Role of Src homology domain binding in signaling complexes assembled by the murid γ-herpesvirus M2 protein

γ-Herpesviruses express proteins that modulate B lymphocyte signaling to achieve persistent latent infections. One such protein is the M2 latency-associated protein encoded by the murid herpesvirus-4. M2 has two closely spaced tyrosine residues, Tyr120 and Tyr129, which are phosphorylated by Src family tyrosine kinases. Here we used mass spectrometry to identify the binding partners of tyrosine-phosphorylated M2. Each M2 phosphomotif is shown to bind directly and selectively to SH2-containing signaling molecules. Specifically, Src family kinases, NCK1 and Vav1, bound to the Tyr(P)120 site, PLCγ2 and the SHP2 phosphatase bound to the Tyr(P)129 motif, and the p85α subunit of PI3K associated with either motif. Consistent with these data, we show that M2 coordinates the formation of multiprotein complexes with these proteins. The effect of those interactions is functionally bivalent, because it can result in either the phosphorylation of a subset of binding proteins (Vav1 and PLCγ2) or in the inactivation of downstream targets (AKT). Finally, we show that translocation to the plasma membrane and subsequent M2 tyrosine phosphorylation relies on the integrity of a C-terminal proline-rich SH3 binding region of M2 and its interaction with Src family kinases. Unlike other γ-herpesviruses, that encode transmembrane proteins that mimic the activation of ITAMs, murid herpesvirus-4 perturbs B cell signaling using a cytoplasmic/membrane shuttling factor that nucleates the assembly of signaling complexes using a bilayered mechanism of phosphotyrosine and proline-rich anchoring motifs.This work was supported by Portuguese Fundação para a Ciência e Tecnologia (FCT) Grant PTDC/SAU-MII/099314/2008 (to J. P. S.) and Spanish Association Against Cancer and Spanish Ministry of Economy and Competitiveness Grants SAF2009-07172 and RD06/0020/0001, respectively (to X. R. B.). Spanish funding is co-sponsored by the European FEDER program. The SPR equipment at the Instituto de Tecnologia Química e Biológica was acquired with FCT Grant PNRC/692/BIO/2264/2005.Peer Reviewe

The Kaposi Sarcoma Herpesvirus latency-associated nuclear antigen DNA binding domain dorsal positive electrostatic patch facilitates DNA replication and episome persistence

© 2015 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.info:eu-repo/semantics/publishedVersio

Cross-species conservation of episome maintenance provides a basis for in vivo investigation of Kaposi's sarcoma herpesvirus LANA

Copyright: © 2017 Habison et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Many pathogens, including Kaposi's sarcoma herpesvirus (KSHV), lack tractable small animal models. KSHV persists as a multi-copy, nuclear episome in latently infected cells. KSHV latency-associated nuclear antigen (kLANA) binds viral terminal repeat (kTR) DNA to mediate episome persistence. Model pathogen murine gammaherpesvirus 68 (MHV68) mLANA acts analogously on mTR DNA. kLANA and mLANA differ substantially in size and kTR and mTR show little sequence conservation. Here, we find kLANA and mLANA act reciprocally to mediate episome persistence of TR DNA. Further, kLANA rescued mLANA deficient MHV68, enabling a chimeric virus to establish latent infection in vivo in germinal center B cells. The level of chimeric virus in vivo latency was moderately reduced compared to WT infection, but WT or chimeric MHV68 infected cells had similar viral genome copy numbers as assessed by immunofluorescence of LANA intranuclear dots or qPCR. Thus, despite more than 60 Ma of evolutionary divergence, mLANA and kLANA act reciprocally on TR DNA, and kLANA functionally substitutes for mLANA, allowing kLANA investigation in vivo. Analogous chimeras may allow in vivo investigation of genes of other human pathogens.This work was supported in part by National Institutes of Health grants CA082036 (NCI), DE025208, and DE024971 (both NIDCR), to KMK, FCT PTDC/IMI-MIC/0980/2014 to JPS, FCT Harvard Medical School Portugal Program in Translational Research (HMSP-ICT/0021/2010) to JPS, KMK, CEM, Instituto de Medicina Molecular Directors Fund to JPS, and iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) FCT/FEDER (PT2020 Partnership Agreement) to CEM. M.P.M is supported by a fellowship from Fundação para a Ciência e Tecnologia (FCT), Portugal.info:eu-repo/semantics/publishedVersio

MLL1 is regulated by KSHV LANA and is important for virus latency

Mixed lineage leukemia 1 (MLL1) is a histone methyltransferase. Kaposi's sarcoma-associated herpesvirus (KSHV) is a leading cause of malignancy in AIDS. KSHV latently infects tumor cells and its genome is decorated with epigenetic marks. Here, we show that KSHV latency-associated nuclear antigen (LANA) recruits MLL1 to viral DNA where it establishes H3K4me3 modifications at the extensive KSHV terminal repeat elements during primary infection. LANA interacts with MLL1 complex members, including WDR5, integrates into the MLL1 complex, and regulates MLL1 activity. We describe the 1.5-A crystal structure of N-terminal LANA peptide complexed with MLL1 complex member WDR5, which reveals a potential regulatory mechanism. Disruption of MLL1 expression rendered KSHV latency establishment highly deficient. This deficiency was rescued by MLL1 but not by catalytically inactive MLL1. Therefore, MLL1 is LANA regulable and exerts a central role in virus infection. These results suggest broad potential for MLL1 regulation, including by non-host factors.info:eu-repo/semantics/publishedVersio

Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein

Vaccinia virus (VACV) encodes many immunomodulatory proteins, including inhibitors of apoptosis and modulators of innate immune signalling. VACV protein N1 is an intracellular homodimer that contributes to virus virulence and was reported to inhibit nuclear factor (NF)-κB signalling. However, analysis of NF-κB signalling in cells infected with recombinant viruses with or without the N1L gene showed no difference in NF-κB-dependent gene expression. Given that N1 promotes virus virulence, other possible functions of N1 were investigated and this revealed that N1 is an inhibitor of apoptosis in cells transfected with the N1L gene and in the context of VACV infection. In support of this finding virally expressed N1 co-precipitated with endogenous pro-apoptotic Bcl-2 proteins Bid, Bad and Bax as well as with Bad and Bax expressed by transfection. In addition, the crystal structure of N1 was solved to 2.9 Å resolution (0.29 nm). Remarkably, although N1 shows no sequence similarity to cellular proteins, its three-dimensional structure closely resembles Bcl-xL and other members of the Bcl-2 protein family. The structure also reveals that N1 has a constitutively open surface groove similar to the grooves of other anti-apoptotic Bcl-2 proteins, which bind the BH3 motifs of pro-apoptotic Bcl-2 family members. Molecular modelling of BH3 peptides into the N1 surface groove, together with analysis of their physico-chemical properties, suggests a mechanism for the specificity of peptide recognition. This study illustrates the importance of the evolutionary conservation of structure, rather than sequence, in protein function and reveals a novel anti-apoptotic protein from orthopoxviruses

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Stochastic Detection of MPSA-Gold Nanoparticles Using a α‑Hemolysin Nanopore Equipped with a Noncovalent Molecular Adaptor

We present the first study of a novel, more sensitive method for the characterization of nanoparticles (NPs). This approach combines detection via a protein nanopore with modification of its interaction behavior using a molecular adaptor. We identify different populations of 3-mercapto-1-propanesulfonate (MPSA)-modified-gold NPs using the biological nanopores α-hemolysin (αHL) and its M113N mutant equipped with a noncovalently bound γ-cyclodextrin molecule as a stochastic sensor. Identification takes place on the basis of the extent of current blockades and residence times. Here, we demonstrate that noncovalently attached adaptors can be used to change the sensing properties of αHL nanopores, allowing the detection and characterization of different populations of MPSA NPs. This is an advance in sensitivity and diversity of NP sensing, as well as a promising and reliable technology to characterize NPs by using protein nanopores

Sensing Single Mixed-Monolayer Protected Gold Nanoparticles by the alpha-Hemolysin Nanopore

Gold nanoparticles are widely used in various applications in fields including chemistry, engineering, biology, medicine, and electronics. These materials can be synthesized and modified with ligands containing different functional groups. Among nanoparticles' characteristics, chemical surface composition is likely to be a crucial feature, demanding robust analytical methodologies for its assessment. Single molecule analysis using the biological nanopores alpha-hemolysin and its E111A mutant is presented here as a promising methodology to stochastically sense organic monolayer protected gold-nanoparticles with different ligand shell compositions. By monitoring the ionic current across a single protein nanopore, differences in the physical and chemical characteristics (e.g., size, ligand shell composition, and arrangement) of individual nanoparticles can be distinguished based on the differences in the current blockade events that they cause. Such differences are observed in the spread of both the amplitude and duration of current blockades. These values cannot be correlated with a single physical characteristic. Instead the spread represents a measure of heterogeneity within the nanoparticle population. While our results compare favorably with the more traditional analytical methodologies, further work will be required to improve the accuracy of identification of the NPs and understand the spread of values within a nanoparticle preparation as well as the overlap between similar preparations

Latency-Associated Nuclear Antigen E3 Ubiquitin Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation

Aerial view, from Nelson Monument; The monument dominates the top of Calton Hill, just to the east of Edinburgh's New Town. Construction started in 1826 and the building was left in its unfinished state in 1829. It is Scotland's national memorial to the Scottish soldiers and sailors who died fighting in the Napoleonic Wars. Playfair was asked to collaborate with Charles Robert Cockerell in the creation of an exact facsimile of the Parthenon to crown Calton Hill. Its verisimilitude extended to the masonry, whose precision was to rival in sandstone the marble original. Financial constraints brought it to a dead halt in a few years, and for Playfair it remained "a striking proof of the pride and poverty of the Scots"; however, the experience provided the final polish to his architectural education, and its exacting construction set a standard of excellence from which he never deviated. Source: Grove Art Online; http://www.oxfordartonline.com/ (accessed 6/30/2012

Overexpression, purification and crystallization of the tetrameric form of SorC sorbitol operon regulator

The sorbitol operon regulator from K. pneumoniae has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 3.2 Å