The Extracellular Matrix as a Determinant of Filovirus Infectivity.

Filoviruses (Ebola [EBOV] and Marburg [MARV]) are emerging global pathogens that cause both acute hemorrhagic disease and chronic persistent infections with long-term sequelae and death. A comprehensive understanding of the filovirus-host interactome is critical for the development of novel host-oriented countermeasures to treat or prevent these deadly viral infections. We have recently identified YAP (Yes- Associated Protein; the downstream transcriptional effector of the Hippo pathway), and filamin A and B, members of a family of actin crosslinking/stabilizing proteins, as novel host determinants of viral infectivity, egress, and spread. Functionally, we find that filamin-A is critical for mediating infectivity of authentic EBOV and MARV in multiple cell types including primary human macrophages, while in contrast, filamin-B restricts filovirus infectivity. Filamin-A forms a mechanosensory complex with β1 integrin to transduce signals from the extracellular matrix (ECM) to internal signaling pathways such as the Hippo pathway, a critical regulator of proliferation, migration, and EMT (epithelial mesenchymal transition) induction. Previously, we found that filamin-A-deficient melanoma cells are softer than wild-type cells and maintain the Hippo pathway in an ON condition in which YAP is predominantly cytoplasmic. Together with our recent data, these findings support the novel hypothesis that ECM composition and stiffness will impact EBOV/MARV infectivity, with EBOV/MARV more easily infecting cells embedded in a stiffer ECM (where YAP is transcriptionally active) than in a softer ECM (where YAP is transcriptionally inactive). In Aim 1, we will test our hypothesis that ECM stiffness is a determinant of filovirus infectivity using innovative approaches, such as atomic force microscopy (AFM), viscoelastic polyacrylamide (PAA) hydrogels, and live filovirus infection assays. In preliminary studies to identify host proteins that functionally contribute to the robust and opposing effects of filamin-A and filamin-B on EBOV/MARV infectivity, we identified 5 ECM-associated proteins, a subset of which are YAP target genes. In Aim 2, we will use innovative approaches including live filovirus infection, pseudotype transductions, CRISPR/Cas9 KO cells, and RNA Seq. to determine whether expression of these 5 candidate ECM-associated proteins modulate filovirus infectivity. Intriguingly, EBOV infection induces EMT, and we have recently identified YAP as a novel EBOV/MARV VP40 interactor and regulator of filovirus egress and spread. Based on these data, in Aim 2 we will also test the novel hypothesis that filovirus infection will reciprocally impact the cellular environment by affecting YAP localization/transcriptional-activity and expression of ECM-associated proteins within the matrisome. Findings from this exploratory proposal will provide a solid foundation for future studies designed to interrogate more mechanistically the role of the ECM and matrisome in regulating filovirus infection, the subsequent host immune response, and the potential to manipulate the ECM to inhibit filovirus infection.