Non‐canonical proline‐tyrosine interactions with multiple host proteins regulate Ebola virus infection
2021; Springer Nature; Volume: 40; Issue: 18 Linguagem: Inglês
10.15252/embj.2020105658
ISSN1460-2075
AutoresJyoti Batra, Hiroyuki Mori, Gabriel I. Small, Manu Anantpadma, Olena Shtanko, Nawneet Mishra, Mengru Zhang, Dandan Liu, Caroline G. Williams, Nadine Biedenkopf, Stephan Becker, Michael L. Gross, Daisy W. Leung, Robert A. Davey, Gaya K. Amarasinghe, Nevan J. Krogan, Christopher F. Basler,
Tópico(s)Hepatitis B Virus Studies
ResumoArticle2 August 2021free access Source DataTransparent process Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection Jyoti Batra Jyoti Batra J. David Gladstone Institutes, San Francisco, CA, USA Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA Quantitative Biosciences Institute, University of California, San Francisco, CA, USA Search for more papers by this author Hiroyuki Mori Hiroyuki Mori Department of Microbiology, NEIDL, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Gabriel I Small Gabriel I Small orcid.org/0000-0002-3506-5256 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA John T. Milliken Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Manu Anantpadma Manu Anantpadma orcid.org/0000-0001-5796-532X Department of Microbiology, NEIDL, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Olena Shtanko Olena Shtanko orcid.org/0000-0002-9848-8870 Host-Pathogen Interactions, Texas Biomedical Research Institute, San Antonio, TX, USA Search for more papers by this author Nawneet Mishra Nawneet Mishra Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Mengru Zhang Mengru Zhang orcid.org/0000-0001-9082-1272 Department of Chemistry, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Dandan Liu Dandan Liu Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Caroline G Williams Caroline G Williams Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA Search for more papers by this author Nadine Biedenkopf Nadine Biedenkopf Institute of Virology, Philipps University of Marburg, Marburg, Germany Search for more papers by this author Stephan Becker Stephan Becker Institute of Virology, Philipps University of Marburg, Marburg, Germany Search for more papers by this author Michael L Gross Michael L Gross orcid.org/0000-0003-1159-4636 Department of Chemistry, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Daisy W Leung Daisy W Leung Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA John T. Milliken Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Robert A Davey Corresponding Author Robert A Davey [email protected] orcid.org/0000-0001-9168-2892 Department of Microbiology, NEIDL, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Gaya K Amarasinghe Corresponding Author Gaya K Amarasinghe [email protected] Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Nevan J Krogan Corresponding Author Nevan J Krogan [email protected] orcid.org/0000-0003-4902-337X J. David Gladstone Institutes, San Francisco, CA, USA Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA Quantitative Biosciences Institute, University of California, San Francisco, CA, USA Search for more papers by this author Christopher F Basler Corresponding Author Christopher F Basler [email protected] orcid.org/0000-0003-4195-425X Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA Search for more papers by this author Jyoti Batra Jyoti Batra J. David Gladstone Institutes, San Francisco, CA, USA Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA Quantitative Biosciences Institute, University of California, San Francisco, CA, USA Search for more papers by this author Hiroyuki Mori Hiroyuki Mori Department of Microbiology, NEIDL, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Gabriel I Small Gabriel I Small orcid.org/0000-0002-3506-5256 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA John T. Milliken Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Manu Anantpadma Manu Anantpadma orcid.org/0000-0001-5796-532X Department of Microbiology, NEIDL, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Olena Shtanko Olena Shtanko orcid.org/0000-0002-9848-8870 Host-Pathogen Interactions, Texas Biomedical Research Institute, San Antonio, TX, USA Search for more papers by this author Nawneet Mishra Nawneet Mishra Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Mengru Zhang Mengru Zhang orcid.org/0000-0001-9082-1272 Department of Chemistry, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Dandan Liu Dandan Liu Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Caroline G Williams Caroline G Williams Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA Search for more papers by this author Nadine Biedenkopf Nadine Biedenkopf Institute of Virology, Philipps University of Marburg, Marburg, Germany Search for more papers by this author Stephan Becker Stephan Becker Institute of Virology, Philipps University of Marburg, Marburg, Germany Search for more papers by this author Michael L Gross Michael L Gross orcid.org/0000-0003-1159-4636 Department of Chemistry, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Daisy W Leung Daisy W Leung Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA John T. Milliken Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Robert A Davey Corresponding Author Robert A Davey [email protected] orcid.org/0000-0001-9168-2892 Department of Microbiology, NEIDL, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Gaya K Amarasinghe Corresponding Author Gaya K Amarasinghe [email protected] Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Nevan J Krogan Corresponding Author Nevan J Krogan [email protected] orcid.org/0000-0003-4902-337X J. David Gladstone Institutes, San Francisco, CA, USA Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA Quantitative Biosciences Institute, University of California, San Francisco, CA, USA Search for more papers by this author Christopher F Basler Corresponding Author Christopher F Basler [email protected] orcid.org/0000-0003-4195-425X Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA Search for more papers by this author Author Information Jyoti Batra1,2,3, Hiroyuki Mori4, Gabriel I Small5,6, Manu Anantpadma4,11, Olena Shtanko7, Nawneet Mishra5, Mengru Zhang8, Dandan Liu5, Caroline G Williams9, Nadine Biedenkopf10, Stephan Becker10, Michael L Gross8, Daisy W Leung5,6, Robert A Davey *,4, Gaya K Amarasinghe *,5, Nevan J Krogan *,1,2,3 and Christopher F Basler *,9 1J. David Gladstone Institutes, San Francisco, CA, USA 2Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA 3Quantitative Biosciences Institute, University of California, San Francisco, CA, USA 4Department of Microbiology, NEIDL, Boston University School of Medicine, Boston, MA, USA 5Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA 6John T. Milliken Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO, USA 7Host-Pathogen Interactions, Texas Biomedical Research Institute, San Antonio, TX, USA 8Department of Chemistry, Washington University School of Medicine, St. Louis, MO, USA 9Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA 10Institute of Virology, Philipps University of Marburg, Marburg, Germany 11Present address: Integrated Research Facility at Fort Detrick, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, Fort Detrick, Frederick, MD, 21702 USA *Corresponding author. Tel: +1 617 358 9166; E-mail: [email protected] *Corresponding author. Tel: +1 314 286 0619; E-mail: [email protected] *Corresponding author. Tel: +1 415 310 4524; E-mail: [email protected] *Corresponding author (Lead contact). Tel: +1 404 413 3651; E-mail: [email protected] The EMBO Journal (2021)40:e105658https://doi.org/10.15252/embj.2020105658 PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, and exert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription. Synopsis Protein mapping studies identified multiple VP30 interacting host protein possessing PPxPxY motifs, including RBBP6, hnRNP L, hnRNPUL1 and PEG10. RBBP6 and hnRNP L prevent VP30-NP interaction, impair VP30 dephosphorylation and inhibit viral RNA synthesis. Multiple host proteins possessing PPxPxY motifs can bind the Ebola virus VP30 protein. Proteins with the motif PxPPPPxY bind tightly to VP30, compete with the viral nucleoprotein for binding to VP30 and inhibit Ebola viral RNA synthesis. RBBP6 and hnRNP L modulate Ebola virus transcription by preventing the dephosphorylation of VP30. RBBP6 and hnRNP L act as restriction factors and can cooperatively modulate Ebola virus replication. Introduction Zaire ebolavirus (Ebola virus or EBOV), a member of the filovirus family of enveloped, non-segmented, negative-sense RNA viruses, is a zoonotic pathogen notable for its propensity to cause outbreaks of severe disease in humans. The public health significance of EBOV is evidenced by past outbreaks where reported case fatality rates ranged up to 90%: by the West Africa EBOV epidemic in 2013–2016, which resulted in more than 28,000 infections and more than 11,000 deaths; by four outbreaks in the Democratic Republic of Congo that have occurred from 2017 to 2021 and the reemergence of EBOV disease in Guinea in 2021 (Bausch et al, 2016; Ilunga Kalenga et al, 2019; Nsio et al, 2019; Adepoju, 2021). The EBOV genomic RNA is ˜19 kilobases in length and has seven separate transcriptional units (genes) that encode distinct mRNAs. The genome is encapsidated by nucleoprotein (NP) with the resulting ribonucleoprotein serving as the template for RNA synthesis reactions that replicate the viral genomic RNA and transcribe the mRNAs that encode the viral proteins. Viral genome replication requires, in addition to NP, the viral proteins VP35 and L, the viral RNA-dependent RNA polymerase. Transcription requires these proteins and the EBOV VP30 protein (Muhlberger et al, 1998; Muhlberger et al, 1999). EBOV VP30 is a zinc- and RNA-binding protein and a component of the viral nucleocapsid complex (Becker et al, 1998; Muhlberger et al, 1999; Modrof et al, 2003; John et al, 2007; Nanbo et al, 2013; Biedenkopf et al, 2016b; Schlereth et al, 2016). VP30 is essential for the virus life cycle (Muhlberger et al, 1999; Enterlein et al, 2006; Halfmann et al, 2008; Biedenkopf et al, 2016a). A critical role for EBOV VP30 is in initiation of viral transcription, a function that is dependent on a stem-loop structure present at the NP gene start site; disruption of this secondary structure leads to VP30-independent transcription (Weik, Modrof et al, 2002). EBOV VP30 also facilitates re-initiation at downstream genes during viral transcription and regulates editing by the viral polymerase during synthesis of nascent mRNAs from the glycoprotein (GP) gene (Martinez et al, 2008; Mehedi et al, 2013). A substantial body of data implicates VP30 phosphorylation as a regulator of its transcriptional function. Dephosphorylation of VP30 N-terminal serine and threonine residues or mutation of these residues to alanine increases pro-transcriptional activity; whereas phosphorylation or mutation to aspartic acid inhibits transcription and promotes viral genome replication (Modrof et al, 2002; Martinez et al, 2011; Biedenkopf et al, 2013; Ilinykh et al, 2014; Biedenkopf et al, 2016a; Lier et al, 2017; Ammosova et al, 2018; Kruse et al, 2018; Tigabu et al, 2018). VP30 interacts with NP, and this influences VP30 phosphorylation levels (Biedenkopf et al, 2013; Kirchdoerfer et al, 2016; Lier et al, 2017; Xu et al, 2017; Kruse et al, 2018). NP recruits the host PP2A-B56 protein phosphatase through a LxxIxE motif, promoting VP30 dephosphorylation and viral transcription (Kruse et al, 2018). This activity likely explains the importance of VP30–NP interaction for RNA synthesis, as was demonstrated in studies that defined the structure of the VP30–NP interaction interface (Kirchdoerfer et al, 2016; Xu et al, 2017). Recently, a comprehensive affinity tag-purification mass spectrometry (AP-MS) analysis of the EBOV–host protein–protein interactome identified a number of VP30-interacting host proteins, including retinoblastoma binding protein 6 (RBBP6) (Batra et al, 2018). RBBP6, a multi-domain protein with E3 ubiquitin ligase activity, has been implicated in cell cycle progression, nucleic acid metabolism, cell proliferation, and differentiation (Ntwasa, 2016). RBBP6 possesses a PPxPxY motif that was demonstrated to interact with VP30 at a site that binds a PPxPxY motif on NP. RBBP6 can compete with NP for binding to VP30, thereby inhibiting viral gene expression. In this study, we characterize VP30 interaction with an additional three host proteins that possess PPxPxY motifs. The data demonstrate that the extended sequence PxPPPPxY mediates optimal binding and that proteins sharing this motif compete with NP for binding to a common site on VP30, alter VP30 phosphorylation, modulate viral mRNA transcription, and influence viral infectivity. These findings reveal a unique virus–host interaction where multiple host factors target the same viral interface to alter replication efficiency. Results Multiple host proteins possessing PPxPxY motifs interact with EBOV VP30 A previous study identified RBBP6 as a VP30-interacting protein and demonstrated that a PPxPxY motif mediates the interaction with VP30; this is reminiscent of a PPxPxY motif present in the EBOV NP protein that binds to VP30 (Fig 1A; Batra et al, 2018). The same study also identified the cellular proteins hnRNP L and hnRNPUL1 in both HEK293T and Huh7 cells and PEG10 in Huh7 cells as VP30 interactors (Batra et al, 2018; Fig 1B). All three of these interactors also contain PPxPxY motifs. To further validate these interactions, FLAG-tagged host proteins were co-expressed with HA-tagged VP30 in HEK293T cells, and pull downs were performed using anti-FLAG beads (Fig 1C). hnRNP L, hnRNPUL1, and PEG10 each interacted robustly with VP30, similar to RBBP6. The interaction between VP30 and the endogenous host proteins was further addressed by co-immunoprecipitation. Endogenous RBBP6, hnRNP L, and hnRNPUL1 were each co-immunoprecipitated in the presence of HA-VP30 but not in the presence of empty vector (Fig 1D). The HA–VP30 interaction with PEG10 was more equivocal in HEK293T cells. PEG10 is highly expressed in hepatocellular carcinoma cells and was previously identified as an interactor in only Huh7 cells; therefore, we confirmed the interaction of VP30 with endogenous PEG10 in these cells (Fig EV1). Figure 1. VP30 interacts with multiple host proteins that contain PPxPxY motifs A model for RBBP6 and NP interaction with VP30 via PPxPxY motifs to regulate viral transcription. Both EBOV NP and human RBBP6 share a common PPxPxY motif for binding to EBOV transcription factor VP30. Interaction between NP and VP30 modulates viral RNA synthesis because RBBP6 outcompetes NP for VP30 binding. An EBOV VP30-human protein–protein interaction network. Purple and green prey nodes indicate that the protein was identified as a VP30 interactor in HEK293T or Huh7 cells, respectively; purple-green bifurcation indicates that the interaction was identified in both cell types. Gray lines correspond to human–human protein–protein interactions curated in the publicly available CORUM database. Host proteins containing PPxPxY motif(s) are circled in red. Co-immunoprecipitation between VP30 and the indicated PPxPxY-containing host factors. Empty expression plasmid (Vector) or FLAG-tagged host protein expression plasmids were co-transfected with an HA-VP30 expression plasmid in HEK293T cells. An anti-FLAG immunoprecipitation (IP: Anti-FLAG) was performed. Western blots of IP and whole-cell lysates (WCL) are shown. Anti-β-tubulin was used as a loading control for the WCLs. Cells were transfected with empty vector or HA-VP30 expression plasmid, and IPs were performed with anti-HA antibody (IP: Anti-HA). Western blotting with anti-HA tag or antibodies to the indicated host proteins was performed on IP and on whole-cell lysates (WCL). Source data are available online for this figure. Source Data for Figure 1 [embj2020105658-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Co-immunoprecipitation between VP30 and endogenous PEG10 Huh7 cells were transfected with empty vector (Vector) or FLAG-VP30 expression plasmid. IPs (IP: Anti-FLAG) and whole-cell lysates (WCL) were analyzed by Western blotting with anti-FLAG or anti-PEG10 antibodies. Download figure Download PowerPoint Molecular dissection of PPxPxY-mediated interactions with VP30 There is only one PPxPxY motif within the proline (Pro)-rich region of RBBP6. However, in hnRNP L, PEG10, and hnRNPUL1, this motif appears two or three times (Fig 2A, left). The amino acid sequences of peptides derived from these host proteins, including 10 amino acids flanking each PPxPxY motif, were aligned (Fig 2A, right). Sequences corresponding to each of these extended peptides (labeled as 1–3) were cloned as N-terminal GFP fusions. The fusion proteins were tested in co-immunoprecipitation assays for their ability to bind VP30. In each case, only one of the tested peptides from hnRNP L (hnRNP L_2), hnRNPUL1 (hnRNPUL1_3), and PEG10 (PEG10_2) strongly interacted with VP30, suggesting that the sequence context of the motif plays a significant role in binding (Fig 2B). Also observed was a weak interaction between hnRNPUL1 peptide 1 and VP30. Figure 2. PPxPxY-containing peptides derived from host proteins interact with VP30 and modulate EBOV RNA synthesis Schematic representation of the arrangement and number of PPxPxY motifs present in the indicated host proteins (left panel). Multiple sequences aligned using ClustalW, of peptides containing PPxPxY motif derived from cellular proteins and from the EBOV NP protein (right panel). Co-immunoprecipitation between HA-VP30 and either GFP or GFP-fused to peptides derived from the host proteins was performed. Western blots of the co-immunoprecipitation and whole-cell lysate (WCL) are shown. Equilibrium dissociation curves of FITC-RBBP6 from eVP30130-272 in the presence of increasing concentrations (0.13–500 μM) of the PPxPxY-containing peptides. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272, at 0.50 μM and 3.8 μM, respectively. Experiments were performed in two independent replicates. Error bars represent standard deviation. Source data are available online for this figure. Source Data for Figure 2 [embj2020105658-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint To quantify the strength of individual peptide binding, and to determine whether the peptides can target the VP30 interface bound by RBBP6 and NP, a fluorescence polarization (FP) assay was employed in which a purified C-terminal domain of EBOV VP30 (eVP30130-272) and a FITC-labeled RBBP6 peptide containing the PPxPxY motif were used (Fig 2C). The RBBP6 peptide displayed the highest binding affinity. hnRNPUL1_3 and PEG10_2 demonstrated affinities similar to the NP peptide, whereas hnRNP L_2 and hnRNPUL1_1 exhibited slightly lower affinities. hnRNP L_1, hnRNPUL1_2, and PEG10_1 exhibited minimal to no binding in the FP assays. Overall, these data support the PPxPxY motif as contributing to binding; however, the extended sequence PxPPPPxY and its composition contribute to the strength of interaction. The EBOV protein VP40 possesses overlapping PTAP and PPEY motifs (PTAPPEY) that function as late domains in viral budding (Licata et al, 2003). To determine whether this sequence mediates an interaction with VP30 and to further address the specificity of Pro-rich motifs to interact with VP30, EBOV VP40/VP30 co-immunoprecipitations were performed. VP40 did not co-precipitate with VP30 (Fig EV2A). Furthermore, fusion of the N-terminal VP40 PTAPPEY sequence to GFP also did not facilitate co-precipitation of VP30. Mutation of the late domain motif to contain five proline residues (PPPPPEY) failed to confer interaction. However, mutation of the VP40 peptide to include an additional sixth proline (PPPPPPEY) resulted in interaction (Fig EV2B). These data further support the extended PxPPPPxY motif as requisite for efficient binding to VP30. Click here to expand this figure. Figure EV2. VP30 does not bind to the native VP40 proline-tyrosine motif but does bind to bat RBBP6 Co-immunoprecipitation assay to assess HA–VP40 interaction with FLAG-VP30. HEK293T cells were transfected with HA-VP40 plasmid and either empty vector or FLAG-VP30 plasmid. An anti-FLAG immunoprecipitation (IP) was performed. IP and WCL were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. An anti-β-tubulin Western blot provided a loading control. Anti-GFP immunoprecipitation of HA-VP30 with GFP fusions to wild-type or mutated VP40-derived peptides (GFP-VP40, GFP-VP40_mut1, or GFP-VP40_mut2). GFP without a fusion partner (GFP) and GFP fused to RBBP6 peptide (GFP-RBBP6) served as controls. Immunoblots with anti-HA, anti-GFP, and anti-β-tubulin are shown. GFP fused to peptides derived from human and bat (Rousettus aegyptiacus) RBBP6 were expressed along with HA-VP30. Co-immunoprecipitation was performed using anti-GFP magnetic beads, and representative immunoblots for IP and WCL are shown. Equilibrium dissociation curves of FITC-RBBP6 peptide to eVP30130-272 as it is outcompeted by increasing concentrations (0.13–500 μM) of human and bat RBBP6 peptides. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272, at 0.50 μM and 3.8 μM, respectively. Experiments were performed in two independent replicates. Error bars represent standard deviation. MG activity upon titration of GFP fused to human and bat-derived RBBP6 peptides (12.5 ng and 125 ng). GFP alone was used as a control. Reporter activity was read at 48 h post-transfection and fold MG activity was calculated relative to a no VP30 control. # denotes statistical significance compared with RBBP6 peptide for each dose. The data represent the mean ± SD from one representative experiment in which each transfection condition was performed in triplicate (n = 3). Each experiment was reproduced in at least two additional, independent experiments (see Appendix Fig S1). Statistical significance was calculated relative to GFP control for each concentration tested using ANOVA with Tukey’s multiple comparisons test. ****P < 0.00005; ##P < 0.005, *P < 0.05 Download figure Download PowerPoint We also tested an RBBP6 peptide derived from a fruit bat (Rousettus aegyptiacus) (bRBBP6 peptide) that serves as MARV reservoir hosts in co-precipitation assays with VP30. bRBBP6 peptide differs at only two positions from the human RBBP6 peptide and binds robustly to VP30 with a near identical affinity, further substantiating the requirement of the PxPPPPxY motif for optimal binding to VP30 (Fig EV2C and D). The RBBP6 peptide was demonstrated to inhibit EBOV RNA synthesis, as measured by a minigenome (MG) assay (Batra et al, 2018). When titrated in the MG assay, bRBBP6 was modestly less inhibitory to MG activity, as compared to the RBBP6 peptide (Fig EV2E, Appendix Fig S1). The binding in solution between VP30 and different host peptides, including the interacting RBBP6, hnRNPUL1_1, hnRNPUL1_3, hnRNP L_2, and PEG10_2, and the non-interacting hnRNPUL1_2, was further characterized with hydrogen-deuterium exchange (HDX) mass spectrometry. RBBP6, hnRNPUL1_1, hnRNPUL1_3, hnRNP L_2, and PEG10_2 peptides bind nearly identically to VP30 as seen in the HDX kinetic curves, indicating that each peptide binds to a similar region on VP30 (Figs 3A and EV3). Statistical analysis of deuterium uptake differences identified two strong peptide-binding regions on VP30 from residue L189 to V210 and from residue A224 to D231. The two regions contain seven and four binding residues, respectively, that were identified by X-ray crystallography (PDB 6E5X; Batra et al, 2018), demonstrating consistency of the two approaches and the ability of HDX to give an accurate picture of the binding interface. The middle region from residue Y211 to E223 presents as a weaker binding region where the differences in HDX are much smaller between bound and unbound, also consistent with the X-ray structure that show binding via only one residue, R213. Although there is clear binding of residues 224–231, the region 230–237 shows different character in the kinetics of HDX. Its extent for the bound state converges at long times with that of the unbound, indicating a larger off rate and weaker binding at the end of the peptide ligand. We further mapped the HDX differences at 4 h onto the VP30 crystal structure, which illustrates common binding regions among different host factor peptides (Fig 3B). Figure 3. HDX-MS analysis of VP30 and host factor peptide interactions HDX kinetics data for VP30 alone and for the indicated host peptides incubated with VP30 are shown. VP30 peptide amino acid residue positions and sequences are indicated at the top of each curve. The error bars indicate mean ± SD (n = 3). Deuterium uptake differences were mapped onto the VP30 structure (PDB 6E5X), revealing similar binding regions for the peptides. Residue-level uptake difference was achieved by overlapping peptides. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. HDX kinetic plots for all VP30 peptides Download figure Download PowerPoint In addition, the HDX studies revealed another region, residues Q248 to E252, that describes tightening dynamics or remote conformational changes upon peptide binding (Figs 3B and EV4). For hnRNPUL_2, differential HDX kinetic analysis in the absence and presence of the ligand yielded similar HDX kinetics, consistent with the lack of its binding in the pull-down and fluorescence polarization assays. Click here to expand this figure. Figure EV4. Statistical analysis of deuterium uptake differences of all time points confirms the binding regions Deuterium uptake differences between different bound VP30 and unbound VP30 were calculated for each time point from 10 s to 4 h, and are depicted by gradient colors. Standard deviation between triplicates was calculated separately for each time point representing the bound and unbound states. Plotted on the graph is a threefold propagation error of each time point as shown by the error bars, giving 99.7% certainty on the observed difference. VP30 bound with RBBP6, hnRNPUL1_1, h
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