(1) Characterization of EBOV protein interactions. Relatively little information exists regarding the molecular details that govern interactions between EBOV proteins. As such, we are actively interested in understanding the determinants of EBOV protein interaction and the functional outcomes of those interactions. The EBOV nucleoprotein (NP) and viral protein (VP) 24, both constituents of the viral nucleocapsid, are the sole factors responsible for EBOV virulence in mice, suggesting that these two proteins play a critical role in the induction of disease. Given their contribution to EBOV virulence, we sought to characterize the physical relationship between NP and VP24. We used confocal microscopy and immunoprecipitations to demonstrate that wild-type NP both co-localizes and interacts with VP24. To determine the region on VP24 responsible for the interaction with NP, we performed bioinformatics analysis to identify the regions most likely to be involved in protein-protein interactions. Based on this prediction, we generated a series of VP24 mutants each with up to eight consecutive amino acids mutated to alanines throughout the protein. Assessing these mutants for their ability to interact with NP revealed a region near the C-terminus of VP24 that plays a critical role in the ability of VP24 to interact with NP. Unlike wild-type VP24, the VP24 mutants that were unable to interact with NP were also unable to support the formation of transcription/replication-competent virus-like particles (trVLPs) and incorporation of viral RNA into viral nucleocapsid suggesting that VP24 plays a critical role in condensing the nucleocapsid, thereby restricting viral replication and transcription and promoting nucleocapsid packaging and egress. Additionally, mutational analyses revealed the 200 amino acids of NP that are critical for its interaction with VP24. Furthermore, in order to examine the significance of the NP-VP24 interaction in the viral replication cycle, recombinant Ebola viruses possessing mutations at the domains that are responsible for NP-VP24 interactions in NP and VP24 will be generated and characterized using our recently developed efficient reverse genetics system. This study, although ongoing, is the first to identify the molecular determinants of the interaction between VP24 and NP, and it suggests that the interaction between these two proteins is critical to the EBOV replication cycle, possibly hinting at new targets for therapeutic intervention. (2) The molecular mechanism for the over-induction of the host pro-inflammatory response by EBOV matrix protein VP40. Several mechanisms have been postulated to contribute to the catastrophic pathology of filoviral hemorrhagic fevers. Induction of an uncontrolled pro-inflammatory response by filoviruses has been proposed as one of the key pathological events that cause coagulation abnormalities, hemorrhagic manifestations, and multi-organ failure in infected humans and animals. For the induction of a pro-inflammatory response by infection, the canonical NF-κB signaling cascade is initiated upon stimulation of various receptors (e.g., toll-like receptor 4), which then leads to the activation of a cellular signaling cascade that finally results in the translocation of NF-κB to the nucleus where it acts as a transcription factor primarily involved in upregulating the expression of pro-inflammatory cytokines. The filovirus matrix protein, VP40 is a membrane-associated protein and promotes formation, budding, and release of filamentous virus particles from infected cells. In addition, MARV VP40 functions to inhibit host type I IFN signaling, suggesting that VP40 is multifunctional protein, although no IFN antagonist function is assigned to EBOV VP40. We uncovered a putative TNF-receptor associated factor (TRAF) 6 binding motif that overlaps with the late domain in EBOV VP40, and we subsequently demonstrated an interaction between TRAF6 and VP40. Following the identification of a putative TRAF6 binding motif in EBOV VP40, we confirmed the ability of VP40 to interact with TRAF6 by a series of co-immunoprecipitations. We have also demonstrated that expression of VP40 activates NF-κB without any stimulation (e.g., LPS, IL-1β etc,), which agrees with our hypothesis that VP40 may, in part, be responsible for driving the uncontrolled expression of pro-inflammatory cytokines during infection. Interestingly, we also have evidence suggesting that TRAF6 expression increases EBOV budding, perhaps implying a novel role for TRAF6 or other downstream signaling molecules in EBOV egress. This finding strongly suggests that VP40 induces the host cellular pro-inflammatory response, and it implies a novel molecular mechanism of Ebola HF pathogenesis. Since the molecular basis of systemic inflammatory response syndrome in filoviral HFs still remains elusive, this finding will facilitate our understanding of pathogenesis and the development of post-exposure interventions targeting the host deleterious response triggered by filovirus infection. (3) Characterization of pathogenic processes in the Syrian hamster model, which recapitulates MHF. While the NHP model is used to evaluate the efficacy of vaccines and therapeutics against filoviruses because it accurately recapitulates disease, rodent models (mice and guinea pigs) are convenient and suitable for elucidating the roles of specific viral proteins in the pathogenic process and have been widely used in numerous aspects of filovirus research. However, rodent models produce only limited and inconsistent coagulation abnormalities, which are a hallmark clinical feature of filoviral HFs. Recently, we developed and characterized a novel lethal Syrian hamster model of EHF based on infection with mouse-adapted EBOV that manifests many of the clinical and pathological findings observed in EBOV-infected NHPs and humans, including coagulation abnormalities. Similarly, we sought to apply our work with EBOV to establishing a rodent model for Marburg virus (MARV) strain Angola, thought to be the most virulent strain of MARV. We demonstrated that infection with hamster-adapted MARV produces severe disease in hamsters, including coagulation abnormalities evidenced by the appearance of a petechial rash that mimics that observed on humans and NHPs. In addition, by using qRT-PCR for monitoring host cytokine/chemokine gene expression, we revealed that early activation of innate immune responses including pro-inflammatory responses in the target organs (e.g., spleen, liver) in infected animals are host response markers associated with a lethal outcome. Furthermore, to identify the molecular determinants of MARV virulence in rodent models, we determined the full-length genome sequences of several lethal rodent-adapted variants and identified three amino acid mutations in VP35, VP40, and VP24. Overall, development of novel small animal models that mimic filoviral HFs provides an excellent experimental system that can be used to illuminate the molecular basis of pathogenesis and virus-host interactions in vivo. In addition, these series of studies will certainly facilitate translational research for the development of medical countermeasures.
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