Ebola (EBOV) and Marburg (MARV) are enveloped, negative-sense RNA viruses belonging to the family Filoviridae. Neither vaccines nor antivirals are currently available to combat these dangerous NIAID Category A pathogens, which cause hemorrhagic syndromes with high mortality rates in humans. Our laboratory focuses on the mechanisms by which filoviruses """"""""hijack"""""""" host proteins to regulate budding, and how the innate immune system counteracts such interactions to block virus egress. Despite the well known role of EBOV VP40 in promoting late stages of virus budding, its potential contributions to early budding stages (e.g. cytoskeletal remodeling and initial bud protrusion) are virtually unknown. Here, we will explore an unanticipated VP40-host interaction that may promote early stages of viral budding and its potential regulation by the innate immune system. Notably, we have identified host IQGAP1 as an interacting partner for EBOV VP40. IQGAP1 is a multidomain protein that orchestrates the formation of protein complexes involved in regulating cell motility, actin filament assembly, and filopodia and lamellipodia formation. Intriguingly, IQGAP1 possesses a WW- domain capable of interacting with a PPxY type L-domain of EBOV VP40, and our preliminary results using siRNA demonstrate that IQGAP1 is required for efficient egress of VP40 virus-like particles (VLPs). Based on these data, we will first test the hypothesis that EBOV VP40 L-domains sequentially recruit IQGAP1 via its WW-domain to initiate early budding events, followed by host Nedd4 and/or Tsg101 to facilitate late stage virus-cell separation (Aim 1). Importantly, IQGAP1 has been implicated as a target of the IFN-stimulated innate immune response that provides a critical first line of defense against invading pathogens. Indeed, interferon stimulated gene-15 (ISG15) is an innate immune response gene which has garnered recent attention due to its broad-range of antiviral activity against a plethora of pathogens including DNA and RNA viruses. As our preliminary results support the hypothesis that IQGAP1 promotes EBOV VLP budding, we hypothesize that ISGylation of host IQGAP1 by ISG15 may disrupt VP40-IQGAP1 complexes, thereby contributing to the subsequent decrease in functional VP40-Nedd4 and/or VP40-Tsg101 interactions required for budding. This unique interplay between host innate immune defenses and viral encoded factors that regulate the budding process will be explored in Aim 2. If our hypotheses are correct, these studies will: 1) challenge the current paradigm that viral L-domain motifs function only at late steps of virus budding, 2) identify novel, functional interactions between EBOV VP40 and host proteins/pathways involved in membrane protrusion, filopodia formation, and/or actin cytoskeletal remodeling that contribute to early budding events, and 3) reveal new host innate immune mechanisms that regulate budding of high priority, emerging pathogens.
Filoviruses cause severe hemorrhagic disease in humans and are classified as high priority, Category A pathogens and potential agents of bioterrorism. There are no vaccines or antiviral drugs currently available for the filoviruses. Here, we will use functional budding assays and live state-of-the-art imaging techniques to understand how novel host proteins and the innate immune system regulate the budding process.
|Harty, Ronald N (2018) Hemorrhagic Fever Virus Budding Studies. Methods Mol Biol 1604:209-215|
|Han, Ziying; Sagum, Cari A; Takizawa, Fumio et al. (2017) Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress. J Virol 91:|
|Liang, Jingjing; Sagum, Cari A; Bedford, Mark T et al. (2017) Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress. PLoS Pathog 13:e1006132|
|Han, Ziying; Sagum, Cari A; Bedford, Mark T et al. (2016) ITCH E3 Ubiquitin Ligase Interacts with Ebola Virus VP40 To Regulate Budding. J Virol 90:9163-71|
|Madara, Jonathan J; Han, Ziying; Ruthel, Gordon et al. (2015) The multifunctional Ebola virus VP40 matrix protein is a promising therapeutic target. Future Virol 10:537-546|
|Han, Ziying; Madara, Jonathan J; Liu, Yuliang et al. (2015) ALIX Rescues Budding of a Double PTAP/PPEY L-Domain Deletion Mutant of Ebola VP40: A Role for ALIX in Ebola Virus Egress. J Infect Dis 212 Suppl 2:S138-45|