A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral in-clusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the ␣ subunit of eukaryotic initiation factor 2 (eIF2␣). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2␣ phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions. IMPORTANCEAlthough progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by many viruses. We show that EBOV does not induce formation of stress granules (SGs) in infected cells and is therefore unrestricted by their concomitant translational arrest. We identified SG proteins sequestered within viral inclusions, which did not impair protein translation. We further show that EBOV is unable to block SG formation triggered by exogenous stress early in infection. These findings provide insight into potential targets of therapeutic intervention. Additionally, we identified a novel function of the interferon antagonist VP35, which is able to disrupt SG formation.

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Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research.

Lassa fever (LF) is a zoonotic viral hemorrhagic fever caused by Lassa virus (LASV), which is endemic to West African countries. Previous studies have suggested an important role for T cell-mediated immunopathology in LF pathogenesis, but the mechanisms by which T cells influence disease severity and outcome are not well understood. Here we present a multiparametric analysis of clinical immunology data collected during the 2017-2018 Lassa fever outbreak in Nigeria. During the acute phase of LF we observed robust activation of the polyclonal T-cell repertoire, which included LASV-specific as well as antigenically-unrelated T cells. However, severe and fatal LF were characterized by poor LASV-specific effector T-cell responses. Severe LF was also characterized by the presence of circulating T cells with homing capacity to inflamed tissues, including the gut mucosa. These findings in LF patients were recapitulated in a mouse model of LASV infection, in which mucosal exposure resulted in remarkably high lethality compared to skin exposure. Taken together, our findings indicate that poor LASV-specific T-cell responses and activation of non-specific T cells with homing capacity to inflamed tissues are associated with severe LF. IMPORTANCE Lassa fever may cause severe disease in humans, in particular in endemic areas such as Sierra Leone and Nigeria. Despite its public health importance, the pathophysiology of Lassa fever in humans is poorly understood. Here we present clinical immunology data obtained in the field during the 2018 Lassa fever outbreak in Nigeria indicating that severe Lassa fever is associated with activation of T cells antigenically unrelated to Lassa virus as well as with poor Lassa virus-specific effector T cell responses. Mechanistically, we show that these bystander T cells express defined tissue homing signatures that suggest their recruitment to inflammed tissues and a putative role of these T cells in immunopathology. These findings open a window of opportunity to consider T cell targeting as a potential post-exposure therapeutic strategy against severe Lassa fever, a hypothesis that could be tested in relevant animal models such as non-human primates.