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New World arenaviruses represent an important group of zoonotic pathogens that pose a serious threat to human health. While some virus species cause severe disease, resulting in hemorrhagic fever and neurological symptoms, other closely related family members exhibit little or no pathogenicity. For instance, Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, while the closely related Tacaribe virus (TCRV) is avirulent in humans. Little is known about host cell responses to infection, or how they contribute to virulence; however, TCRV strongly induces caspase-dependent apoptosis (i.e. non-inflammatory programmed cell death) in infected cells, whereas JUNV does not.
In order to better understand the connection between apoptosis and pathogenesis, we sought to unravel the regulation of pro- and anti-apoptotic signaling in response to arenavirus infection. We demonstrated that apoptosis induced by TCRV proceeds over the mitochondrial-regulated intrinsic pathway and involves activation of p53 (accumulation and phosphorylation), activation of the pro-apoptotic BH3-only factors Puma and Noxa (accumulation), as well as inactivation of another pro-apoptotic factor called Bad (phosphorylation). The regulation of these factors in response to TCRV infection is accompanied by other classical hallmarks of intrinsic apoptosis, such as disorganization of the mitochondrial network, cytochrome c release, PS flipping, caspase cleavage and nuclear condensation. The involvement of the BH3-only factors as key players in regulating TCRV-induced apoptosis could also be validated in knockout cells, which showed either suppressed or increased apoptosis depending on the respective activation (i.e. Puma and Noxa) or inactivation (i.e. Bad) status of the respective BH3 protein. Interestingly, while JUNV does not trigger late stages of apoptosis induction (i.e. caspase activation, nuclear condensation and cell death), we could show that it activates similar upstream pro-apoptotic signaling events including activation of p53, Puma and Noxa. This supports the current hypothesis that JUNV actively evades the induction of apoptosis through the involvement of a mechanism targeting late steps in the apoptotic cascade. Specifically, this model proposes that intrinsic activation is suppressed at the level of caspase activation by JUNV NP, which serves as an alternative substrate for caspase cleavage.
Additionally, in order to identify viral factors associated with the induction of apoptosis, a full genome sequencing of TCRV was performed and contributed to the validation and correction of substantial errors reported in existing sequences for TCRV. With the help of this sequence, correct expression plasmids containing the viral genes for NP, GP and Z were constructed and tested for their ability to induce apoptosis in vitro. This revealed that both TCRV and JUNV Z are triggers for apoptosis, which further supports our finding that JUNV also induces activation of pro-apoptotic factors. Again, consistent with a model where JUNV NP blocks caspase activation directly, co-expression of JUNV Z and NP abrogated caspase activation, while simultaneous expression of TCRV NP and Z still resulted in cell death.
Finally, identification of the specific apoptotic factors involved in regulating TCRV-induced apoptosis (i.e. Bad, Puma and Noxa) and the generation of the respective knockout cell lines allowed us to investigate what influence apoptosis induction has on virus infection. Interestingly, knockout of these factors showed no direct impact on virus growth in Vero cells. However, TCRV particles produced in cells with the individual pro-apoptotic (i.e. Puma and Noxa) or anti-apoptotic (i.e. Bad) factors knocked out showed altered infectivity in primary human monocytes and macrophages, which represent important target cells for arenaviruses. Since TCRV particles that originate from the different knockout cells would be expected to contain different amounts of PS in their envelope (depending on the level of apoptosis taking place), this suggests a role of apoptosis in facilitating PS-receptor-mediated entry and/or PS-receptor signaling through downstream kinases, either of which could be contributing to successful infection in professional phagocytic cells. In particular, phosphorylation of some of the identified factors involved in regulating TCRV-induced apoptosis indicates the involvement of upstream kinases from diverse signaling pathways, some of which also play a role in regulating cytokine production – another host cell reaction that differs significantly between TCRV- and JUNV-infected monocytes and macrophages. As such, these findings represent an exciting basis for a possible connection between apoptotic responses and the regulation of pro- and anti-inflammatory cytokine responses via their associated upstream signaling processes and provide a starting point for future studies that will help us to better understand how these processes contribute to arenavirus pathogenicity.
Ebolaviruses are dependent on host cell proteins for almost all steps in their viral life cycle. While some cellular factors with crucial roles in the ebolavirus life cycle have been identified, many of them remain to be identified or fully characterised. This thesis focuses on the characterisation and identification of host cell interactions of the highly pathogenic Ebola virus (EBOV), probing host-virus interaction at various stages of the viral life cycle. Beginning with viral budding, the function of a recently proposed late domain motif within the EBOV matrix protein VP40 was examined using an EBOV transcription and replication-competent virus-like particle (trVLP) system. Although this motif has been suggested to interact with the endosomal sorting complex required for transport (ESCRT), we could show that this late domain motif does not contribute to EBOV budding.
While many host cell proteins have been identified so far that are important for viral budding, only a few proteins are known that are necessary for EBOV RNA synthesis. Thus, to identify host proteins that are involved in viral replication and transcription, we performed a genome-wide siRNA screen in the context of an EBOV minigenome assay. Using this approach, we identified several proteins that appear to be important for viral RNA synthesis or protein expression. Two of the most prominent hits in our screen were CAD (Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase) and NXF1 (nuclear RNA export factor 1). CAD catalyses the first three steps in the de novo pyrimidine biosynthesis, while NXF1 is the main nuclear export protein for cellular mRNAs. In subsequent characterisation studies, using a range of life cycle modelling systems as well as molecular analyses, we could demonstrate that the canonical function of CAD during the pyrimidine biosynthesis is necessary for EBOV replication and transcription. In contrast to this, for NXF1 we discovered a so-far unknown function: Again, by applying different life cycle modelling alongside with molecular assays, we provided evidence that the EBOV nucleoprotein recruits NXF1 into inclusion bodies, the site of EBOV RNA synthesis, where it binds viral mRNAs to export them from these structures. Importantly, for both CAD and NXF1 we were able to recapitulate key data in the context of live EBOV infection, confirming their roles in the viral life cycle.
Both of these identified host factors are promising targets for antiviral therapies and indeed de novo pyrimidine synthesis is emerging as a possible antiviral target for a number of viruses. Similarly, as we could show NXF1 to be important in the life cycle of the highly pathogenic Junín virus, this raises the possibility that disruption of this interaction may result in broad-spectrum antiviral activity. Moreover, for an increasing number of negative-sense RNA viruses inclusion bodies as site of viral RNA synthesis are described to have a liquid organelle character. Therefore, our findings on NXF1 also provide an intriguing model to explain how negative-sense RNA viruses in general overcome this obstacle and export viral mRNAs from inclusion bodies.