RESUMO
BACKGROUND: The developing field of osteoimmunology supports importance of an interferon (IFN) response pathway in osteoblasts. Clarifying osteoblast-IFN interactions is important because IFN is used as salvage anti-tumor therapy but systemic toxicity is high with variable clinical results. In addition, osteoblast response to systemic bursts and disruptions of IFN pathways induced by viral infection may influence bone remodeling. ZIKA virus (ZIKV) infection impacts bone development in humans and IFN response in vitro. Consistently, initial evidence of permissivity to ZIKV has been reported in human osteoblasts. HYPOTHESIS: Osteoblast-like Saos-2 cells are permissive to ZIKV and responsive to IFN. METHODS: Multiple approaches were used to assess whether Saos-2 cells are permissive to ZIKV infection and exhibit IFN-mediated ZIKV suppression. Proteomic methods were used to evaluate impact of ZIKV and IFN on Saos-2 cells. RESULTS: Evidence is presented confirming Saos-2 cells are permissive to ZIKV and support IFN-mediated suppression of ZIKV. ZIKV and IFN differentially impact the Saos-2 proteome, exemplified by HELZ2 protein which is upregulated by IFN but non responsive to ZIKV. Both ZIKV and IFN suppress proteins associated with microcephaly/pseudo-TORCH syndrome (BI1, KI20A and UBP18), and ZIKV induces potential entry factor PLVAP. CONCLUSIONS: Transient ZIKV infection influences osteoimmune state, and IFN and ZIKV activate distinct proteomes in Saos-2 cells, which could inform therapeutic, engineered, disruptions.
Assuntos
Antivirais/imunologia , Interferon Tipo I/imunologia , Osteoblastos/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Antivirais/farmacologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferon Tipo I/farmacologia , Camundongos Knockout , Osteoblastos/metabolismo , Osteoblastos/virologia , Proteoma/imunologia , Proteoma/metabolismo , Proteômica/métodos , Células Vero , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologia , Zika virus/fisiologia , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologiaRESUMO
Beta genus human papillomaviruses (ß-HPVs) cause cutaneous squamous cell carcinomas (cSCCs) in a subset of immunocompromised patients. However, ß-HPVs are not necessary for tumor maintenance in the general population. Instead, they may destabilize the genome in the early stages of cancer development. Supporting this idea, ß-HPV's 8E6 protein attenuates p53 accumulation after failed cytokinesis. This paper offers mechanistic insight into how ß-HPV E6 causes this change in cell signaling. An in silico screen and characterization of HCT 116 cells lacking p300 suggested that the histone acetyltransferase is a negative regulator of Hippo pathway (HP) gene expression. HP activation restricts growth in response to stimuli, including failed cytokinesis. Loss of p300 resulted in increased HP gene expression, including proproliferative genes associated with HP inactivation. ß-HPV 8E6 expression recapitulates some of these phenotypes. We used a chemical inhibitor of cytokinesis (dihydrocytochalasin B [H2CB]) to induce failed cytokinesis. This system allowed us to show that ß-HPV 8E6 reduced activation of large tumor suppressor kinase (LATS), an HP kinase. LATS is required for p53 accumulation following failed cytokinesis. These phenotypes were dependent on ß-HPV 8E6 destabilizing p300 and did not completely attenuate the HP. It did not alter H2CB-induced nuclear exclusion of the transcription factor YAP. ß-HPV 8E6 also did not decrease HP activation in cells grown to a high density. Although our group and others have previously described inhibition of DNA repair, to the best of our knowledge, this marks the first time that a ß-HPV E6 protein has been shown to hinder HP signaling.IMPORTANCE ß-HPVs contribute to cSCC development in immunocompromised populations. However, it is unclear if these common cutaneous viruses are tumorigenic in the general population. Thus, a more thorough investigation of ß-HPV biology is warranted. If ß-HPV infections do promote cSCCs, they are hypothesized to destabilize the cellular genome. In vitro data support this idea by demonstrating the ability of the ß-HPV E6 protein to disrupt DNA repair signaling events following UV exposure. We show that ß-HPV E6 more broadly impairs cellular signaling, indicating that the viral protein dysregulates the HP. The HP protects genome fidelity by regulating cell growth and apoptosis in response to a myriad of deleterious stimuli, including failed cytokinesis. After failed cytokinesis, ß-HPV 8E6 attenuates phosphorylation of the HP kinase (LATS). This decreases some, but not all, HP signaling events. Notably, ß-HPV 8E6 does not limit senescence associated with failed cytokinesis.
Assuntos
Citocinese/genética , Interações Hospedeiro-Patógeno/genética , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocalasina B/análogos & derivados , Citocalasina B/farmacologia , Citocinese/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Proteína p300 Associada a E1A/deficiência , Proteína p300 Associada a E1A/genética , Regulação da Expressão Gênica , Células HCT116 , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/virologia , Proteínas Oncogênicas Virais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/virologia , Papillomaviridae/metabolismo , Fenótipo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mayaro virus (MAYV) is an emerging arthritogenic alphavirus belonging to the Togaviridae family. Infection leads to a dengue-like illness accompanied by severe polyarthralgia. However, the molecular and cellular mechanisms of arthritis as a result of MAYV infection remain poorly understood. In the present study, we assess the susceptibility of human chondrocytes (HC), fibroblast-like synoviocytes and osteoblasts that are the major cell types involved in osteoarthritis, to infection with MAYV. We show that these cells are highly permissive to MAYV infection and that viral RNA copy number and viral titers increase over time in infected cells. Knowing that HC are the primary cells in articular cartilage and are essential for maintaining the cartilaginous matrix, gene expression studies were conducted in MAYV-infected primary HC using polymerase chain reaction (PCR) arrays. The infection of the latter cells resulted in an induction in the expression of several matrix metalloproteinases (MMP) including MMP1, MMP7, MMP8, MMP10, MMP13, MMP14 and MMP15 which could be involved in the destruction of articular cartilage. Infected HC were also found to express significantly increased levels of various IFN-stimulated genes and arthritogenic mediators such as TNF-α and IL-6. In conclusion, MAYV-infected primary HC overexpress arthritis-related genes, which may contribute to joint degradation and pathogenesis.
Assuntos
Infecções por Alphavirus/virologia , Alphavirus/fisiologia , Artrite/genética , Condrócitos/virologia , Alphavirus/imunologia , Infecções por Alphavirus/genética , Infecções por Alphavirus/imunologia , Adesão Celular/genética , Sobrevivência Celular , Células Cultivadas , Condrócitos/imunologia , Citocinas/genética , Citocinas/metabolismo , Matriz Extracelular/genética , Perfilação da Expressão Gênica , Humanos , Metaloproteinases da Matriz/genética , Osteoblastos/virologia , RNA Viral/metabolismo , Sinoviócitos/virologiaRESUMO
Ribosomal frameshifting during the translation of RNA is implicated in human disease and viral infection. While previous work has uncovered many details about single RNA frameshifting kinetics in vitro, little is known about how single RNA frameshift in living systems. To confront this problem, we have developed technology to quantify live-cell single RNA translation dynamics in frameshifted open reading frames. Applying this technology to RNA encoding the HIV-1 frameshift sequence reveals a small subset (â¼8%) of the translating pool robustly frameshift. Frameshifting RNA are translated at similar rates as non-frameshifting RNA (â¼3 aa/s) and can continuously frameshift for more than four rounds of translation. Fits to a bursty model of frameshifting constrain frameshifting kinetic rates and demonstrate how ribosomal traffic jams contribute to the persistence of the frameshifting state. These data provide insight into retroviral frameshifting and could lead to alternative strategies to perturb the process in living cells.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , HIV-1/genética , Fases de Leitura Aberta , Osteoblastos/metabolismo , RNA Viral/genética , Imagem Individual de Molécula/métodos , Pareamento de Bases , Linhagem Celular Tumoral , HIV-1/metabolismo , Humanos , Modelos Genéticos , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos/síntese química , Sondas de Oligonucleotídeos/genética , Sondas de Oligonucleotídeos/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Osteoblastos/virologia , RNA Viral/química , RNA Viral/metabolismo , Coloração e Rotulagem/métodosRESUMO
BACKGROUND: The arenavirus Junin virus (JUNV), causative agent of the argentine hemorrhagic fever, is able to modulate several signaling pathways involved in cell survival and multiplication. OBJECTIVES: We aimed to characterize the infection of rat osteoblasts (OBCs) with JUNV and its consequence on the modulation of osteogenic genes expression, thus studying the ability of this virus to induce cell differentiation. In addition, we evaluated the effect of purinergic agonists on viral replication. METHOD: Quantification of infectivity by plaque forming unit (PFU) assay, synthesis of viral proteins by western blot and immunofluorescence, and expression of osteogenic differentiation markers (ODM) by quantitative real-time polymerase chain reaction were employed. RESULTS: Infection of OBCs with JUNV (MOI 0.01 PFU/cell) showed a peak of infectivity, reaching 1.5 × 105 PFU/mL at the second day post-infection (p.i.). A marked restriction in multiplication was detected at day 7 p.i. that did not impair the establishment of a persistent stage of infection in OBCs. Analysis of mRNAs corresponding to ODM such as alkaline phosphatase, bone sialo-protein, and bone morphogenetic proteins (BMPs) 4 and 6 revealed that only the levels of BMP-6 were significantly higher in infected cells. Treatment with the purinergic agonists ATPγS, UTP, ADP, or UDP diminished viral titer and reduced the expression of the viral nucleoprotein. Also, treatment with 10 µM ATPγS reduced the stimulation of BMP-6 expression induced by the infection. CONCLUSIONS: These data demonstrate that JUNV is capable of infecting OBCs and point out BMP-6 as a key factor during this process.
Assuntos
Proteína Morfogenética Óssea 6/genética , Vírus Junin/fisiologia , Osteoblastos/virologia , Osteogênese/genética , Animais , Diferenciação Celular , Células Cultivadas , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Agonistas Purinérgicos/farmacologia , Ratos , Transdução de Sinais , Replicação Viral/efeitos dos fármacosRESUMO
Tripartite motif (TRIM) proteins belong to a large family with many roles in host biology, including restricting virus infection. Here, we found that TRIM2, which has been implicated in cases of Charcot-Marie-Tooth disease (CMTD) in humans, acts by blocking hemorrhagic fever New World arenavirus (NWA) entry into cells. We show that Trim2-knockout mice, as well as primary fibroblasts from a CMTD patient with mutations in TRIM2, are more highly infected by the NWAs Junín and Tacaribe virus than wild-type mice or cells are. Using mice with different Trim2 gene deletions and TRIM2 mutant constructs, we demonstrate that its antiviral activity is uniquely independent of the RING domain encoding ubiquitin ligase activity. Finally, we show that one member of the TRIM2 interactome, signal regulatory protein α (SIRPA), a known inhibitor of phagocytosis, also restricts NWA infection and conversely that TRIM2 limits phagocytosis of apoptotic cells. In addition to demonstrating a novel antiviral mechanism for TRIM proteins, these studies suggest that the NWA entry and phagocytosis pathways overlap.
Assuntos
Antígenos de Diferenciação/genética , Arenavirus do Novo Mundo/genética , Doença de Charcot-Marie-Tooth/genética , Interações Hospedeiro-Patógeno/genética , Proteínas Nucleares/genética , Receptores Imunológicos/genética , Animais , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Apoptose , Arenavirus do Novo Mundo/crescimento & desenvolvimento , Arenavirus do Novo Mundo/patogenicidade , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/virologia , Linhagem Celular Tumoral , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Chlorocebus aethiops , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/imunologia , Proteínas de Neurofilamentos/metabolismo , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Osteoblastos/imunologia , Osteoblastos/metabolismo , Osteoblastos/virologia , Cultura Primária de Células , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Transdução de Sinais , Células Vero , Internalização do VírusRESUMO
Zika virus (ZIKV) infection is typically characterized by a mild self-limiting disease presenting with fever, rash, myalgia and arthralgia and severe fetal complications during pregnancy such as microcephaly, subcortical calcifications and arthrogyropsis. Virus-induced arthralgia due to perturbed osteoblast function has been described for other arboviruses. In case of ZIKV infection, the role of osteoblasts in ZIKV pathogenesis and bone related pathology remains unknown. Here, we study the effect of ZIKV infection on osteoblast differentiation, maturation and function by quantifying activity and gene expression of key biomarkers, using human bone marrow-derived mesenchymal stromal cells (MSCs, osteoblast precursors). MSCs were induced to differentiate into osteoblasts and we found that osteoblasts were highly susceptible to ZIKV infection. While infection did not cause a cytopathic effect, a significant reduction of key osteogenic markers such as ALP, RUNX2, calcium contents and increased expression of IL6 in ZIKV-infected MSCs implicated a delay in osteoblast development and maturation, as compared to uninfected controls. In conclusion, we have developed and characterized a new in vitro model to study the role of bone development in ZIKV pathogenesis, which will help to identify possible new targets for developing therapeutic and preventive measures.
Assuntos
Osteoblastos/patologia , Infecção por Zika virus/patologia , Adulto , Fosfatase Alcalina/genética , Animais , Diferenciação Celular , Chlorocebus aethiops , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Efeito Citopatogênico Viral , Marcadores Genéticos , Humanos , Interleucina-6/genética , Masculino , Células-Tronco Mesenquimais/virologia , Osteoblastos/virologia , Células Vero , Adulto Jovem , Zika virus/isolamento & purificação , Zika virus/patogenicidade , Infecção por Zika virus/genéticaRESUMO
A link between inflammatory disease and bone loss is now recognized. However, limited data exist on the impact of virus infection on bone loss and regeneration. Bone loss results from an imbalance in remodeling, the physiological process whereby the skeleton undergoes continual cycles of formation and resorption. The specific molecular and cellular mechanisms linking virus-induced inflammation to bone loss remain unclear. In the current study, we provide evidence that infection of mice with either lymphocytic choriomeningitis virus (LCMV) or pneumonia virus of mice (PVM) resulted in rapid and substantial loss of osteoblasts from the bone surface. Osteoblast ablation was associated with elevated levels of circulating inflammatory cytokines, including TNF-α, IFN-γ, IL-6, and CCL2. Both LCMV and PVM infections resulted in reduced osteoblast-specific gene expression in bone, loss of osteoblasts, and reduced serum markers of bone formation, including osteocalcin and procollagen type 1 N propeptide. Infection of Rag-1-deficient mice (which lack adaptive immune cells) or specific depletion of CD8+ T lymphocytes limited osteoblast loss associated with LCMV infection. By contrast, CD8+ T cell depletion had no apparent impact on osteoblast ablation in association with PVM infection. In summary, our data demonstrate dramatic loss of osteoblasts in response to virus infection and associated systemic inflammation. Further, the inflammatory mechanisms mediating viral infection-induced bone loss depend on the specific inflammatory condition.
Assuntos
Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Pneumonia Murina/imunologia , Osteoblastos/virologia , Infecções por Pneumovirus/imunologia , Infecções por Pneumovirus/virologia , Animais , Biomarcadores , Medula Óssea/patologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Proteínas de Homeodomínio/genética , Depleção Linfocítica , Camundongos , Camundongos Knockout , Osteoblastos/imunologia , OsteogêneseRESUMO
Historically, bone was thought to be immunologically inactive with the sole function of supporting locomotion and ensuring stromaness functions as a major lymphoid organ. However, a myriad of pathogens (bacteria such as staphylococcus as well as viruses including alphaviruses, HIV or HCV) can invade the bone. These pathogens can cause apoptosis, autophagy and necrosis of osteoblasts and lead to lymphopenia and immune paralysis. There are now several detailed studies on how osteoblasts contribute to innate immune and inflammatory responses; indeed, osteoblasts in concert with resident macrophages can engage an armory of defense mechanisms capable of detecting and controlling pathogen evasion mechanisms. Osteoblasts can express the so-called pattern recognition receptors such as TOLL-like receptors involved in the detection for example of lipids and unique sugars (polysaccharides and polyriboses) expressed by bacteria or viruses (e.g. LPS and RNA respectively). Activated osteoblasts can produce interferon type I, cytokines, chemokines and interferon-stimulated proteins through autocrine and paracrine mechanisms to control for viral replication and to promote phagocytosis or lysis of bacteria for example by defensins. Uncontrolled and sustained innate immune activation of infected osteoblasts will also lead to an imbalance in the production of osteoclastogenic factors such as RANKL and osteoprotegerin involved in bone repair.
Assuntos
Infecções Bacterianas/imunologia , Osso e Ossos/citologia , Imunidade Inata , Osteoblastos/imunologia , Viroses/imunologia , Animais , Infecções Bacterianas/microbiologia , Infecções Bacterianas/fisiopatologia , Osso e Ossos/imunologia , Osso e Ossos/microbiologia , Osso e Ossos/virologia , Quimiocinas/biossíntese , Quimiocinas/imunologia , Citocinas/biossíntese , Citocinas/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/fisiologia , Camundongos , Osteoblastos/microbiologia , Osteoblastos/fisiologia , Osteoblastos/virologia , Osteoclastos/imunologia , Osteoclastos/fisiologia , Transdução de Sinais , Receptores Toll-Like/imunologia , Viroses/fisiopatologia , Viroses/virologiaRESUMO
Strain-specific factors contribute in significant but undefined ways to the variable incidence of herpes simplex virus (HSV) recrudescence. Studies that investigate these strain-specific factors are needed. Here, we used qPCR, in vitro assays, and genomic sequencing to identify important relationships between in vitro and clinical phenotypes of unique HSV-1 clinical isolates. Nine HSV-1 isolates from individuals displaying varying reactivation patterns were studied. Isolates associated with frequent recurrent herpes labialis (RHL) (1) displayed higher rates of viral shedding in the oral cavity than those associated with rare RHL and (2) tended to replicate more efficiently at 33 °C than 39 °C. HSV-1 isolates also displayed a more stable phenotype during propagation in U2OS cells than in Vero cells. Draft genome sequences of four isolates and one variant spanning 95.6 to 97.2 % of the genome were achieved, and whole-genome alignment demonstrated that the majority of these isolates clustered with known North American/European isolates. These findings revealed procedures that could help identify unique genotypes and phenotypes associated with HSV-1 isolates, which can be important for determining viral factors critical for regulating HSV-1 reactivation.
Assuntos
Genoma Viral , Genótipo , Herpesvirus Humano 1/genética , Fenótipo , Filogenia , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Animais , Sequência de Bases , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Herpes Simples/virologia , Herpesvirus Humano 1/classificação , Herpesvirus Humano 1/crescimento & desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Osteoblastos/patologia , Osteoblastos/virologia , Alinhamento de Sequência , Células Vero , Ativação ViralRESUMO
UNLABELLED: Adenovirus E4-ORF3 and E1B-55K converge in subverting critical overlapping cellular pathways to facilitate virus replication. Here, we show that E1B-55K and E4-ORF3 induce sumoylation and the assembly of SUMO2/3 viral genome replication domains. Using a conjugation-deficient SUMO2 construct, we demonstrate that SUMO2/3 is recruited to E2A viral genome replication domains through noncovalent interactions. E1B-55K and E4-ORF3 have critical functions in inactivating MRN and ATM to facilitate viral genome replication. We show that ATM kinase inhibitors rescue ΔE1B-55K/ΔE4-ORF3 viral genome replication and that the assembly of E2A domains recruits SUMO2/3 independently of E1B-55K and E4-ORF3. However, the morphology and organization of SUMO2/3-associated E2A domains is strikingly different from that in wild-type Ad5-infected cells. These data reveal that E1B-55K and E4-ORF3 specify the nuclear compartmentalization and structure of SUMO2/3-associated E2A domains, which could have important functions in viral replication. We show that E4-ORF3 specifically targets and sequesters the cellular E3 SUMO ligase PIAS3 but not PIAS1, PIAS2, or PIAS4. The assembly of E4-ORF3 into a multivalent nuclear matrix is required to target PIAS3. In contrast to MRN, PIAS3 is targeted by E4-ORF3 proteins from disparate adenovirus subgroups. Our studies reveal that PIAS3 is a novel and evolutionarily conserved target of E4-ORF3 in human adenovirus infections. Furthermore, we reveal that viral proteins not only disrupt but also usurp SUMO2/3 to transform the nucleus and assemble novel genomic domains that could facilitate pathological viral replication. IMPORTANCE: SUMO is a key posttranslational modification that modulates the function, localization, and assembly of protein complexes. In the ever-escalating host-pathogen arms race, viruses have evolved strategies to subvert sumoylation. Adenovirus is a small DNA tumor virus that is a global human pathogen and key biomedical agent in basic research and therapy. We show that adenovirus infection induces global changes in SUMO localization and conjugation. Using virus and SUMO mutants, we demonstrate that E1B-55K and E4-ORF3 disrupt and usurp SUMO2/3 interactions to transform the nucleus and assemble highly structured and compartmentalized viral genome domains. We reveal that the cellular E3 SUMO ligase PIAS3 is a novel and conserved target of E4-ORF3 proteins from disparate adenovirus subgroups. The induction of sumoylation and SUMO2/3 viral replication domains by early viral proteins could play an important role in determining the outcome of viral infection.
Assuntos
Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Adenovírus Humanos/metabolismo , Genoma Viral , Chaperonas Moleculares/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteínas E1B de Adenovirus/genética , Proteínas E4 de Adenovirus/genética , Adenovírus Humanos/genética , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Osteoblastos/metabolismo , Osteoblastos/virologia , Proteínas Inibidoras de STAT Ativados/genética , Alinhamento de Sequência , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Ubiquitinas/genética , Replicação ViralRESUMO
UNLABELLED: Latent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requires trans-acting elements, both viral and cellular, such as ORCs, MCMs, and latency-associated nuclear antigen (LANA). However, how cellular proteins are recruited to the viral genome is not very clear. Here, we demonstrated that the host cellular protein, Bub1, is involved in KSHV latent DNA replication. We show that Bub1 constitutively interacts with proliferating cell nuclear antigen (PCNA) via a highly conserved PIP box motif within the kinase domain. Furthermore, we demonstrated that Bub1 can form a complex with LANA and PCNA in KSHV-positive cells. This strongly indicated that Bub1 serves as a scaffold or molecular bridge between LANA and PCNA. LANA recruited PCNA to the KSHV genome via Bub1 to initiate viral replication in S phase and interacted with PCNA to promote its monoubiquitination in response to UV-induced damage for translesion DNA synthesis. This resulted in increased survival of KSHV-infected cells. IMPORTANCE: During latency in KSHV-infected cells, the viral episomal DNA replicates once each cell cycle. KSHV does not express DNA replication proteins during latency. Instead, KSHV LANA recruits the host cell DNA replication machinery to the replication origin. However, the mechanism by which LANA mediates replication is uncertain. Here, we show that LANA is able to form a complex with PCNA, a critical protein for viral DNA replication. Furthermore, our findings suggest that Bub1, a spindle checkpoint protein, serves as a scaffold or molecular bridge between LANA and PCNA. Our data further support a role for Bub1 and LANA in PCNA-mediated cellular DNA replication processes as well as monoubiquitination of PCNA in response to UV damage. These data reveal a therapeutic target for inhibition of KSHV persistence in malignant cells.
Assuntos
Antígenos Virais/genética , DNA Viral/genética , Genoma Viral , Herpesvirus Humano 8/genética , Proteínas Nucleares/genética , Osteoblastos/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Antígenos Virais/metabolismo , Linhagem Celular , Sobrevivência Celular , Replicação do DNA , DNA Viral/metabolismo , Regulação da Expressão Gênica , Herpesvirus Humano 8/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Osteoblastos/virologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fase S , Transdução de Sinais , Sequências Repetidas Terminais , Ubiquitinação , Latência Viral , Replicação ViralRESUMO
UNLABELLED: Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family. During infection, an array of viral proteins manipulates the host cell cycle. We have previously shown that expression of HCMV pUL27 results in increased levels of the cyclin-dependent kinase (CDK) inhibitor p21(Cip1). In addition, pUL27 is necessary for the full antiviral activity of the pUL97 kinase inhibitor maribavir (MBV). The purpose of this study was to define the relationship between pUL27 and pUL97 and its role in MBV antiviral activity. We observed that expression of wild-type but not kinase-inactive pUL97 disrupted pUL27-dependent induction of p21(Cip1). Furthermore, pUL97 associated with and promoted the phosphorylation of pUL27. During infection, inhibition of the kinase resulted in elevated levels of p21(Cip1) in wild-type virus but not a pUL27-deficient virus. We manipulated the p21(Cip1) levels to evaluate the functional consequence to MBV. Overexpression of p21(Cip1) restored MBV activity against a pUL27-deficient virus, while disruption reduced activity against wild-type virus. We provide evidence that the functional target of p21(Cip1) in the context of MBV activity is CDK1. One CDK-like activity of pUL97 is to phosphorylate nuclear lamin A/C, resulting in altered nuclear morphology and increased viral egress. In the presence of MBV, we observed that infection using a pUL27-deficient virus still altered the nuclear morphology. This was prevented by the addition of a CDK inhibitor. Overall, our results demonstrate an antagonistic relationship between pUL27 and pUL97 activities centering on p21(Cip1) and support the idea that CDKs can complement some activities of pUL97. IMPORTANCE: HCMV infection results in severe disease upon immunosuppression and is a leading cause of congenital birth defects. Effective antiviral compounds exist, yet they exhibit high levels of toxicity, are not approved for use during pregnancy, and can result in antiviral resistance. Our studies have uncovered new information regarding the antiviral efficacy of the HCMV pUL97 kinase inhibitor MBV as it relates to the complex interplay between pUL97 and a second HCMV protein, pUL27. We demonstrate that pUL97 functions antagonistically against pUL27 by phosphorylation-dependent inactivation of pUL27-mediated induction of p21(Cip1). In contrast, we provide evidence that p21(Cip1) functions to antagonize overlapping activities between pUL97 and cellular CDKs. In addition, these studies further support the notion that CDK inhibitors or p21(Cip1) activators might be useful in combination with MBV to effectively inhibit HCMV infections.
Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Citomegalovirus/efeitos dos fármacos , Ribonucleosídeos/farmacologia , Proteínas Virais/genética , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/virologia , Proteína Quinase CDC2 , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Citomegalovirus/genética , Citomegalovirus/metabolismo , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/virologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Proteínas Virais/metabolismoRESUMO
UNLABELLED: The vaccinia virus B1R gene encodes a highly conserved protein kinase that is essential for the poxviral life cycle. As demonstrated in many cell types, B1 plays a critical role during viral DNA replication when it inactivates the cellular host defense effector barrier to autointegration factor (BAF or BANF1). To better understand the role of B1 during infection, we have characterized the growth of a B1-deficient temperature-sensitive mutant virus (Cts2 virus) in U2OS osteosarcoma cells. In contrast to all other cell lines tested to date, we found that in U2OS cells, Cts2 viral DNA replication is unimpaired at the nonpermissive temperature. However, the Cts2 viral yield in these cells was reduced more than 10-fold, thus indicating that B1 is required at another stage of the vaccinia virus life cycle. Our results further suggest that the host defense function of endogenous BAF may be absent in U2OS cells but can be recovered through either overexpression of BAF or fusion of U2OS cells with mouse cells in which the antiviral function of BAF is active. Interestingly, examination of late viral proteins during Cts2 virus infection demonstrated that B1 is required for optimal processing of the L4 protein. Finally, execution point analyses as well as electron microscopy studies uncovered a role for B1 during maturation of poxviral virions. Overall, this work demonstrates that U2OS cells are a novel model system for studying the cell type-specific regulation of BAF and reveals a role for B1 beyond DNA replication during the late stages of the viral life cycle. IMPORTANCE: The most well characterized role for the vaccinia virus B1 kinase is to facilitate viral DNA replication by phosphorylating and inactivating BAF, a cellular host defense responsive to foreign DNA. Additional roles for B1 later in the viral life cycle have been postulated for decades but are difficult to examine directly due to the importance of B1 during DNA replication. Here, we demonstrate that in U2OS cells, a B1 mutant virus escapes the block in DNA replication observed in other cell types and, instead, this mutant virus exhibits impaired late protein accumulation and incomplete maturation of new virions. These data provide the clearest evidence to date that B1 is needed for multiple critical junctures in the poxviral life cycle in a manner that is both dependent on and independent of BAF.
Assuntos
Replicação do DNA , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Osteoblastos/virologia , Proteínas Quinases/genética , Vaccinia virus/genética , Proteínas Virais/genética , Animais , Fusão Celular , Linhagem Celular Tumoral , DNA Viral/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/imunologia , Osteoblastos/imunologia , Proteínas Quinases/imunologia , Transdução de Sinais , Vaccinia virus/imunologia , Proteínas Virais/imunologia , Vírion/genética , Vírion/crescimento & desenvolvimento , Vírion/imunologia , Replicação Viral/genéticaRESUMO
BACKGROUND: Arthritogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) have caused widespread outbreaks of chronic polyarthritis. The inflammatory responses in alphavirus-induced arthritis and osteoarthritis (OA) share many similar features, which suggests the possibility of exacerbated alphavirus-induced bone pathology in individuals with pre-existing OA. Here, we investigated the susceptibility of osteoblasts (OBs) from OA patients to RRV infection and dissected the immune mechanisms elicited from infection. METHODS: Primary hOBs obtained from trabecular bone of healthy donors and OA patients were infected with RRV. Infectivity and viral replication were determined using flow cytometry and plaque assay, respectively. Real-time PCR was performed to determine expression kinetics of type I interferon (IFN)-related immune mediators and osteotropic factors. RESULTS: OA hOBs showed enhanced RRV infectivity and replication during infection, which was associated with delayed induction of IFN-ß and RIG-I expression. Enhanced susceptibility of OA hOBs to RRV was associated with a more pronounced increase in RANKL/OPG ratio and expression of osteotropic factors (IL-6, IL-1ß, TNF-α and CCL2) in comparison to RRV-infected healthy hOBs. CONCLUSIONS: Delayed activation of type I IFN-signalling pathway may have contributed to enhanced susceptibility to RRV infection in hOBs from OA patients. RRV-induced increases in RANKL/OPG ratio and expression of osteotropic factors that favour bone resorption, which may be exacerbated during osteoarthritis. This study provides the novel insight that osteoarthritis may be a risk factor for exacerbated arthritogenic alphaviral infection.
Assuntos
Interferon Tipo I/metabolismo , Osteoartrite , Osteoblastos/imunologia , Osteoblastos/virologia , Ross River virus/fisiologia , Replicação Viral , Células Cultivadas , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Ross River virus/crescimento & desenvolvimento , Ensaio de Placa ViralRESUMO
In this work we characterized the infection of a primary culture of rat osteoblastic lineage cells (OBCs) with measles virus (MeV) and the effect of infection on cell differentiation and maturation. Infection of OBCs with MeV led to high titers of infectivity released early after infection. Also, analysis of mRNAs corresponding to osteogenic differentiation markers like alkaline phosphatase (ALP), bone sialo-protein (BSP) and bone morphogenetic proteins (BMPs) 1-4-5-7 in OBCs revealed higher values (2-75-fold of increment) for infected cells in comparison with uninfected controls. Differentiation of OBCs in osteogenic medium prior to infection influenced the level of stimulation induced by MeV. Furthermore, treatment of OBCs with Ly294002, a PI3K/AKT inhibitor, increased viral titers, whereas treatment with 10µM or 100µM ATPγS diminished MeV multiplication. In addition, increments of osteogenic differentiation markers induced by MeV infection were not modified either by treatment with Ly294002 or ATPγS. These data provide the first evidence demonstrating that MeV can infect osteoblasts in vitro leading to osteoblastic differentiation, a key feature in bone pathogenic processes like otosclerosis.
Assuntos
Vírus do Sarampo/fisiologia , Osteoblastos/virologia , Osteogênese/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Fosfatase Alcalina/biossíntese , Animais , Proteína Morfogenética Óssea 4/biossíntese , Osso e Ossos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Morfolinas/farmacologia , Osteoblastos/fisiologia , Otosclerose/etiologia , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Replicação ViralRESUMO
Arthritogenic alphaviruses including Ross River virus (RRV), Sindbis virus, and chikungunya virus cause worldwide outbreaks of musculoskeletal disease. The ability of alphaviruses to induce bone pathologies remains poorly defined. Here we show that primary human osteoblasts (hOBs) can be productively infected by RRV. RRV-infected hOBs produced high levels of inflammatory cytokine including IL-6. The RANKL/OPG ratio was disrupted in the synovial fluid of RRV patients, and this was accompanied by an increase in serum Tartrate-resistant acid phosphatase 5b (TRAP5b) levels. Infection of bone cells with RRV was validated using an established RRV murine model. In wild-type mice, infectious virus was detected in the femur, tibia, patella, and foot, together with reduced bone volume in the tibial epiphysis and vertebrae detected by microcomputed tomographic (µCT) analysis. The RANKL/OPG ratio was also disrupted in mice infected with RRV; both this effect and the bone loss were blocked by treatment with an IL-6 neutralizing antibody. Collectively, these findings provide previously unidentified evidence that alphavirus infection induces bone loss and that OBs are capable of producing proinflammatory mediators during alphavirus-induced arthralgia. The perturbed RANKL/OPG ratio in RRV-infected OBs may therefore contribute to bone loss in alphavirus infection.
Assuntos
Infecções por Alphavirus/patologia , Infecções por Alphavirus/virologia , Artrite/virologia , Reabsorção Óssea/patologia , Reabsorção Óssea/virologia , Osteoblastos/patologia , Ross River virus/fisiologia , Fosfatase Ácida/sangue , Adulto , Infecções por Alphavirus/sangue , Animais , Anticorpos Neutralizantes/farmacologia , Artrite/sangue , Artrite/patologia , Reabsorção Óssea/sangue , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Osso e Ossos/virologia , Feminino , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/patologia , Lâmina de Crescimento/virologia , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-6/biossíntese , Isoenzimas/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Osteoblastos/efeitos dos fármacos , Osteoblastos/virologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Osteoclastos/virologia , Osteogênese/efeitos dos fármacos , Osteoprotegerina/metabolismo , Fenótipo , Ligante RANK/metabolismo , Ross River virus/efeitos dos fármacos , Líquido Sinovial/metabolismo , Fosfatase Ácida Resistente a Tartarato , Replicação Viral/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
Oncolytic virotherapy using adenoviruses has potential therapeutic benefits for a variety of cancers. We recently developed MOA5, a tumor-specific midkine promoter-regulated oncolytic vector based on human adenovirus serotype 5 (Ad5). We modified the binding tropism of MOA5 by replacing the cell-binding domain of the Ad5 fiber knob with that from another adenovirus serotype 35 (Ad35); the resulting vector was designated MOA35. Here we evaluated the therapeutic efficacies of MOA5 and MOA35 for human osteosarcoma. Midkine mRNA expression and its promoter activity was significantly high in five human osteosarcoma cell lines, but was restricted in normal cells. Very low levels of adenovirus cellular receptor coxsackievirus/adenovirus receptor (CAR) (Ad5 receptor) expression were observed in MNNG-HOS and MG-63 cells, whereas high levels of CAR expression were seen in the other osteosarcoma cell lines. By contrast, CD46 (Ad35 receptor) was highly expressed in all osteosarcoma cell lines. Infectivity and in vitro cytocidal effect of MOA35 was significantly enhanced in MNNG-HOS and MG-63 cells compared with MOA5, although the cytocidal effects of MOA5 were sometimes higher in high CAR-expressing cell lines. In MG-63 xenograft models, MOA35 significantly enhanced antitumor effects compared with MOA5. Our findings indicate that MOA5 and MOA35 allow tailored virotherapy and facilitate more effective treatments for osteosarcoma.
Assuntos
Fatores de Crescimento Neural/genética , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Osteossarcoma/terapia , Osteossarcoma/virologia , Infecções por Adenoviridae/genética , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Feminino , Vetores Genéticos/genética , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Midkina , Vírus Oncolíticos/genética , Osteoblastos/virologia , Osteossarcoma/genética , Regiões Promotoras Genéticas , Receptores Virais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bone mineralization is a normal physiological process, whereas ectopic calcification of soft tissues is a pathological process that leads to irreversible tissue damage. We have established a coxsackievirus B3 (CVB3)-infected mouse model that manifests both osteoporosis and ectopic calcification specifically in heart, pancreas, and lung. The CVB3-infected mice showed increased serum concentrations of both cytokines including IL-1ß, TNF-α, and the receptor activator of NF-κB ligand (RANKL) that stimulate osteoclast formation and of the osteoclast-derived protein tartrate-resistant acid phosphatase 5b. They exhibited more osteoclasts in bone, with no change in the number of osteoblasts, and a decrease in bone formation and the serum concentration of osteoblast-produced osteocalcin. These results indicate that CVB3-induced osteoporosis is likely due to upregulation of osteoclast formation and function, in addition to decreased osteoblast activity. In addition, the serum in the CVB3-infected mice contained a high inorganic phosphate content, which causes ectopic calcification. RANKL treatment induced an increase in the in vitro cardiac fibroblast calcification by inorganic phosphate via the upregulation of osteogenic BMP2, SPARC, Runx2, Fra-1, and NF-κB signaling. We finally observed that i.p. administration of RANK-Fc, a recombinant antagonist of RANKL, prevented bone loss as well as ectopic calcification in CVB3-infected mice. Thus, our results indicate that RANKL may contribute to both abnormal calcium deposition in soft tissues and calcium depletion in bone. In addition, our animal model should provide a tool for the development of new therapeutic agents for calcium disturbance in soft and hard tissues.
Assuntos
Calcinose/prevenção & controle , Infecções por Coxsackievirus/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoporose/metabolismo , Osteoporose/prevenção & controle , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Animais , Calcinose/patologia , Calcinose/virologia , Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/patologia , Modelos Animais de Doenças , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ossificação Heterotópica/metabolismo , Ossificação Heterotópica/patologia , Ossificação Heterotópica/virologia , Osteoblastos/patologia , Osteoblastos/virologia , Osteoclastos/patologia , Osteoclastos/virologia , Osteoporose/virologia , Ligante RANK/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Receptor Ativador de Fator Nuclear kappa-B/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genéticaRESUMO
PURPOSE: Biomaterial-associated infections (BAI) remain a major concern in modern health care. BAI is difficult to treat and often results in implant replacement or removal. Pathogens can be introduced on implant surfaces during surgery and compete with host cells attempting to integrate the implant. Here we studied the influence of prophylactically given cephatholin in the competition between highly virulent Staphylococcus aureus and human osteoblast-like cells (U-2 OS, ATCC HTB-94) for a poly(methyl methacrylate) surface in vitro using a peri-operative contamination model. METHOD: S. aureus was seeded on the acrylic surface in a parallel plate flow chamber prior to adhesion of U-2 OS cells. Next, S. aureus and U-2 OS cells were allowed to grow simultaneously under shear (0.14 1/s) in a modified culture medium containing cephatholin for 8 h, the time period this drug is supposed to be active in situ. Subsequently, the flow was continued with modified culture medium for another 64 h. RESULTS: In the absence of cephatholin, highly virulent S. aureus caused U-2 OS cell death within 18 h. In contrast, the presence of cephatholin for 8 h resulted in survival of U-2 OS cell up to 72 h during simultaneous growth of U-2 OS cells and bacteria. Not all adhering bacteria were killed however, but they showed a delayed growth. CONCLUSIONS: These findings are in line with the recalcitrance of biofilms against antibiotic treatment observed clinically, and represent another support for the use of in vitro co-culture models in mimicking the clinical situation.