Nuclear Localization and Function of the Ebola Virus VP40 Protein

Summary: Ebola (EBOV) and Marburg (MARV) are emerging zoonotic viruses that cause sporadic outbreaks of both acute hemorrhagic fever and chronic persistent infections, from which they can re-emerge to cause long- term sequelae or death. Filovirus-host interactions can be exploited as potential targets for the much-needed development of novel antiviral countermeasures. As such, we have reported on modular interactions mediated by filoviral PPxY Late (L) domain motifs conserved in EBOV/MARV VP40 matrix proteins and WW-domains of select host proteins such as Nedd4, WWP1, WWOX, and BAG3. Recently, one of the more intriguing "hits" from our screen of ~115 WW-domain containing host proteins using WT or mutant PPxY motifs from eVP40 and mVP40 as bait was Yes-Associated Protein (YAP). YAP traffics into the nucleus where it functions as the key downstream transcriptional effector of the Hippo signaling pathway; a pivotal pathway controlling organ size/development, cell proliferation/migration, cellular homeostasis, and the induction of EMT (epithelial mesenchymal transition); a process promoted by EBOV infection. Intriguingly, nuclear shuttling of YAP is regulated in part by PPxY/WW-domain interactions mediated by the PPxY motifs within the LATS1/2 kinases that are identical to those of eVP40 and mVP40. We and others have noted detectable levels of EBOV VP40 in the nuclei of both VP40-transfected and live EBOV infected cells, including Huh7, Hela, HUVEC and monocyte- derived macrophages (MDMs). Viral/host proteins that lack a nuclear localization signal (NLS) can interact with another protein to hitch a ride into the nucleus via a broad strategy known as "nuclear hitchhiking". Based on these findings, the lack of an NLS on eVP40, and our surprising findings linking EBOV infection and eVP40 with YAP, we hypothesize that eVP40 hitchhikes into the nucleus with YAP as the driver. Moreover, as YAP-bound host proteins (e.g. angiomotin {Amot}) that enter the nucleus can skew YAP-mediated transcriptional programming, we hypothesize that and once in the nucleus, eVP40 impacts YAP-mediated transcription to promote the establishment of a cellular environment conducive for a productive infection and efficient virus dissemination. We will address each aspect of our hypotheses in two Aims. In Aim 1, we will test our hypothesis that eVP40 hitches a ride into the nucleus with YAP in a PPxY/WW-domain dependent manner and in Aim 2, we will investigate the biological significance of nuclear eVP40 by determining whether a nuclear eVP40-YAP interaction skews YAP-dependent transcription patterns to those that might favor EBOV production and dissemination. We believe that this project fits well with the R21 format, as our Aims originate from a firm scientific premise, while also being exploratory in nature. Indeed, identification of a biologically relevant VP40 nuclear phase of EBOV infection could potentially open new and exciting areas of investigation into EBOV-nuclear interactions, EBOV pathogenesis, and novel antiviral targets/strategies.