RESUMO
OBJECTIVES: The objective of our study was to determine the effects of science-based communications on the attitude toward pneumococcal vaccination and understand how nonwhite racial and ethnic populations respond to these messages. DESIGN: Our team tested several science-based communications using a nationally representative survey, and validated them in a local community pharmacy as a field experiment. SETTING AND PARTICIPANTS: The nationally representative sample phase was a survey of 3276 participants, conducted by YouGov, a leading online survey firm. The field experiment was conducted at a community pharmacy in the northeastern United States and included 86 participants. OUTCOME MEASURES: In the national survey, participants were assigned to treatment groups or a control group to determine the effects of messaging strategies on influencing favorable views of pneumococcal vaccination. In the field experiment, participants were assigned to treatment or control groups to determine if the messaging strategies affected intent to ask a medical professional about the vaccine. RESULTS: The nationally representative sample survey identified that messaging that focused on community and family duty had statistically significant treatment effects toward increasing individuals' perception of personal importance to have the vaccine in both the nonwhite (increase of 12.2% points relative to control) and white respondents (increase of 8.7% points relative to control). These results were validated through a field experiment, which showed that a combination message, emphasizing duty, increased the individual's intent to vaccinate by 25% points in a diverse ethnic population as compared with the control. CONCLUSIONS: Messaging focused on appeals to community and family duty produced statistically significant increases in favorable attitudes toward pneumococcal vaccines and behavioral intent to seek medical advice about the vaccine in white and nonwhite populations across both the nationally representative survey and the field experiment. Medical professionals should highlight the duty to family and community when communicating with patients, as it may motivate vaccination in all populations.
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Vacinas Pneumocócicas , Vacinação , Adulto , Comunicação , Humanos , Streptococcus pneumoniae , Inquéritos e Questionários , Estados UnidosRESUMO
Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a gammaherpesvirus etiologically associated with KS, a highly disseminated angiogenic tumor of hyperproliferative spindle endothelial cells. KSHV encodes 25 mature microRNAs but their roles in KSHV-induced tumor dissemination and angiogenesis remain unknown. Here, we investigated KSHV-encoded miR-K12-6-3p (miR-K6-3p) promotion of endothelial cell migration and angiogenesis, which are the underlying mechanisms of tumor dissemination and angiogenesis. We found that ectopic expression of miR-K6-3p promoted endothelial cell migration and angiogenesis. Mass spectrometry, bioinformatics and luciferase reporter analyses revealed that miR-K6-3p directly targeted sequence in the 3' untranslated region (UTR) of SH3 domain binding glutamate-rich protein (SH3BGR). Overexpression of SH3BGR reversed miR-K6-3p induction of cell migration and angiogenesis. Mechanistically, miR-K6-3p downregulated SH3BGR, hence relieved STAT3 from SH3BGR direct binding and inhibition, which was required for miR-K6-3p maximum activation of STAT3 and induction of cell migration and angiogenesis. Finally, deletion of miR-K6 from the KSHV genome abrogated its effect on the SH3BGR/STAT3 pathway, and KSHV-induced migration and angiogenesis. Our results illustrated that, by inhibiting SH3BGR, miR-K6-3p enhances cell migration and angiogenesis by activating the STAT3 pathway, and thus contributes to the dissemination and angiogenesis of KSHV-induced malignancies.
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MicroRNAs , Proteínas Musculares/metabolismo , Neovascularização Patológica/metabolismo , RNA Viral , Fator de Transcrição STAT3/metabolismo , Sarcoma de Kaposi/patologia , Animais , Western Blotting , Movimento Celular/fisiologia , Herpesvirus Humano 8/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Nus , Microscopia Confocal , Neovascularização Patológica/genética , Reação em Cadeia da Polimerase , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
KSHV is a DNA tumor virus that causes Kaposi's sarcoma. Upon KSHV infection, only a limited number of latent genes are expressed. We know that KSHV infection regulates host gene expression, and hypothesized that latent genes also modulate the expression of host miRNAs. Aberrant miRNA expression contributes to the development of many types of cancer. Array-based miRNA profiling revealed that all six miRNAs of the oncogenic miR-17-92 cluster are up-regulated in KSHV infected endothelial cells. Among candidate KSHV latent genes, we found that vFLIP and vCyclin were shown to activate the miR-17-92 promoter, using luciferase assay and western blot analysis. The miR-17-92 cluster was previously shown to target TGF-ß signaling. We demonstrate that vFLIP and vCyclin induce the expression of the miR-17-92 cluster to strongly inhibit the TGF-ß signaling pathway by down-regulating SMAD2. Moreover, TGF-ß activity and SMAD2 expression were fully restored when antagomirs (inhibitors) of miR-17-92 cluster were transfected into cells expressing either vFLIP or vCyclin. In addition, we utilized viral genetics to produce vFLIP or vCyclin knock-out viruses, and studied the effects in infected TIVE cells. Infection with wildtype KSHV abolished expression of SMAD2 protein in these endothelial cells. While single-knockout mutants still showed a marked reduction in SMAD2 expression, TIVE cells infected by a double-knockout mutant virus were fully restored for SMAD2 expression, compared to non-infected TIVE cells. Expression of either vFLIP or vCycIin was sufficient to downregulate SMAD2. In summary, our data demonstrate that vFLIP and vCyclin induce the oncogenic miR-17-92 cluster in endothelial cells and thereby interfere with the TGF-ß signaling pathway. Manipulation of the TGF-ß pathway via host miRNAs represents a novel mechanism that may be important for KSHV tumorigenesis and angiogenesis, a hallmark of KS.
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Herpesvirus Humano 8 , MicroRNAs/genética , Sarcoma de Kaposi/virologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Linhagem Celular , Regulação para Baixo , Células Endoteliais/virologia , Humanos , Neovascularização Patológica/genética , Neovascularização Patológica/virologia , RNA Longo não Codificante , Sarcoma de Kaposi/irrigação sanguíneaRESUMO
Kaposi's sarcoma (KS) is a highly disseminated angiogenic tumor of endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced tumor dissemination and metastasis remain unknown. Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion. Bioinformatics and luciferase reporter analyses showed that miR-K3 directly targeted G protein-coupled receptor (GPCR) kinase 2 (GRK2, official gene symbol ADRBK1). Importantly, overexpression of GRK2 reversed miR-K3 induction of cell migration and invasion. Furthermore, the chemokine receptor CXCR2, which was negatively regulated by GRK2, was upregulated in miR-K3-transduced endothelial cells. Knock down of CXCR2 abolished miR-K3-induced cell migration and invasion. Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT. Both CXCR2 induction and the release of AKT from GRK2 were required for miR-K3 maximum activation of AKT and induction of cell migration and invasion. Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion. Our data provide the first-line evidence that, by repressing GRK2, miR-K3 facilitates cell migration and invasion via activation of CXCR2/AKT signaling, which likely contribute to the dissemination of KSHV-induced tumors.
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Endotélio Vascular/virologia , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Herpesvirus Humano 8/fisiologia , Interações Hospedeiro-Patógeno , MicroRNAs/metabolismo , RNA Viral/metabolismo , Internalização do Vírus , Movimento Celular , Células Cultivadas , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Repressão Enzimática , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Deleção de Genes , Herpesvirus Humano 8/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Mutação , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA/metabolismo , Interferência de RNA , Receptores de Interleucina-8B/agonistas , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia , Transdução de SinaisRESUMO
UNLABELLED: The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 gene product is essential for lytic KSHV replication and virion production. Recombinant ORF57-null mutants fail to accumulate several lytic cycle mRNAs at wild-type levels, leading to decreased production of lytic proteins necessary for efficient replication. Several mechanisms by which ORF57 may enhance expression of lytic KSHV mRNAs have been proposed, including mRNA stabilization, mRNA nuclear export, increased polyadenylation, and transcriptional activation. ORF57 activity is also gene specific, with some genes being highly dependent on ORF57, whereas others are relatively independent. Most experiments have utilized transfection models for ORF57 and have not systematically examined the gene specificity and potential mechanisms of action of ORF57 in the context of KSHV-infected cells. In this study, the KSHV genes that are most highly upregulated by ORF57 during KSHV lytic replication were identified by a combination of high-throughput deep RNA sequencing, quantitative PCR, Northern blotting, and rapid amplification of cDNA ends methods. Comparison of gene expression from a ΔORF57 KSHV recombinant, a rescued ΔORF57 KSHV recombinant, and wild-type KSHV revealed that two clusters of lytic genes are most highly dependent on ORF57 for efficient expression. Despite contiguous location in the genome and shared polyadenylation of several of the ORF57-dependent genes, ORF57 regulation was promoter and polyadenylation signal independent, suggesting that the mRNAs are stabilized by ORF57. The eight genes identified to critically require ORF57 belong to both early and late lytic temporal classes, and seven are involved in DNA replication, virion assembly, or viral infectivity, explaining the essential role of ORF57 in infectious KSHV production. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus involved in the causation of several human cancers. The KSHV ORF57 protein is required for KSHV to replicate and produce infectious virus. We have identified several KSHV genes whose expression is highly dependent on ORF57 and shown that ORF57 increases expression of these genes specifically. These genes code for proteins that are required for the virus to replicate its DNA and to infect other cells. Identifying the targets and mechanism of action of ORF57 provides further approaches to discover antiviral therapy.
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Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Proteínas Virais/metabolismo , Montagem de Vírus , Internalização do Vírus , Liberação de Vírus , Replicação Viral , Linhagem Celular , Deleção de Genes , Perfilação da Expressão Gênica , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/crescimento & desenvolvimento , Humanos , Estabilidade de RNA , Proteínas Virais/genéticaRESUMO
Regulation of the positive transcription elongation factor, P-TEFb, plays a major role in controlling mammalian transcription and this is accomplished in part by controlled release of P-TEFb from the 7SK snRNP that sequesters the kinase in an inactive state. We demonstrate here that a similar P-TEFb control system exists in Drosophila. We show that an RNA previously suggested to be a 7SK homolog is, in fact, associated with P-TEFb, through the action of a homolog of the human HEXIM1/2 proteins (dHEXIM). In addition, a Drosophila La related protein (now called dLARP7) is shown to be the functional homolog of human LARP7. The Drosophila 7SK snRNP (d7SK snRNP) responded to treatment of cells with P-TEFb inhibitors and to nuclease treatment of cell lysates by releasing P-TEFb. Supporting a critical role for the d7SK snRNP in Drosophila development, dLARP7 and dHEXIM were found to be ubiquitously expressed throughout embryos and tissues at all stages. Importantly, knockdown of dHEXIM was embryonic lethal, and reduction of dHEXIM in specific tissues led to serious developmental defects. Our results suggest that regulation of P-TEFb by the d7SK snRNP is essential for the growth and differentiation of tissues required during Drosophila development.
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Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Ligação a RNA/fisiologia , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Desenvolvimento Embrionário/genética , Dados de Sequência Molecular , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genéticaRESUMO
BACKGROUND: University spring break carries a two-pronged SARS-CoV-2 variant transmission risk. Circulating variants from universities can spread to spring break destinations, and variants from spring break destinations can spread to universities and surrounding communities. Therefore, it is critical to implement SARS-CoV-2 variant surveillance and testing strategies to limit community spread before and after spring break to mitigate virus transmission and facilitate universities safely returning to in-person teaching. METHODS: We examined the SARS-CoV-2 positivity rate and changes in variant lineages before and after the university spring break for two consecutive years. 155 samples were sequenced across four time periods: pre- and post-spring break 2021 and pre- and post-spring break 2022; following whole genome sequencing, samples were assigned clades. The clades were then paired with positivity and testing data from over 50,000 samples. RESULTS: In 2021, the number of variants in the observed population increased from four to nine over spring break, with variants of concern being responsible for most of the cases; Alpha percent composition increased from 22.2% to 56.4%. In 2022, the number of clades in the population increased only from two to three, all of which were Omicron or a sub-lineage of Omicron. However, phylogenetic analysis showed the emergence of distantly related sub-lineages. 2022 saw a greater increase in positivity than 2021, which coincided with a milder mitigation strategy. Analysis of social media data provided insight into student travel destinations and how those travel events may have impacted spread. CONCLUSIONS: We show the role that repetitive testing can play in transmission mitigation, reducing community spread, and maintaining in-person education. We identified that distantly related lineages were brought to the area after spring break travel regardless of the presence of a dominant variant of concern.
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COVID-19 , SARS-CoV-2 , Viagem , Humanos , COVID-19/transmissão , COVID-19/prevenção & controle , COVID-19/epidemiologia , COVID-19/virologia , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Universidades , Sequenciamento Completo do Genoma , Filogenia , Estações do AnoAssuntos
Genoma Humano , Síndromes de Imunodeficiência/genética , Mutação , NADPH Oxidases/genética , Proteínas Nucleares/genética , Processamento Alternativo , Linfócitos B/imunologia , Linfócitos B/patologia , Sequência de Bases , Criança , Proteínas de Ligação a DNA , Endonucleases , Éxons , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina A/genética , Imunoglobulina G/genética , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Contagem de Linfócitos , Masculino , Dados de Sequência Molecular , NADPH Oxidases/imunologia , Proteínas Nucleares/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Regulation of the elongation phase of RNA polymerase II transcription by P-TEFb is a critical control point for gene expression. The activity of P-TEFb is regulated, in part, by reversible association with one of two HEXIMs and the 7SK snRNP. A recent proteomics survey revealed that P-TEFb and the HEXIMs are tightly connected to two previously-uncharacterized proteins, the methyphosphate capping enzyme, MEPCE, and a La-related protein, LARP7. Glycerol gradient sedimentation analysis of lysates from cells treated with P-TEFb inhibitors, suggested that the 7SK snRNP reorganized such that LARP7 and 7SK remained associated after P-TEFb and HEXIM1 were released. Immunodepletion of LARP7 also depleted most of the 7SK regardless of the presence of P-TEFb, HEXIM or hnRNP A1 in the complex. Small interfering RNA knockdown of LARP7 in human cells decreased the steady-state level of 7SK, led to an initial increase in free P-TEFb and increased Tat transactivation of the HIV-1 LTR. Knockdown of LARP7 or 7SK ultimately caused a decrease in total P-TEFb protein levels. Our studies have identified LARP7 as a 7SK-binding protein and suggest that free P-TEFb levels are determined by a balance between release from the large form and reduction of total P-TEFb.
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Fator B de Elongação Transcricional Positiva/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular , Produtos do Gene tat/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Interferência de RNA , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/análise , Ribonucleoproteínas/antagonistas & inibidores , Fatores de Transcrição , Ativação TranscricionalRESUMO
The benefits of using high flow rates in preparative subcritical fluid chromatography are explored. It is demonstrated that chromatograms loaded to onset of peak coalescence do not deteriorate as flow increases. This allows separation of material in very short time periods leading to dramatically increased production rates. A key factor to accessing elevated flows is the use of shorter columns and the resulting decrease in pressure drop.
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Cromatografia Líquida/métodos , Acetaminofen/isolamento & purificação , Antipirina/isolamento & purificação , PiridinasRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 viral microRNAs (miRNAs) that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red recombination system together with a new bacmid background, BAC16, to create mutants for all known KSHV miRNAs. The specific miRNA deletions or mutations and the integrity of the bacmids have been strictly quality controlled using PCR, restriction digestion, and sequencing. In addition, stable viral producer cell lines based on iSLK cells have been created for wildtype KSHV, for 12 individual miRNA knock-out mutants (ΔmiR-K12-1 through -12), and for mutants deleted for 10 of 12 (ΔmiR-cluster) or all 12 miRNAs (ΔmiR-all). NGS, in combination with SureSelect technology, was employed to sequence the entire latent genome within all producer cell lines. qPCR assays were used to verify the expression of the remaining viral miRNAs in a subset of mutants. Induction of the lytic cycle leads to efficient production of progeny viruses that have been used to infect endothelial cells. Wt BAC16 and miR mutant iSLK producer cell lines are now available to the research community.
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Herpesvirus Humano 8/genética , MicroRNAs/genética , RNA Viral/genética , Sarcoma de Kaposi/virologia , Deleção de Sequência , Herpesvirus Humano 8/metabolismo , Humanos , MicroRNAs/metabolismo , RNA Viral/metabolismoRESUMO
Recent intense investigations have uncovered important functions for a diverse array of novel noncoding RNA (ncRNA) species, including microRNAs (miRNAs) and long noncoding RNAs. Not surprisingly, viruses from multiple families have evolved to encode their own regulatory RNAs; however, the specific in vivo functions of these ncRNAs are largely unknown. The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are highly ubiquitous pathogens that are associated with the development of a wide range of malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, and Kaposi's sarcoma. Like EBV and KSHV, murine gammaherpesvirus 68 (MHV68) establishes lifelong latency in B cells and is associated with lymphoproliferative disease and lymphoma. Similar to the EBV-encoded small RNA (EBER)-1 and -2, MHV68 encodes eight 200- to 250-nucleotide polymerase III-transcribed ncRNAs called TMERs (tRNA-miRNA-encoded RNAs), which are highly expressed in latently infected cells and lymphoproliferative disease. To define the in vivo contribution of TMERs to MHV68 biology, we generated a panel of individual TMER mutant viruses. Through comprehensive in vivo analyses, we identified TMER4 as a key mediator of virus dissemination. The TMER4 mutant virus replicated normally in lungs and spread with normal kinetics and distribution to lung-draining lymph nodes, but it was significantly attenuated for infection of circulating blood cells and for latency establishment at peripheral sites. Notably, TMER4 stem-loops but not miRNAs were essential for wild-type TMER4 activity. Thus, these findings revealed a crucial miRNA-independent function of the TMER4 ncRNA in MHV68 hematogenous dissemination and latency establishment. IMPORTANCE: Noncoding RNAs (ncRNAs) represent an intriguing and diverse class of molecules that are now recognized for their participation in a wide array of cellular processes. Viruses from multiple families have evolved to encode their own such regulatory RNAs; however, the specific in vivo functions of these ncRNAs are largely unknown. Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are ubiquitous human pathogens that are associated with the development of numerous malignancies. Like EBV and KSHV, murine gammaherpesvirus 68 (MHV68) establishes lifelong latency in B cells and is associated with lymphomagenesis. The work described here reveals that the MHV68 ncRNA TMER4 acts at a critical bottleneck in local lymph nodes to facilitate hematogenous dissemination of the virus and establishment of latency at peripheral sites.
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UNLABELLED: Kaposi's sarcoma (KS), a highly angiogenic and invasive tumor often involving different organ sites, including the oral cavity, is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV). Diverse cell markers have been identified on KS tumor cells, but their origin remains an enigma. We previously showed that KSHV could efficiently infect, transform, and reprogram rat primary mesenchymal stem cells (MSCs) into KS-like tumor cells. In this study, we showed that human primary MSCs derived from diverse organs, including bone marrow (MSCbm), adipose tissue (MSCa), dental pulp, gingiva tissue (GMSC), and exfoliated deciduous teeth, were permissive to KSHV infection. We successfully established long-term cultures of KSHV-infected MSCa, MSCbm, and GMSC (LTC-KMSCs). While LTC-KMSCs had lower proliferation rates than the uninfected cells, they expressed mixtures of KS markers and displayed differential angiogenic, invasive, and transforming phenotypes. Genetic analysis identified KSHV-derived microRNAs that mediated KSHV-induced angiogenic activity by activating the AKT pathway. These results indicated that human MSCs could be the KSHV target cells in vivo and established valid models for delineating the mechanism of KSHV infection, replication, and malignant transformation in biologically relevant cell types. IMPORTANCE: Kaposi's sarcoma is the most common cancer in AIDS patients. While KSHV infection is required for the development of Kaposi's sarcoma, the origin of KSHV target cells remains unclear. We show that KSHV can efficiently infect human primary mesenchymal stem cells of diverse origins and reprogram them to acquire various degrees of Kaposi's sarcoma-like cell makers and angiogenic, invasive, and transforming phenotypes. These results indicate that human mesenchymal stem cells might be the KSHV target cells and establish models for delineating the mechanism of KSHV-induced malignant transformation.
Assuntos
Transformação Celular Neoplásica , Herpesvirus Humano 8/crescimento & desenvolvimento , Herpesvirus Humano 8/fisiologia , Células-Tronco Mesenquimais/virologia , Neovascularização Patológica , Proliferação de Células , Células Cultivadas , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , MicroRNAs/metabolismo , RNA Viral/metabolismo , Cultura de VírusRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Most tumor cells in these malignancies are latently infected by KSHV. Thus, viral latency is critical for the development of tumor and induction of tumor-associated angiogenesis. KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced angiogenesis remains unknown. We have recently shown that miR-K12-3 (miR-K3) promoted cell migration and invasion by targeting GRK2/CXCR2/AKT signaling (PLoS Pathog, 2015;11(9):e1005171). Here, we further demonstrated a role of miR-K3 and its induced signal pathway in KSHV latency and KSHV-induced angiogenesis. We found that overexpression of miR-K3 not only promoted viral latency by inhibiting viral lytic replication, but also induced angiogenesis. Further, knockdown of GRK2 inhibited KSHV replication and enhanced KSHV-induced angiogenesis by enhancing the CXCR2/AKT signals. As a result, blockage of CXCR2 or AKT increased KSHV replication and decreased angiogenesis induced by PEL cells in vivo. Finally, deletion of miR-K3 from viral genome reduced KSHV-induced angiogenesis and increased KSHV replication. These findings indicate that the miR-K3/GRK2/CXCR2/AKT axis plays an essential role in KSHV-induced angiogenesis and promotes KSHV latency, and thus may be a potential therapeutic target of KSHV-associated malignancies.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Herpesvirus Humano 8/genética , Linfoma de Células B/enzimologia , MicroRNAs/genética , Neovascularização Patológica , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Viral/genética , Receptores de Interleucina-8B/metabolismo , Latência Viral , Animais , Linhagem Celular Tumoral , Movimento Celular , Feminino , Quinase 2 de Receptor Acoplado a Proteína G/genética , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patologia , Linfoma de Células B/virologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Interferência de RNA , Transdução de Sinais , TransfecçãoRESUMO
OBJECTIVE: To elucidate the functional consequences of epileptic encephalopathy-causing de novo mutations in DNM1 (A177P, K206N, G359A), which encodes a large mechanochemical GTPase essential for neuronal synaptic vesicle endocytosis. METHODS: HeLa and COS-7 cells transfected with wild-type and mutant DNM1 constructs were used for transferrin assays, high-content imaging, colocalization studies, Western blotting, and electron microscopy (EM). EM was also conducted on the brain sections of mice harboring a middle-domain Dnm1 mutation (Dnm1 (Ftfl)). RESULTS: We demonstrate that the expression of each mutant protein decreased endocytosis activity in a dominant-negative manner. One of the G-domain mutations, K206N, decreased protein levels. The G359A mutation, which occurs in the middle domain, disrupted higher-order DNM1 oligomerization. EM of mutant DNM1-transfected HeLa cells and of the Dnm1 (Ftfl) mouse brain revealed vesicle defects, indicating that the mutations likely interfere with DNM1's vesicle scission activity. CONCLUSION: Together, these data suggest that the dysfunction of vesicle scission during synaptic vesicle endocytosis can lead to serious early-onset epilepsies.
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Genetically targeted therapies for rare Mendelian conditions are improving patient outcomes. Here, we present the case of a 20-mo-old female suffering from a rapidly progressing neurological disorder. Although diagnosed initially with a possible autoimmune condition, analysis of the child's exome resulted in a diagnosis of Brown-Vialetto-Van Laere syndrome 2 (BVVLS2). This new diagnosis led to a change in the therapy plan from steroids and precautionary chemotherapy to high-dose riboflavin. Improvements were reported quickly, including in motor strength after 1 mo. In this case, the correct diagnosis and appropriate treatment would have been unlikely in the absence of exome sequencing and careful interpretation. This experience adds to a growing list of examples that emphasize the importance of early genome-wide diagnostics.
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Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. We report the results of a moderate-scale sequencing study aimed at increasing the number of genes known to contribute to predisposition for ALS. We performed whole-exome sequencing of 2869 ALS patients and 6405 controls. Several known ALS genes were found to be associated, and TBK1 (the gene encoding TANK-binding kinase 1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention.
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Esclerose Lateral Amiotrófica/genética , Autofagia/genética , Exoma/genética , Predisposição Genética para Doença , Proteínas Serina-Treonina Quinases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ciclo Celular , Feminino , Genes , Estudos de Associação Genética , Humanos , Masculino , Proteínas de Membrana Transportadoras , Pessoa de Meia-Idade , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Risco , Análise de Sequência de DNA , Proteína Sequestossoma-1 , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismo , Adulto JovemRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) microRNAs are encoded in the latency-associated region. Knockdown of KSHV miR-K12-3 and miR-K12-11 increased expression of lytic genes in BC-3 cells, and increased virus production from latently infected BCBL-1 cells. Furthermore, iSLK cells infected with miR-K12-3 and miR-K12-11 deletion mutant viruses displayed increased spontaneous reactivation and were more sensitive to inducers of reactivation than cells infected with wild type KSHV. Predicted binding sites for miR-K12-3 and miR-K12-11 were found in the 3'UTRs of the cellular transcription factors MYB, Ets-1, and C/EBPα, which activate RTA, the KSHV replication and transcription activator. Targeting of MYB by miR-K12-11 was confirmed by cloning the MYB 3'UTR downstream from the luciferase reporter. Knockdown of miRK12-11 resulted in increased levels of MYB transcript, and knockdown of miR-K12-3 increased both C/EBPα and Ets-1 transcripts. Thus, miR-K12-11 and miR-K12-3 contribute to maintenance of latency by decreasing RTA expression indirectly, presumably via down-regulation of MYB, C/EBPα and Ets-1, and possibly other host transcription factors.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , MicroRNAs/genética , Proteínas Virais/metabolismo , Linhagem Celular , Regulação para Baixo , Células Endoteliais/virologia , Técnicas de Silenciamento de Genes , Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/metabolismo , Humanos , MicroRNAs/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Receptores Virais/fisiologia , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/genética , Internalização do Vírus , Latência ViralRESUMO
UNLABELLED: Gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV68, γHV68, or MuHV-4), are B cell-tropic pathogens that each encode at least 12 microRNAs (miRNAs). It is predicted that these regulatory RNAs facilitate infection by suppressing host target genes involved in a wide range of key cellular pathways. However, the precise contribution that gammaherpesvirus miRNAs make to viral life cycle and pathogenesis in vivo is unknown. MHV68 infection of mice provides a highly useful system to dissect the function of specific viral elements in the context of both asymptomatic infection and disease. Here, we report (i) analysis of in vitro and in vivo MHV68 miRNA expression, (ii) generation of an MHV68 miRNA mutant with reduced expression of all 14 pre-miRNA stem-loops, and (iii) comprehensive phenotypic characterization of the miRNA mutant virus in vivo. The profile of MHV68 miRNAs detected in infected cell lines varied with cell type and did not fully recapitulate the profile from cells latently infected in vivo. The miRNA mutant virus, MHV68.Zt6, underwent normal lytic replication in vitro and in vivo, demonstrating that the MHV68 miRNAs are dispensable for acute replication. During chronic infection, MHV68.Zt6 was attenuated for latency establishment, including a specific defect in memory B cells. Finally, MHV68.Zt6 displayed a striking attenuation in the development of lethal pneumonia in mice deficient in IFN-γ. These data indicate that the MHV68 miRNAs may facilitate virus-driven maturation of infected B cells and implicate the miRNAs as a critical determinant of gammaherpesvirus-associated disease. IMPORTANCE: Gammaherpesviruses such as EBV and KSHV are widespread pathogens that establish lifelong infections and are associated with the development of numerous types of diseases, including cancer. Gammaherpesviruses encode many small noncoding RNAs called microRNAs (miRNAs). It is predicted that gammaherpesvirus miRNAs facilitate infection and disease by suppressing host target transcripts involved in a wide range of key cellular pathways; however, the precise contribution that these regulatory RNAs make to in vivo virus infection and pathogenesis is unknown. Here, we generated a mutated form of murine gammaherpesvirus (MHV68) to dissect the function of gammaherpesvirus miRNAs in vivo. We demonstrate that the MHV68 miRNAs were dispensable for short-term virus replication but were important for establishment of lifelong infection in the key virus reservoir of memory B cells. Moreover, the MHV68 miRNAs were essential for the development of virus-associated pneumonia, implicating them as a critical component of gammaherpesvirus-associated disease.
Assuntos
Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , MicroRNAs/genética , RNA Viral , Latência Viral/genética , Animais , Linfócitos B/virologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Regulação Viral da Expressão Gênica , Ordem dos Genes , Genoma Viral , Interferon gama/deficiência , Camundongos , Camundongos Knockout , MicroRNAs/química , Mutação , Conformação de Ácido Nucleico , Pneumonia Viral/genética , Pneumonia Viral/mortalidade , Pneumonia Viral/virologia , Ativação Viral , Replicação ViralRESUMO
BACKGROUND: The positive transcription elongation factor, P-TEFb, is required for the production of mRNAs, however the majority of the factor is present in the 7SK snRNP where it is inactivated by HEXIM1. Expression of HIV-1 Tat leads to release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo, but the release mechanisms are unclear. METHODOLOGY/PRINCIPAL FINDINGS: We developed an in vitro P-TEFb release assay in which the 7SK snRNP immunoprecipitated from HeLa cell lysates using antibodies to LARP7 was incubated with potential release factors. We found that P-TEFb was directly released from the 7SK snRNP by HIV-1 Tat or the P-TEFb binding region of the cellular activator Brd4. Glycerol gradient sedimentation analysis was used to demonstrate that the same Brd4 protein transfected into HeLa cells caused the release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo. Although HEXIM1 binds tightly to 7SK RNA in vitro, release of P-TEFb from the 7SK snRNP is accompanied by the loss of HEXIM1. Using a chemical modification method, we determined that concomitant with the release of HEXIM1, 7SK underwent a major conformational change that blocks re-association of HEXIM1. CONCLUSIONS/SIGNIFICANCE: Given that promoter proximally paused polymerases are present on most human genes, understanding how activators recruit P-TEFb to those genes is critical. Our findings reveal that the two tested activators can extract P-TEFb from the 7SK snRNP. Importantly, we found that after P-TEFb is extracted a dramatic conformational change occurred in 7SK concomitant with the ejection of HEXIM1. Based on our findings, we hypothesize that reincorporation of HEXIM1 into the 7SK snRNP is likely the regulated step of reassembly of the 7SK snRNP containing P-TEFb.