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

Características sociodemográficas, hábitos de vida e condições de saúde de pessoas privadas de liberdade

The deprivation of liberty, due to its characteristics, imposes on people differentiated habits and customs that can influence their health. In that sense, the objective of this is to verify the prevalence of chronic diseases in the prison population. This is a cross-sectional descriptive study, carried out in four prison unity in a city in southern Brazil. Data collection was performed by a semi-structured instrument and descriptive statistics were used for analysis. Participated 326 people deprived of freedom, 90.8% were male, 53.4% young, aged between 18 and 29 years, 43.3% single, 55.8% with less than nine years of schooling, 61.3% performed some activity in the penal unit, 63.2% were smokers or former smokers, 28.2% drank alcohol and 60.4% used or ex used illicit drugs, 71.2% practiced physical activities, 86.1% positively evaluated their health status and 52.5% reported some chronic disease. The most prevalent self-reported diseases were respiratory, gastrointestinal, mental, cardiovascular, and musculoskeletal. People deprived of freedom have chronic diseases and risk factors prevalent in the general population. Knowing the epidemiological profile of this population group can contribute to health-promoting actions, prevention, and control of risk factors.La privación de la libertad, por sus características, impone a las personas hábitos y costumbres diferenciados que pueden influir en su salud. En ese sentido, el propósito de este estudio es describir las características sociodemográficas, hábitos de vida y condiciones de salud de las personas privadas de libertad. Se trata de un estudio transversal y descriptivo, realizado en cuatro centros penitenciarios de una ciudad del sur de Brasil. La recopilación de datos se realizó mediante un instrumento semiestructurado y se utilizó la estadística descriptiva para el análisis. Participaron 326 personas privadas de libertad, 90,8% eran hombres, 53,4% jóvenes, entre 18 y 29 años, 43,3% solteros, 55,8% con menos de nueve años de escolaridad, 61,3% realizaban alguna actividad en la unidad carcelaria, 63,2% eran fumadores o exfumadores, 28,2% ingerían bebidas alcohólicas y 60,4% eran usuarios o exusuarios de drogas ilícitas, 71,2% practicaban actividades físicas, 86,1% evaluaban positivamente su estado de salud y 52,5% reportaban alguna enfermedad crónica. Las enfermedades más frecuentes declaradas en sus relatos fueron las respiratorias, las gastrointestinales, las mentales, las cardiovasculares y las musculoesqueléticas. Las personas privadas de libertad tienen enfermedades crónicas y factores de riesgo prevalentes en la población general. Conocer el perfil epidemiológico de este grupo de población puede contribuir a las acciones de promoción de la salud, prevención y control de los factores de riesgo.A privação de liberdade, por suas características, impõe as pessoas hábitos e costumes diferenciados que podem influenciar em sua saúde. Nesse sentido, o objetivo deste é descrever as características sociodemográficas, hábitos de vida e condições de saúde de pessoas privadas de liberdade. Trata-se de um estudo transversal, descritivo, realizado em quatro unidades penais de um município do sul do Brasil. A coleta de dados foi realizada por instrumento semiestruturado e utilizou-se estatística descritiva para análise. Participaram 326 pessoas privadas de liberdade, dessas 90,8% eram do sexo masculino, 53,4% jovens, com idade entre 18 e 29 anos, 43,3% solteiras, 55,8% com escolaridade inferior a nove anos, 61,3% realizavam alguma atividade na unidade penal, 63,2% eram fumantes ou ex-fumantes, 28,2% ingeriam bebida alcoólica e 60,4% usuários ou ex-usuários de drogas ilícitas, 71,2% praticavam atividades físicas, 86,1% avaliaram positivamente o estado de saúde e 52,5% relatou alguma doença crônica. As doenças que prevaleceram nos autorrelatos foram as respiratórias, gastrointestinais, psíquicas, cardiovasculares e osteomusculares. As pessoas privadas de liberdade possuem as doenças crônicas e fatores de risco prevalentes na população em geral. Conhecer o perfil epidemiológico desse grupo populacional pode contribuir com ações promotoras de saúde, prevenção e controle dos fatores de risco

Gamma-herpesvirus colonization of the spleen requires lytic replication in B cells

Gamma-herpesviruses infect lymphocytes and cause lymphocytic cancers. Murid Herpesvirus-4 (MuHV-4), Epstein-Barr virus and the Kaposi's Sarcoma-associated Herpesvirus all infect B cells. Latent infection can spread by B cell recirculation and proliferation, but whether this alone achieves systemic infection is unclear. To test the need of MuHV-4 for lytic infection in B cells we flanked its essential ORF50 lytic transactivator with loxP sites, then infected mice with B cell-specific cre expression (CD19-cre). The floxed virus replicated normally in cre- mice. In CD19-cre mice, nasal and lymph node infections were maintained but there was little splenomegaly and splenic virus loads remained low. Cre-mediated removal of other essential lytic genes gave a similar phenotype. CD19-cre spleen infection by intraperitoneal virus was also impaired. Therefore MuHV-4 had to emerge lytically from B cells to colonize the spleen. An important role for B cell lytic infection in host colonization is consistent with the large CD8+ T cell responses made to gamma-herpesvirus lytic antigens during infectious mononucleosis, and suggests that vaccine-induced immunity capable of suppressing B cell lytic infection might reduce long-term virus loads.IMPORTANCE Gamma-herpesviruses cause B cell cancers. Most models of host colonization derive from cell cultures with continuous, virus-driven B cell proliferation. However vaccines based on these models have worked poorly. To test whether proliferating B cells suffice for host colonization, we inactivated the capacity of MuHV-4, a gamma-herpesvirus of mice, to re-emerge from B cells. The modified virus was able to colonize a first wave of B cells in lymph nodes, but spread poorly to B cells in secondary sites such as the spleen. Consequently viral loads remained low. These results were consistent with virus-driven B cell proliferation exploiting normal host pathways, and so having to transfer lytically to new B cells for new proliferation. We conclude that viral lytic infection is a potential target to reduce B cell proliferation

KSHV LANA acetylation-selective acidic domain reader sequence mediates virus persistence

Viruses modulate biochemical cellular pathways to permit infection. A recently described mechanism mediates selective protein interactions between acidic domain readers and unacetylated, lysine-rich regions, opposite of bromodomain function. Kaposi´s sarcoma (KS)-associated herpesvirus (KSHV) is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman’s disease. KSHV latently infects cells, and its genome persists as a multicopy, extrachromosomal episome. During latency, KSHV expresses a small subset of genes, including the latency-associated nuclear antigen (LANA), which mediates viral episome persistence. Here we show that LANA contains two tandem, partially overlapping, acidic domain sequences homologous to the SET oncoprotein acidic domain reader. This domain selectively interacts with unacetylated p53, as evidenced by reduced LANA interaction after overexpression of CBP, which acetylates p53, or with an acetylation mimicking carboxyl-terminal domain p53 mutant. Conversely, the interaction of LANA with an acetylation-deficient p53 mutant is enhanced. Significantly, KSHV LANA mutants lacking the acidic domain reader sequence are deficient for establishment of latency and persistent infection. This deficiency was confirmed under physiological conditions, on infection of mice with a murine gammaherpesvirus 68 chimera expressing LANA, where the virus was highly deficient in establishing latent infection in germinal center B cells. Therefore, LANA’s acidic domain reader is critical for viral latency. These results implicate an acetylation-dependent mechanism mediating KSHV persistence and expand the role of acidic domain readers.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

De novo human angiotensin-converting enzyme 2 decoy NL-CVX1 protects mice from severe disease after severe acute respiratory syndrome Coronavirus 2 infection

© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.The emergence of novel variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need to investigate alternative approaches to prevent infection and treat patients with coronavirus disease 2019. Here, we report the preclinical efficacy of NL-CVX1, a de novo decoy that blocks virus entry into cells by binding with nanomolar affinity and high specificity to the receptor-binding domain of the SARS-CoV-2 spike protein. Using a transgenic mouse model of SARS-CoV-2 infection, we showed that a single prophylactic intranasal dose of NL-CVX1 conferred complete protection from severe disease following SARS-CoV-2 infection. Multiple therapeutic administrations of NL-CVX1 also protected mice from succumbing to infection. Finally, we showed that infected mice treated with NL-CVX1 developed both anti-SARS-CoV-2 antibodies and memory T cells and were protected against reinfection a month after treatment. Overall, these observations suggest NL-CVX1 is a promising therapeutic candidate for preventing and treating severe SARS-CoV-2 infections.This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreements number 852985.info:eu-repo/semantics/publishedVersio

De novo human angiotensin - converting enzyme 2 Decoy NL-CVX1 protects mice from severe disease after severe acute respiratory syndrome coronavirus 2 infection

The emergence of novel variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need to investigate alternative approaches to prevent infection and treat patients with coronavirus disease 2019. Here, we report the preclinical efficacy of NL-CVX1, a de novo decoy that blocks virus entry into cells by binding with nanomolar affinity and high specificity to the receptor-binding domain of the SARS-CoV-2 spike protein. Using a transgenic mouse model of SARS-CoV-2 infection, we showed that a single prophylactic intranasal dose of NL-CVX1 conferred complete protection from severe disease following SARS-CoV-2 infection. Multiple therapeutic administrations of NL-CVX1 also protected mice from succumbing to infection. Finally, we showed that infected mice treated with NL-CVX1 developed both anti-SARS-CoV-2 antibodies and memory T cells and were protected against reinfection a month after treatment. Overall, these observations suggest NL-CVX1 is a promising therapeutic candidate for preventing and treating severe SARS-CoV-2 infections.info:eu-repo/semantics/publishedVersio

Proximity-Induced Nucleic Acid Degrader (PINAD) approach to targeted RNA degradation using small molecules

Nature has evolved intricate machinery to target and degrade RNA, and some of these molecular mechanisms can be adapted for therapeutic use. Small interfering RNAs and RNase H-inducing oligonucleotides have yielded therapeutic agents against diseases that cannot be tackled using protein-centered approaches. Because these therapeutic agents are nucleic acid-based, they have several inherent drawbacks which include poor cellular uptake and stability. Here we report a new approach to target and degrade RNA using small molecules, proximity-induced nucleic acid degrader (PINAD). We have utilized this strategy to design two families of RNA degraders which target two different RNA structures within the genome of SARS-CoV-2: G-quadruplexes and the betacoronaviral pseudoknot. We demonstrate that these novel molecules degrade their targets using in vitro, in cellulo, and in vivo SARS-CoV-2 infection models. Our strategy allows any RNA binding small molecule to be converted into a degrader, empowering RNA binders that are not potent enough to exert a phenotypic effect on their own. PINAD raises the possibility of targeting and destroying any disease-related RNA species, which can greatly expand the space of druggable targets and diseases.info:eu-repo/semantics/publishedVersio

The Gammaherpesvirus m2 Protein Manipulates the Fyn/Vav Pathway through a Multidocking Mechanism of Assembly

To establish latent infections in B-cells, gammaherpesviruses express proteins in the infected B-cells of the host that spuriously activate signalling pathways located downstream of the B-cell receptor. One such protein is M2, a murine gammaherpesvirus 68-encoded molecule that activates the Vav1/Rac1 pathway via the formation of trimolecular complexes with Scr family members. Previous reports have shown that the formation of this heteromolecular complex involves interactions between a proline rich region of M2 and the Vav1 and Fyn SH3 domains. Here, we show that the optimal association of these proteins requires a second structural motif encompassing two tyrosine residues (Tyr120 and 129). These residues are inducibly phosphorylated by Fyn in non-hematopoietic cells and constitutively phosphorylated in B-cells. We also demonstrate that the phosphorylation of Tyr120 creates specific docking sites for the SH2 domains of both Vav1 and Fyn, a condition sine qua non for the optimal association of these two signalling proteins in vivo. Interestingly, signaling experiments indicate that the expression of M2 in B-cells promotes the tyrosine phosphorylation of Vav1 and additional signaling proteins, a biological process that requires the integrity of both the M2 phosphotyrosine and proline rich region motifs. By infecting mice with viruses mutated in the m2 locus, we show that the integrity of each of these two M2 docking motifs is essential for the early steps of murine gammaherpesvirus-68 latency. Taken together, these results indicate that the M2 phosphotyrosine motif and the previously described M2 proline rich region work in a concerted manner to manipulate the signaling machinery of the host B-cell