Pilot project to elucidate the role of DDX60L in Ebola virus disease

SUMMARY Preliminary Data, based on siRNA knockdown studies, suggests that host encoded DExD/H box family protein DDX60L exerts anti-Ebola virus (EBOV) activity. We therefore hypothesize that DDX60L is a negative regulator of EBOV replication that can influence EBOV disease (EVD). While constitutively expressed in a number of cell types, DXX60L is an interferon (IFN) stimulated gene (ISG) and is related to DDX60, which is reported to augment production of IFNα/β. Therefore, DDX60L may also contribute to the anti-EBOV effects of IFNs and promote upregulation of IFN responses. As a negative regulator of infection, understanding the mechanisms of DDX60L-mediated inhibition may suggest strategies to mitigate EVD. We proposed to define how DDX60L exerts its anti-EBOV effects using a combination of siRNA knockdown and CRISPR-Cas9-based approaches. Studies will be performed in cell lines that correspond to cell types infected by EBOV in vivo. These are the Huh7 (hepatocyte), A549 (epithelial) and THP-1 (monocyte/macrophage) cell lines. Because siRNA knockdown measurably impairs EBOV growth in cell culture, we will use this approach to define effects on a transfection- based EBOV replication cycle modeling system that can assay all the major steps in the virus replication cycle. In parallel, we will use in knockdown studies a recombinant EBOV in which the viral VP30 coding sequences have been replaced with GFP (EBOV-GFPΔVP30). This virus only replicates in cells that provide VP30 in trans, and can be used at reduced biocontainment levels. Finally, we will assay the effects of knockdown in the context of fully replication competent EBOV. For each system, we will perform assays in the absence and presence of IFNα and define steps in the replication cycle affected by knockdown using a combination of approaches that include measuring viral RNA synthesis, viral protein production and host IFNα/β responses. We will also build additional experimental systems to further characterize the role of DDX60L in EBOV infection. First, we will generate knockout cells using CRISPR-Cas9. This will allow us to define the impact of DDX60L more definitively. Second, because DDX60L is an enzyme, we will use CRISPR base editing to mutate the ATP binding site in the endogenous gene, to determine whether enzymatic activity is required for anti-EBOV effects. Finally, we will use a CRISPR-Cas9 activation approach to induce expression of endogenous DDX60L and determine whether this enhances its antiviral effects. This latter approach will enable over-expression studies in difficult to transfect cell types and overcome reported difficulties of expressing DDX60L from mammalian expression plasmids. Multiple alternatively spliced DDX60L mRNAs have been described. By inducing expression of DDX60L from its endogenous promoter, the CRISPR activation approach is expected to yield normal splicing patterns, overcoming the need to select a specific isoform for study. As these new cell-based systems become available, they will be used in the assays developed to study DDX60L-EBOV interactions in the context of siRNA knockdown.