RESUMO
Interferons (IFNs) and the products of interferon-stimulated genes (ISGs) play crucial roles in host defense against virus infections. Although many ISGs have been characterized with respect to their antiviral activity, their target specificities and mechanisms of action remain largely unknown. Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that is linked to several human malignancies. Here, we used the genetically and biologically related virus, murine gammaherpesvirus 68 (MHV-68) and screened for ISGs with anti-gammaherpesvirus activities. We found that overexpression of RNF213 dramatically inhibited MHV-68 infection, whereas knockdown of endogenous RNF213 significantly promoted MHV-68 proliferation. Importantly, RNF213 also inhibited KSHV de novo infection, and depletion of RNF213 in the latently KSHV-infected iSLK-219 cell line significantly enhanced lytic reactivation. Mechanistically, we demonstrated that RNF213 targeted the Replication and Transcription Activator (RTA) of both KSHV and MHV-68, and promoted the degradation of RTA protein through the proteasome-dependent pathway. RNF213 directly interacted with RTA and functioned as an E3 ligase to ubiquitinate RTA via K48 linkage. Taken together, we conclude that RNF213 serves as an E3 ligase and inhibits the de novo infection and lytic reactivation of gammaherpesviruses by degrading RTA through the ubiquitin-proteasome pathway.
Assuntos
Gammaherpesvirinae , Infecções por Herpesviridae , Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Humanos , Adenosina Trifosfatases/metabolismo , Gammaherpesvirinae/genética , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/genética , Herpesvirus Humano 8/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Latência Viral/genética , Replicação ViralRESUMO
Urinary tract infection (UTI) is the most common type of urogenital disease. UTI affects the urethra, bladder, ureter, and kidney. A total of 13.3% of women, 2.3% of men, and 3.4% of children in the United States will require treatment for UTI. Traditionally, bladder (cystitis) and kidney (pyelonephritis) infections are considered independently. However, both infections induce host defenses that are either shared or coordinated across the urinary tract. Here, we review the chemical and biophysical mechanisms of bacteriostasis, which limit the duration and severity of the illness. Urinary bacteria attempt to overcome each of these defenses, complicating description of the natural history of UTI.
Assuntos
Cistite , Infecções Urinárias , Sistema Urinário , Criança , Cistite/complicações , Cistite/microbiologia , Feminino , Humanos , Rim , MasculinoRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family, which can cause human malignancies including Kaposi sarcoma, primary effusion lymphoma, and multicentric Castleman's diseases. KSHV typically maintains a persistent latent infection within the host. However, after exposure to intracellular or extracellular stimuli, KSHV lytic replication can be reactivated. The reactivation process of KSHV triggers the innate immune response to limit viral replication. Here, we found that the transcriptional regulator RUNX3 is transcriptionally upregulated by the NF-κB signaling pathway in KSHV-infected SLK cells and B cells during KSHV reactivation. Notably, knockdown of RUNX3 significantly promotes viral lytic replication as well as the gene transcription of KSHV. Consistent with this finding, overexpression of RUNX3 impairs viral lytic replication. Mechanistically, RUNX3 binds to the KSHV genome and limits viral replication through transcriptional repression, which is related to its DNA- and ATP-binding ability. However, KSHV has also evolved corresponding strategies to antagonize this inhibition by using the viral protein RTA to target RUNX3 for ubiquitination and proteasomal degradation. Altogether, our study suggests that RUNX3, a novel host-restriction factor of KSHV that represses the transcription of viral genes, may serve as a potential target to restrict KSHV transmission and disease development.IMPORTANCEThe reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latent infection to lytic replication is important for persistent viral infection and tumorigenicity. However, reactivation is a complex event, and the regulatory mechanisms of this process are not fully elucidated. Our study revealed that the host RUNX3 is upregulated by the NF-κB signaling pathway during KSHV reactivation, which can repress the transcription of KSHV genes. At the late stage of lytic replication, KSHV utilizes a mechanism involving RTA to degrade RUNX3, thus evading host inhibition. This finding helps elucidate the regulatory mechanism of the KSHV life cycle and may provide new clues for the development of therapeutic strategies for KSHV-associated diseases.
Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core , Herpesvirus Humano 8 , Infecção Latente , Humanos , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica , Genoma Viral , Herpesvirus Humano 8/fisiologia , NF-kappa B/metabolismo , Ativação Viral , Latência Viral , Replicação Viral , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismoRESUMO
TRIM32 is often aberrantly expressed in many types of cancers. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked with several human malignancies, including Kaposi's sarcoma and primary effusion lymphomas (PELs). Increasing evidence has demonstrated the crucial role of KSHV lytic replication in viral tumorigenesis. However, the role of TRIM32 in herpesvirus lytic replication remains unclear. Here, we reveal that the expression of TRIM32 is upregulated by KSHV in latency, and reactivation of KSHV lytic replication leads to the inhibition of TRIM32 in PEL cells. Strikingly, RTA, the master regulator of lytic replication, interacts with TRIM32 and dramatically promotes TRIM32 for degradation via the proteasome systems. Inhibition of TRIM32 induces cell apoptosis and in turn inhibits the proliferation and colony formation of KSHV-infected PEL cells and facilitates the reactivation of KSHV lytic replication and virion production. Thus, our data imply that the degradation of TRIM32 is vital for the lytic activation of KSHV and is a potential therapeutic target for KSHV-associated cancers. IMPORTANCE: TRIM32 is associated with many cancers and viral infections; however, the role of TRIM32 in viral oncogenesis remains largely unknown. In this study, we found that the expression of TRIM32 is elevated by Kaposi's sarcoma-associated herpesvirus (KSHV) in latency, and RTA (the master regulator of lytic replication) induces TRIM32 for proteasome degradation upon viral lytic reactivation. This finding provides a potential therapeutic target for KSHV-associated cancers.
Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Proteólise , Transativadores , Fatores de Transcrição , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Ativação Viral , Replicação Viral , Humanos , Apoptose , Linhagem Celular , Herpesvirus Humano 8/crescimento & desenvolvimento , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidade , Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/genética , Linfoma de Efusão Primária/virologia , Linfoma de Efusão Primária/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sarcoma de Kaposi/virologia , Sarcoma de Kaposi/metabolismo , Transativadores/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Latência ViralRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition.
Assuntos
Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , Genoma Viral , Herpesvirus Humano 8 , Regiões Promotoras Genéticas , Ativação Viral , Latência Viral , Humanos , Antígenos Virais/genética , Antígenos Virais/metabolismo , Cromatina/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Regulação Viral da Expressão Gênica/genética , Genoma Viral/genética , Células HEK293 , Herpesvirus Humano 8/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Sequências Repetidas Terminais/genética , Transativadores/metabolismo , Ativação Viral/genética , Latência Viral/genéticaRESUMO
The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi's sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi's sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma-secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. Reduction of KSHV production is associated with gene-specific reduction of viral transcription in both Vero and PEL cells. Specific inhibition of Notch1 by siRNA partially reduces the production of infectious KSHV, and NICD1 forms promoter-specific complexes with viral DNA during reactivation. We conclude that constitutive Notch activity is required for the robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk/DNA complex during viral reactivation. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA-binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces the expression of specific viral genes yet does not affect the growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta's ability to redistribute RBP-Jk DNA binding to the virus during reactivation.
Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Receptor Notch1 , Transativadores , Ativação Viral , Latência Viral , Herpesvirus Humano 8/fisiologia , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/genética , Humanos , Animais , Transativadores/metabolismo , Transativadores/genética , Receptor Notch1/metabolismo , Receptor Notch1/genética , Células Vero , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/genética , Chlorocebus aethiops , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regulação Viral da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Ligação a DNARESUMO
Diabetic cardiomyopathy (dCM) is a major complication of diabetes; however, specific treatments for dCM are currently lacking. RTA 408, a semisynthetic triterpenoid, has shown therapeutic potential against various diseases by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. We established in vitro and in vivo models using high glucose toxicity and db/db mice, respectively, to simulate dCM. Our results demonstrated that RTA 408 activated Nrf2 and alleviated various dCM-related cardiac dysfunctions, both in vivo and in vitro. Additionally, it was found that silencing the Nrf2 gene eliminated the cardioprotective effect of RTA 408. RTA 408 ameliorated oxidative stress in dCM mice and high glucose-exposed H9C2 cells by activating Nrf2, inhibiting mitochondrial fission, exerting anti-inflammatory effects through the Nrf2/NF-κB axis, and ultimately suppressing apoptosis, thereby providing cardiac protection against dCM. These findings provide valuable insights for potential dCM treatments.NEW & NOTEWORTHY We demonstrated first that the nuclear factor erythroid 2-related factor 2 (Nrf2) activator RTA 408 has a protective effect against diabetic cardiomyopathy. We found that RTA 408 could stimulate the nuclear entry of Nrf2 protein, regulate the mitochondrial fission-fusion balance, and redistribute p65, which significantly alleviated the oxidative stress level in cardiomyocytes, thereby reducing apoptosis and inflammation, and protecting the systolic and diastolic functions of the heart.
Assuntos
Diabetes Mellitus , Cardiomiopatias Diabéticas , Triterpenos , Camundongos , Animais , NF-kappa B/genética , Cardiomiopatias Diabéticas/tratamento farmacológico , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Dinâmica Mitocondrial , Estresse Oxidativo , Inflamação/metabolismo , Triterpenos/metabolismo , Triterpenos/farmacologia , Triterpenos/uso terapêutico , Miócitos Cardíacos/metabolismo , Glucose/metabolismo , Diabetes Mellitus/metabolismoRESUMO
Corn leaf aphids (Rhopalosiphum maidis) are highly destructive pests of maize (Zea mays) that threaten growth and seed yield, but resources for aphid resistance are scarce. Here, we identified an aphid-resistant maize mutant, resistance to aphids 1 (rta1), which is allelic to LIGULELESS1 (LG1). We confirmed LG1's role in aphid resistance using the independent allele lg1-2, allelism tests and LG1 overexpression lines. LG1 interacts with, and increases the stability of ZINC-FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM (ZIM1), a central component of the jasmonic acid (JA) signalling pathway, by disturbing its interaction with the F-box protein CORONATINE INSENSITIVE 1a (COI1a). Natural variation in the LG1 promoter was associated with aphid resistance among inbred lines. Moreover, a loss-of-function mutant in the LG1-related gene SPL8 in the dicot Arabidopsis thaliana conferred aphid resistance. This study revealed the aphid resistance mechanism of lg1, providing a theoretical basis and germplasm for breeding aphid-resistant crops.
RESUMO
Epstein-Barr virus (EBV) is a human oncogenic γ-herpesvirus that establishes persistent infection in more than 90% of the world's population. EBV has two life cycles, latency and lytic replication. Reactivation of EBV from latency to the lytic cycle is initiated and controlled by two viral immediate-early transcription factors, Zta and Rta, encoded by BZLF1 and BRLF1, respectively. In this study, we found that IQGAP2 expression was elevated in EBV-infected B cells and identified Rta as a viral gene responsible for the IQGAP2 upregulation in both B cells and nasopharyngeal carcinoma cell lines. Mechanistically, we showed that Rta increases IQGAP2 expression through direct binding to the Rta-responsive element in the IQGAP2 promoter. We also demonstrated the direct interaction between Rta and IQGAP2 as well as their colocalization in the nucleus. Functionally, we showed that the induced IQGAP2 is required for the Rta-mediated Rta promoter activation in the EBV lytic cycle progression and may influence lymphoblastoid cell line clumping morphology through regulating E-cadherin expression. IMPORTANCE Elevated levels of antibodies against EBV lytic proteins and increased EBV DNA copy numbers in the sera have been reported in patients suffering from Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma, indicating that EBV lytic cycle progression may play an important role in the pathogenesis of EBV-associated diseases and highlighting the need for a more complete mechanistic understanding of the EBV lytic cycle. Rta acts as an essential transcriptional activator to induce lytic gene expression and thus trigger EBV reactivation. In this study, scaffolding protein IQGAP2 was found to be upregulated prominently following EBV infection via the direct binding of Rta to the RRE in the IQGAP2 promoter but not in response to other biological stimuli. Importantly, IQGAP2 was demonstrated to interact with Rta and promote the EBV lytic cycle progression. Suppression of IQGAP2 was also found to decrease E-cadherin expression and affect the clumping morphology of lymphoblastoid cell lines.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Proteínas Imediatamente Precoces , Neoplasias Nasofaríngeas , Humanos , Infecções por Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ativação ViralRESUMO
IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing human herpesvirus that establishes a persistent infection in humans. The lytic viral cycle plays a crucial part in lifelong infection as it is involved in the viral dissemination. The master regulator of the KSHV lytic replication cycle is the viral replication and transcription activator (RTA) protein, which is necessary and sufficient to push the virus from latency into the lytic phase. Thus, the identification of host factors utilized by RTA for controlling the lytic cycle can help to find novel targets that could be used for the development of antiviral therapies against KSHV. Using a proteomics approach, we have identified a novel interaction between RTA and the cellular E3 ubiquitin ligase complex RNF20/40, which we have shown to be necessary for promoting RTA-induced KSHV lytic cycle.
Assuntos
Herpesvirus Humano 8 , Interações entre Hospedeiro e Microrganismos , Proteínas Imediatamente Precoces , Ubiquitina-Proteína Ligases , Proteínas Virais , Ativação Viral , Latência Viral , Replicação Viral , Humanos , Herpesvirus Humano 8/crescimento & desenvolvimento , Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Ligação Proteica , Proteômica , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Infectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain's gC, TK, gG, gD, and gE genes. RESULTS: The study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/µL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28. CONCLUSIONS: The established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.
Assuntos
Alphaherpesvirinae , Rinotraqueíte Infecciosa Bovina , Reação em Cadeia da Polimerase em Tempo Real , Rinotraqueíte Infecciosa Bovina/virologia , Animais , Bovinos , Alphaherpesvirinae/classificação , Alphaherpesvirinae/genética , Alphaherpesvirinae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e Especificidade , Manejo de Espécimes/veterinária , FilogeniaRESUMO
The progressive neurodegenerative disease amyotrophic lateral sclerosis (ALS) is caused by a decline in motor neuron function, resulting in worsened motor impairments, malnutrition, respiratory failure and mortality, and there is a lack of effective clinical treatments. The exact mechanism of motor neuronal degeneration remains unclear. Previously, we reported that ferroptosis, which is characterized by the accumulation of lipid peroxide and glutathione depletion in an iron-dependent manner, contributed to motor neuronal death in ALS cell models with the hSOD1G93A (human Cu/Zn-superoxide dismutase) gene mutation. In this study, we further explored the role of ferroptosis in motor neurons and its regulation in mutant hSOD1G93A cell and mouse models. Our results showed that ferroptosis was activated in hSOD1G93A NSC-34 cells and mouse models, which was accompanied by decreased nuclear retention of nuclear factor erythroid 2-related factor 2 (NRF2) and downregulation of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) levels. Moreover, RTA-408, an NRF2 activator, inhibited ferroptosis in hSOD1G93A NSC-34 cells by upregulating the protein expression of SLC7A11 and GPX4. Moreover, hSOD1G93A mice treated with RTA-408 showed obvious improvements in body weight and motor function. Our study demonstrated that ferroptosis contributed to the toxicity of motor neurons and that activating NRF2 could alleviate neuronal degeneration in ALS with the hSOD1G93A mutation.
Assuntos
Esclerose Lateral Amiotrófica , Ferroptose , Doenças Neurodegenerativas , Animais , Humanos , Camundongos , Esclerose Lateral Amiotrófica/metabolismo , Modelos Animais de Doenças , Camundongos Transgênicos , Neurônios Motores/metabolismo , Mutação/genética , Doenças Neurodegenerativas/metabolismo , Neuroproteção , Fator 2 Relacionado a NF-E2/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is responsible for the pathogenesis of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. The expression of immunosuppressive genes, such as IL-10 and CD274/PD-L1 is observed during KSHV-associated pathogenesis, and the modulation of the host immune system by KSHV contributes to establishing viral persistence in the host. Understanding the mechanism that allows the virus to evade host cell immunity would be helpful in order to develop therapeutic strategies for KSHV malignancy. In this study, we show that KSHV replication and transcriptional activator (K-RTA), an essential activator of the viral lytic cycle, transactivates the CD274/PD-L1 gene promoter. Mechanistically, we demonstrate that the binding of K-RTA to the cellular specificity protein 1 (SP1) is critical for K-RTA-mediated CD274/PD-L1 promoter activation. These findings suggest that K-RTA cooperates with intracellular SP1 to activate the expression of CD274/PD-L1, which helps the virus regulate immune checkpoints to escape and survive.
Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Humanos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Proteínas Imediatamente Precoces/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Replicação Viral/genética , Regiões Promotoras GenéticasRESUMO
Gammaherpesviruses (GHVs) are lymphotropic tumor viruses with a biphasic infectious cycle. Lytic replication at the primary site of infection is necessary for GHVs to spread throughout the host and establish latency in distal sites. Dissemination is mediated by infected B cells that traffic hematogenously from draining lymph nodes to peripheral lymphoid organs, such as the spleen. B cells serve as the major reservoir for viral latency, and it is hypothesized that periodic reactivation from latently infected B cells contributes to maintaining long-term chronic infection. While fundamentally important to an understanding of GHV biology, aspects of B cell infection in latency establishment and maintenance are incompletely defined, especially roles for lytic replication and reactivation in this cell type. To address this knowledge gap and overcome limitations of replication-defective viruses, we generated a recombinant murine gammaherpesvirus 68 (MHV68) in which ORF50, the gene that encodes the essential immediate-early replication and transcription activator protein (RTA), was flanked by loxP sites to enable conditional ablation of lytic replication by ORF50 deletion in cells that express Cre recombinase. Following infection of mice that encode Cre in B cells with this virus, splenomegaly and viral reactivation from splenocytes were significantly reduced; however, the number of latently infected splenocytes was equivalent to WT MHV68. Despite ORF50 deletion, MHV68 latency was maintained over time in spleens of mice at levels approximating WT, reactivation-competent MHV68. Treatment of infected mice with lipopolysaccharide (LPS), which promotes B cell activation and MHV68 reactivation ex vivo, yielded equivalent increases in the number of latently infected cells for both ORF50-deleted and WT MHV68, even when mice were simultaneously treated with the antiviral drug cidofovir to prevent reactivation. Together, these data demonstrate that productive viral replication in B cells is not required for MHV68 latency establishment and support the hypothesis that B cell proliferation facilitates latency maintenance in vivo in the absence of reactivation. IMPORTANCE Gammaherpesviruses establish lifelong chronic infections in cells of the immune system and place infected hosts at risk for developing lymphomas and other diseases. It is hypothesized that gammaherpesviruses must initiate acute infection in these cells to establish and maintain long-term infection, but this has not been directly tested. We report here the use of a viral genetic system that allows for cell-type-specific deletion of a viral gene that is essential for replication and reactivation. We employ this system in an in vivo model to reveal that viral replication is not required to initiate or maintain infection within B cells.
Assuntos
Linfócitos B , Infecções por Herpesviridae , Proteínas Imediatamente Precoces , Ativação Viral , Animais , Linfócitos B/virologia , Gammaherpesvirinae/genética , Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , Proteínas Imediatamente Precoces/genética , Camundongos , Camundongos Endogâmicos C57BL , Latência Viral , Replicação ViralRESUMO
The immediate early viral protein replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for activating the lytic cycle of KSHV. RTA induces the KSHV lytic cycle by several mechanisms, acting as a viral transcription factor that directly induces viral and host genes and acting as a viral E3 ubiquitin ligase by degrading host proteins that block viral lytic replication. Recently, we have characterized the global gene expression changes in primary effusion lymphoma (PEL) upon lytic reactivation of KSHV, which also led to the identification of rapidly downregulated genes such as ID2, an inhibitor of basic helix-loop-helix transcription factors. Here, we demonstrate that ID2 overexpression in PEL ablates KSHV lytic reactivation, indicating that ID2 inhibits the KSHV lytic cycle. Furthermore, we show that while ID2 is highly expressed during latency, its protein level is rapidly reduced by 4 h postinduction during lytic reactivation. Our results indicate that RTA binds to ID2 and induces its degradation during the KSHV lytic cycle by N-terminal ubiquitination through the ubiquitin-proteasome pathway. Importantly, we found that not only KSHV RTA but also its Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) homologs interact with ID2, and they can induce the degradation of all four members of the ID protein family, suggesting an evolutionarily conserved interplay between gammaherpesvirus RTAs and ID proteins. Taken together, we propose that ID2 acts as a repressor of the KSHV lytic cycle, which is counteracted by its RTA-mediated degradation. We also predict that ID proteins may act as restriction factors of the lytic phase of the other gammaherpesviruses as well. IMPORTANCE In addition to its transcription regulatory role, RTA is also known to have an E3 ubiquitin ligase activity, which RTA utilizes for inducing protein degradation. However, it is still largely unknown what host factors are downregulated during KSHV lytic reactivation by RTA-mediated protein degradation and what the biological significance of the degradation of these host factors is. In this study, we discovered that RTA employs N-terminal ubiquitination to induce degradation of ID2, a potent transcription repressor of host genes, via the ubiquitin-proteasome pathway to promote KSHV lytic reactivation in PEL cells. Furthermore, we found that not only KSHV RTA but also RTA of EBV and MHV68 gammaherpesviruses can induce the degradation of all four human ID proteins, indicating that the interplay between gammaherpesvirus RTAs and ID proteins is evolutionarily conserved.
Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Proteína 2 Inibidora de Diferenciação , Transativadores , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo , Replicação ViralRESUMO
The HBV rtA181T mutation is associated with an increased risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). This study aimed to evaluate the mechanism by which rtA181T mutation increases the risk of HCC. We enrolled 470 CHB patients with rtA181T and rtA181V mutation in this study; 68 (22.15%) of the 307 patients with rtA181T mutation and 22 (13.5%) of the 163 patients with rtA181V mutation developed HCC (p < .05). The median follow-up periods were 8.148 and 8.055 years (p > .05). Serum HBV DNA and HBsAg levels in rtA181T-positive patients were similar to that in rtA181V-positive patients. However, the serum HBeAg levels in the rtA181T-positive patients were significantly higher than that in rtA181V-positive patients. In situ hybridization experiments showed that the HBV cccDNA and HBV RNA levels were significantly higher in the liver cancer tissues of patients with the rtA181T mutation compared to that in the tissues of patients with the rtA181V mutation. The percentage of anti-tumour hot-gene site mutations was significantly higher in the rtA181T-positive HCC liver tissue compared to that in the rtA181T-negative HCC liver tissue (7.65% and 4.3%, p < .05). This is the first study to use a large cohort and a follow-up of more than 5 years (average 8 years) to confirm that the rtA181T mutation increased the risk of HCC, and that it could be related to the increase in the mutation rate of hotspots of tumour suppressor genes (CTNNB1, TP53, NRAS and PIK3CA).
Assuntos
Carcinoma Hepatocelular , Hepatite B Crônica , Neoplasias Hepáticas , Humanos , Vírus da Hepatite B/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/tratamento farmacológico , Hepatite B Crônica/tratamento farmacológico , Antivirais/uso terapêutico , Taxa de Mutação , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamento farmacológico , Mutação , Genes Supressores de Tumor , DNA Viral/genética , Antígenos de Superfície da Hepatite B/genéticaRESUMO
The palearctic spider genus Mastigusa Menge, 1854 is characterized by a remarkable morphology and wide ecological variability, with free-living, cave dwelling and myrmecophile populations known. This genus has a long and tangled taxonomic history and was placed in different families in the past, all belonging to the "marronoid clade", an informal grouping of families characterized by the lack of strong synapomorphies. Three species are currently recognized, but their identity and circumscription has been long debated. A molecular approach was never applied for trying to solve these uncertainties, and doubts still remain both about its phylogenetic placement and about the taxonomic status of the described species. For the first time the genus Mastigusa is included in a molecular phylogenetic analysis and strong support is found for its placement within the family Cybaeidae, in sister relationship with the genus Cryphoeca Thorell, 1870. An analysis of Mastigusa populations spanning across the distribution range of the genus identifies a high and previously overlooked genetic diversity, with six distinct genetic lineages showing a strong geographic pattern. Divergence times between Mastigusa and its sister genus and between the distinct Mastigusa lineages are estimated, and the groundwork is laid for a taxonomic revision of the species belonging to the genus.
Assuntos
Aranhas , Animais , Filogenia , Aranhas/genética , IncertezaRESUMO
BACKGROUND: The relationship between hypertension (HT) and serum liver enzymes was reported in a few studies, but the findings were inconsistent. Therefore, the present study aimed to identify the association between elevated serum liver enzymes and raised BP through the use of a large sample of Royal Thai Army (RTA) personnel. METHODS: The dataset obtained from the annual health examination database of RTA personnel in Thailand was utilized. A total of 244,281 RTA personnel aged 35-60 were included in the current study. Elevated serum liver enzymes were defined as aspartate aminotransferase (AST) or alanine aminotransferase (ALT) ≥ 40 U/L in males and ≥ 35 U/L in females. HT was defined as systolic BP ≥ 140 or diastolic BP ≥ 90 mmHg. A multivariable linear regression model was used to estimate the coefficient and 95% confidence intervals (CI), whereas a multivariable logistic regression model was applied to estimate adjusted odds ratios (AORs) and 95% CI for the association between raised BP and serum liver enzymes. RESULTS: Compared to individuals with SBP < 120 and DBP < 80 mmHg, the ß coefficients of log-transformed AST and ALT were 0.13 (95% CI: 0.12-0.13) and 0.11 (95% CI: 0.11-0.12) in males with HT. Meanwhile, the ß coefficients of log-transformed AST and ALT were 0.03 (95% CI: 0.02-0.04) and 0.07 (95% CI: 0.05-0.08) in females with HT. In males, HT was associated with elevated AST (AOR: 1.92; 95% CI: 1.85-2.01) and elevated ALT (AOR: 1.43; 95% CI: 1.38-1.48). On the other hand, in females, HT was associated with elevated AST (AOR: 1.42; 95% CI: 1.21-1.66) and elevated ALT (AOR: 1.38; 95% CI: 1.21-1.57). CONCLUSION: Raised BP was positively correlated with elevated AST and ALT in active-duty RTA personnel. Moreover, HT was independently attributed to higher odds of elevated AST and ALT in comparison to optimal BP in both males and females. Furthermore, the relationship between serum liver enzymes and BP was modified by sex.
Assuntos
Hipertensão , Hepatopatias , Militares , Masculino , Feminino , Humanos , Fígado , Pressão Sanguínea , Tailândia/epidemiologia , População do Sudeste Asiático , Hipertensão/diagnóstico , Alanina Transaminase , Aspartato AminotransferasesRESUMO
BACKGROUND: Tubulointerstitial nephritis with IgM-positive plasma cells (IgMPC-TIN) is a newer disease about which there are many unclear points. Glucocorticoid therapy is effective in many cases of IgMPC-TIN; however, relapse during glucocorticoid tapering has been reported. Relapse and its treatment are poorly defined. CASE PRESENTATION: Case 1 was a 61-year-old man with renal dysfunction and proteinuria. Tubulointerstitial nephritis and IgM-positive plasma cells were observed in a renal biopsy. He was diagnosed with IgMPC-TIN accompanied by Fanconi syndrome and distal renal tubular acidosis (d-RTA). Prednisolone (PSL; 30 mg daily, 0.45 mg/kg/day) treatment was highly effective, and PSL was gradually tapered and discontinued after 1 year. However, 1 month after PSL discontinuation, therapeutic markers were elevated. Therefore, PSL (10 mg daily, 0.15 mg/kg/day) was administered, and the markers indicated improvement. Case 2 was a 43-year-old woman referred for renal dysfunction and proteinuria. Laboratory data revealed that she had primary biliary cholangitis (PBC), d-RTA, and Fanconi syndrome. A renal biopsy showed accumulation of IgM-positive plasma cells in the tubulointerstitium without any glomerular changes. A diagnosis of IgMPC-TIN was made and the patient was started on PSL (35 mg daily, 0.6 mg/kg/day). Therapeutic markers decreased immediately and PSL was discontinued after 1 year. Three months later, the proteinuria and Fanconi syndrome worsened. PSL treatment was restarted (20 mg daily, 0.35 mg/kg/day) and markers indicated improvement. Case 3 was a 45-year-old woman with renal dysfunction and proteinuria. Tubulointerstitial nephritis and IgM-positive plasma cells were observed in a renal biopsy. The patient had PBC, Sjögren syndrome, d-RTA, and Fanconi syndrome, and the diagnosis of IgMPC-TIN was made. The patient was started on PSL (30 mg daily, 0.4 mg/kg/day) and disease markers decreased immediately. However, when PSL was tapered to 15 mg daily (0.2 mg/kg/day), the patient's serum IgM levels increased; therefore, we maintained the PSL at 15 mg daily (0.2 mg/kg/day). CONCLUSION: We report three cases of relapsed IgMPC-TIN associated with reduction or discontinuation of glucocorticoid therapy. In these cases, elevation of serum IgM preceded that of other markers such as urinary ß2-microglobulin, proteinuria, and glycosuria. We recommend monitoring serum IgM levels while tapering glucocorticoids; a maintenance dose of glucocorticoid should be considered if relapse is suspected or anticipated.
Assuntos
Acidose Tubular Renal , Síndrome de Fanconi , Glucocorticoides , Nefrite Intersticial , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Acidose Tubular Renal/diagnóstico , Síndrome de Fanconi/complicações , Glucocorticoides/uso terapêutico , Imunoglobulina M/sangue , Nefrite Intersticial/diagnóstico , Nefrite Intersticial/tratamento farmacológico , Nefrite Intersticial/complicações , Plasmócitos , Proteinúria/tratamento farmacológico , RecidivaRESUMO
RTA dh404 is a novel synthetic oleanolic acid derivative that has been reported to possess anti-allergic, neuroprotective, antioxidative, and anti-inflammatory properties, and exerts therapeutic effects on various cancers. Although CDDO and its derivatives have anticancer effects, the actual anticancer mechanism has not been fully explored. Therefore, in this study, glioblastoma cell lines were exposed to different concentrations of RTA dh404 (0, 2, 4, and 8 µM). Cell viability was evaluated using the PrestoBlue™ reagent assay. The role of RTA dh404 in cell cycle progression, apoptosis, and autophagy was analyzed using flow cytometry and Western blotting. The expression of cell cycle-, apoptosis-, and autophagy-related genes was detected by next-generation sequencing. RTA dh404 reduces GBM8401 and U87MG glioma cell viability. RTA dh404 treated cells had a significant increase in the percentage of apoptotic cells and caspase-3 activity. In addition, the results of the cell cycle analysis showed that RTA dh404 arrested GBM8401 and U87MG glioma cells at the G2/M phase. Autophagy was observed in RTA dh404-treated cells. Subsequently, we found that RTA dh404-induced cell cycle arrest, apoptosis, and autophagy were related to the regulation of associated genes using next-generation sequencing. Our data indicated that RTA dh404 causes G2/M cell cycle arrest and induces apoptosis and autophagy by regulating the expression of cell cycle-, apoptosis-, and autophagy-related genes in human glioblastoma cells, suggesting that RTA dh404 is a potential drug candidate for the treatment of glioblastoma.