Interaction between Ebola Virus Glycoprotein and Host Toll-Like Receptor 4 Leads to Induction of Proinflammatory Cytokines and SOCS1

Ebola virus initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal Ebola virus infections. Here we report that host Toll-like receptor 4 (TLR4) is a sensor for Ebola virus glycoprotein (GP) on virus-like particles (VLPs) and that resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines and suppressor of cytokine signaling 1 (SOCS1) in a human monocytic cell line and in HEK293-TLR4/MD2 cells stably expressing the TLR4/MD2 complex. Ebola virus GP was found to interact with TLR4 by immunoprecipitation/Western blot analyses, and Ebola virus GP on VLPs was able to stimulate expression of NF-κB in a TLR4-dependent manner. Interestingly, we found that budding of Ebola virus VLPs was more pronounced in TLR4-stimulated cells than in unstimulated control cells. In sum, these findings identify the host innate immune protein TLR4 as a sensor for Ebola virus GP which may play an important role in the immunopathogenesis of Ebola virus infection.Ebola virus and Marburg virus comprise the Filoviridae family and represent important human pathogens and potential agents of bioterrorism. Currently there are no approved vaccines or specific treatments available to prevent or treat filovirus infections. The filoviruses are the cause of severe hemorrhagic disease in humans (7). Ebola virus initially targets monocytes/macrophages and dendritic cells (DCs), which can lead to the release of proinflammatory cytokines and chemokines (3, 7). A better understanding of the physical and functional interactions between Ebola virus proteins and cellular factors regulating the host innate immune response may reveal novel insights into the pathogenesis of Ebola virus and offer new strategies to inhibit Ebola virus replication.The VP40 matrix protein of Ebola virus is a key structural protein critical for budding virus-like particles (VLPs) and virion egress. Interactions between late budding domains of VP40 and specific host proteins facilitate efficient release of VLPs and infectious virus. Viral proteins other than VP40 also contribute to efficient budding of VLPs. Ebola virus glycoprotein (GP), when coexpressed with VP40, is incorporated into budding VLPs and enhances VLP egress (15), possibly by antagonizing the function of host proteins (12).Several studies have reported the induction of an innate immune response following infection or stimulation of macrophages/monocytes and DCs with Ebola virus or VLPs, respectively (2, 31). For example, incubation of Ebola virus VP40+GP VLPs with DCs led to the induction of interleukin-6 (IL-6), IL-8, NF-κB and ERK1/2 (18, 31). The triggering mechanism by which Ebola virus VLPs stimulate cytokine production is unknown. Here, we present evidence that Ebola virus VLPs stimulate induction of proinflammatory cytokines as well as SOCS1 (a ubiquitin ligase and negative feedback regulator of cytokine production) by interacting with host Toll-like receptor 4 (TLR4). Importantly, Ebola virus VP40+GP VLPs, but not VP40 VLPs, induced cytokine and SOCS1 expression in a TLR4/MD2 dependent manner both in a human monocytic cell line (THP-1 cells) and in 293T cells expressing a functional TLR4/MD2 receptor. These results indicate that the stimulation of TLR4 by Ebola virus envelope GP results in an innate host response, induction of SOCS1 protein, and potential enhancement of virus egress.