Modular mimicry and engagement of the Hippo pathway by Marburg virus VP40: Implications for filovirus biology and budding

Han, Ziying; Dash, Sanjit Kumar; Sagum, Cari A.; Ruthel, Gordon; Jaladanki, Chaitanya K.; Berry, Corbett T.; Schwoerer, Michael P.; Harty, Nina M.; Freedman, Bruce D.; Bedford, Mark T.; Fan, Hao; Sidhu, Sachdev S.; Sudol, Marius; Shtanko, Olena; Harty, Ronald N.

doi:10.1371/journal.ppat.1008231

Cited by 13 publications

(27 citation statements)

References 96 publications

(122 reference statements)

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“…Expression and purification of GST fusion protein from pGEX-ZO1-2/3 plasmid was performed as described previously [35,36]. Briefly, the plasmids were transformed into E. coli BL21(DE3) cells and single colonies were cultured in 10ml of LB media overnight with shaking at 37˚C.…”

Section: Expression and Purification Of Gst-zo1-pdz-2 Fusion Proteinmentioning

confidence: 99%

SARS-CoV-2 Envelope (E) protein interacts with PDZ-domain-2 of host tight junction protein ZO1

et al. 2021

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Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread.

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Section: Expression and Purification Of Gst-zo1-pdz-2 Fusion Proteinmentioning

confidence: 99%

SARS-CoV-2 Envelope (E) protein interacts with PDZ-domain-2 of host tight junction protein ZO1

et al. 2021

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“…We recently revealed a key role for endogenous Amot in positively regulating egress and spread of PPxY-containing filoviruses (32, 33). Amotp130 contains multiple PPxY motifs that mediate interactions with WW-domains of YAP, BAG3, and WWOX (negative regulators of budding), and in doing so, function as a master regulator of several physiologically relevant pathways/processes, including transcription (Hippo pathway), actin polymerization, and tight junction (TJ) formation/integrity (45–54).…”

Section: Discussionmentioning

confidence: 99%

Angiomotin Counteracts the Negative Regulatory Effect of Host WWOX on Viral PPxY-Mediated Egress

Liang

Ruthel

Sagum

et al. 2021

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Filoviridae family members Ebola (EBOV) and Marburg (MARV) viruses and Arenaviridae family member Lassa virus (LASV) are emerging pathogens that can cause hemorrhagic fever and high rates of mortality in humans. A better understanding of the interplay between these viruses and the host will inform about the biology of these pathogens, and may lead to the identification of new targets for therapeutic development. Notably, expression of the filovirus VP40 and LASV Z matrix proteins alone drives assembly and egress of virus-like particles (VLPs). The conserved PPxY Late (L) domain motifs in the filovirus VP40 and LASV Z proteins play a key role in the budding process by mediating interactions with select host WW-domain containing proteins that then regulate virus egress and spread. To identify the full complement of host WW-domain interactors, we utilized WT and PPxY mutant peptides from EBOV and MARV VP40 and LASV Z proteins to screen an array of GST-WW-domain fusion proteins. We identified WW domain-containing oxidoreductase (WWOX) as a novel PPxY-dependent interactor, and we went on to show that full-length WWOX physically interacts with eVP40, mVP40 and LASV Z to negatively regulate egress of VLPs and of a live VSV/Ebola recombinant virus (M40). Interestingly, WWOX is a versatile host protein that regulates multiple signaling pathways and cellular processes via modular interactions between its WW-domains and PPxY motifs of select interacting partners, including host angiomotin (AMOT). Notably, we demonstrated recently that expression of endogenous AMOT not only positively regulates egress of VLPs, but also promotes egress and spread of live EBOV and MARV. Toward the mechanism of action, we show that the competitive and modular interplay among WWOX-AMOT-VP40/Z regulates VLP and M40 virus egress. Thus, WWOX is the newest member of an emerging group of host WW-domain interactors (e.g. BAG3; YAP/TAZ) that negatively regulate viral egress. These findings further highlight the complex interplay of virus-host PPxY/WW-domain interactions and their potential impact on the biology of both the virus and the host during infection.Author SummaryFiloviruses (Ebola [EBOV] and Marburg [MARV]) and arenavirus (Lassa virus; LASV) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we identified host WW-domain containing protein WWOX as a novel interactor with VP40 and Z, and showed that WWOX inhibited budding of VP40/Z virus-like particles (VLPs) and live virus in a PPxY/WW-domain dependent manner. Our findings are important to the field as they expand the repertoire of host interactors found to regulate PPxY-mediated budding of RNA viruses, and further highlight the competitive interplay and modular virus-host interactions that impact both the virus lifecycle and the host cell.

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“…MARV, like many other enveloped RNA viruses, relies on its matrix protein (mVP40) to direct and promote budding of infectious virions. We and others have demonstrated that mVP40 completes the budding process, in part, by using its highly conserved PPxY L-domain motif to hijack host proteins/pathways that then facilitate efficient virus-cell separation (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). One of the best characterized host interactors is WWdomain containing E3 ubiquitin ligase, Nedd4 (7,(16)(17)(18).…”

mentioning

confidence: 99%

“…Notably, we and others have shown that Nedd4 and Nedd4 family members physically interact with viral PPxY motifs via one or more of their WWdomains, and functionally interact with viral PPxY-containing proteins to enhance or promote budding of virus-like particles (VLPs) and live virus (7,(16)(17)(18)(19)(20)(21)(22)(23)(24). The highly conserved physical and functional nature of the PPxY motif in a wide array of RNA viruses, makes it an attractive target for the development of antivirals (2,(25)(26)(27)(28)(29)(30). Indeed, compounds targeting the PPxY/WW-domain virus-host interaction would be predicted to dampen or reduce the ability of the virus to bud or pinch-off from infected cells, thus allowing an individual's immune system more time to combat and clear the virus.…”

mentioning

confidence: 99%

“…Collected samples were analyzed by LC-MS/MS. PK parameters were calculated using Prism Graphpad.VLP Budding AssaysMARV VP40 VLP budding assays in HEK293T cells were described previously(2). For VLP budding, HEK293T cells were transfected with 0.5µg of mVP40, and cells were treated with DMSO alone or theindicated concentration of inhibitor for 24 hours post transfection.…”

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confidence: 99%

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Compound FC-10696 Inhibits Egress and Spread of Marburg Virus

Han

Liang

et al. 2021

Preprint

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Marburg virus (MARV) VP40 protein (mVP40) directs egress and spread of MARV, in part, by recruiting specific host WW-domain containing proteins via its conserved PPxY Late (L) domain motif to facilitate efficient virus-cell separation. We reported previously that small molecule compounds targeting the viral PPxY/host WW-domain interaction inhibited VP40-mediated egress and spread. Here, we report on the antiviral potency of novel compound FC-10696, which emerged from extensive structure activity relationship (SAR) of a previously described series of PPxY inhibitors. We show that FC-10696 inhibits egress of both mVP40 VLPs and egress and spread of authentic MARV from HeLa cells and primary human macrophages. Moreover, FC-10696 treated mice displayed delayed onset of weight loss, clinical signs, and significantly lower viral loads compared to controls, with 14% of animals surviving 21 days following a lethal MARV challenge. Thus, FC-10696 represents a first-in-class, host-oriented inhibitor effectively targeting late stages of the MARV lifecycle.

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Modular mimicry and engagement of the Hippo pathway by Marburg virus VP40: Implications for filovirus biology and budding

Cited by 13 publications

References 96 publications

SARS-CoV-2 Envelope (E) protein interacts with PDZ-domain-2 of host tight junction protein ZO1

SARS-CoV-2 Envelope (E) protein interacts with PDZ-domain-2 of host tight junction protein ZO1

Angiomotin Counteracts the Negative Regulatory Effect of Host WWOX on Viral PPxY-Mediated Egress

Compound FC-10696 Inhibits Egress and Spread of Marburg Virus

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