 3 and PARP, as well as chromatin condensation and the formation of apoptotic bodies, could be detected during infection. Further, characterisation of TCRV-induced apoptosis revealed an induction of apoptosis at late stages of infection and was dependent on cell type. Studies with UV-inactivated TCRV further demonstrated that apoptosis-induction is mediated not by binding or entry of the virus into the cell, but by down-stream steps in virus replication (e.g. viral replication/transcription and/or the accumulation of viral proteins in the cytoplasm). Cleavage of caspases 8 and 9 however, indicated that both, the extrinsic as well as the intrinsic apoptosis pathways are involved in signal transduction during the induction of apoptosis. Interestingly, caspase inhibition during TCRV infection results in a reduced virus titre, while caspase activation increased the virus titre. Therefore, the absence of NP-cleavage and its corresponding anti-apoptotic effect is unlikely to present a disadvantage for TCRV during infection. In contrast, TCRV seems to rely on apoptotic processes to yield efficient virus replication and release. The data presented in this work show for the first time that, in some species, the arenavirus nucleoprotein exerts a novel anti-apoptotic function related to its cleavage by caspases. In addition, this work has shown that JUNV and TCRV, despite their close phylogenetic relationship, are using different mechanisms to modulate apoptosis in the infected host cell to achieve efficient virus production and spread. While JUNV inhibits apoptosis by using NP as a decoy substrate for caspases, TCRV depends on apoptotic processes in the infected cell to replicate efficiently and reach virus titres equivalent to those achieved by JUNV. Based on the comparative analysis used in this study, differences between JUNV and TCRV NP that have a crucial effect on virus-host cell-interaction were identified and may help in future to explain the fact that JUNV and TCRV differ dramatically in their pathogenicity, as well as to predict the virulence of newly emerging arenavirus species.