RESUMEN
Individuals infected with human immunodeficiency virus type-1 (HIV-1) show metabolic alterations of CD4+ T cells through unclear mechanisms with undefined consequences. We analyzed the transcriptome of CD4+ T cells from patients with HIV-1 and revealed that the elevated oxidative phosphorylation (OXPHOS) pathway is associated with poor outcomes. Inhibition of OXPHOS by the US Food and Drug Administration-approved drug metformin, which targets mitochondrial respiratory chain complex-I, suppresses HIV-1 replication in human CD4+ T cells and humanized mice. In patients, HIV-1 peak viremia positively correlates with the expression of NLRX1, a mitochondrial innate immune receptor. Quantitative proteomics and metabolic analyses reveal that NLRX1 enhances OXPHOS and glycolysis during HIV-1-infection of CD4+ T cells to promote viral replication. At the mechanistic level, HIV infection induces the association of NLRX1 with the mitochondrial protein FASTKD5 to promote expression of mitochondrial respiratory complex components. This study uncovers the OXPHOS pathway in CD4+ T cells as a target for HIV-1 therapy.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Genómica , Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , Metaboloma , Metabolómica , Fosforilación Oxidativa , Proteoma , Transcriptoma , Replicación Viral , Animales , Antivirales/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Células HEK293 , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/inmunología , VIH-1/metabolismo , Interacciones Huésped-Patógeno , Humanos , Células Jurkat , Masculino , Metformina/farmacología , Ratones , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Proteómica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Carga Viral , Replicación Viral/efectos de los fármacosRESUMEN
NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-kB and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.
Asunto(s)
Hepacivirus/inmunología , Hepatitis C/inmunología , Inmunidad Innata/inmunología , Factor 1 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/inmunología , Proteínas Mitocondriales/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Células Cultivadas , Activación Enzimática/inmunología , Células HEK293 , Hepatitis C/virología , Hepatocitos/inmunología , Hepatocitos/virología , Humanos , Factor 1 Regulador del Interferón/metabolismo , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , FN-kappa B/metabolismo , ARN Viral/genética , Virus Sendai/inmunología , eIF-2 Quinasa/metabolismoRESUMEN
Appropriate immune responses require a fine balance between immune activation and attenuation. NLRC3, a non-inflammasome-forming member of the NLR innate immune receptor family, attenuates inflammation in myeloid cells and proliferation in epithelial cells. T lymphocytes express the highest amounts of Nlrc3 transcript where its physiologic relevance is unknown. We show that NLRC3 attenuated interferon-γ and TNF expression by CD4+ T cells and reduced T helper 1 (Th1) and Th17 cell proliferation. Nlrc3-/- mice exhibited increased and prolonged CD4+ T cell responses to lymphocytic choriomeningitis virus infection and worsened experimental autoimmune encephalomyelitis (EAE). These functions of NLRC3 were executed in a T-cell-intrinsic fashion: NLRC3 reduced K63-linked ubiquitination of TNF-receptor-associated factor 6 (TRAF6) to limit NF-κB activation, lowered phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and diminished glycolysis and oxidative phosphorylation. This study reveals an unappreciated role for NLRC3 in attenuating CD4+ T cell signaling and metabolism.
Asunto(s)
Autoinmunidad/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Inmunidad Innata/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Autoinmunidad/genética , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Encefalomielitis Autoinmune Experimental/genética , Factores Eucarióticos de Iniciación , Humanos , Inmunidad Innata/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/microbiología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/inmunología , Factor 6 Asociado a Receptor de TNF/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/inmunología , Células Th17/metabolismoRESUMEN
HIV-1 expresses several accessory proteins to counteract host anti-viral restriction factors to facilitate viral replication and disease progression. One such protein, Vpr, has been implicated in affecting multiple cellular processes, but its mechanism remains elusive. Here we report that Vpr targets TET2 for polyubiquitylation by the VprBP-DDB1-CUL4-ROC1 E3 ligase and subsequent degradation. Genetic inactivation or Vpr-mediated degradation of TET2 enhances HIV-1 replication and substantially sustains expression of the pro-inflammatory cytokine interleukin-6 (IL-6). This process correlates with reduced recruitment of histone deacetylase 1 and 2 to the IL-6 promoter, thus enhancing its histone H3 acetylation level during resolution phase. Blocking IL-6 signaling reduced the ability of Vpr to enhance HIV-1 replication. We conclude that HIV-1 Vpr degrades TET2 to sustain IL-6 expression to enhance viral replication and disease progression. These results suggest that disrupting the Vpr-TET2-IL6 axis may prove clinically beneficial to reduce both viral replication and inflammation during HIV-1 infection.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , VIH-1/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Monocitos/virología , Proteínas Proto-Oncogénicas/metabolismo , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Dioxigenasas , Células HEK293 , VIH-1/genética , VIH-1/crecimiento & desarrollo , VIH-1/patogenicidad , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Interacciones Huésped-Patógeno , Humanos , Interleucina-6/genética , Células Jurkat , Monocitos/enzimología , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas , Proteolisis , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Células THP-1 , Ubiquitina-Proteína Ligasas , Ubiquitinación , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.
Asunto(s)
Virus de la Hepatitis B , Hepatitis B , Virus de la Hepatitis Delta , Replicación Viral , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Virus de la Hepatitis B/inmunología , Humanos , Virus de la Hepatitis Delta/genética , Virus de la Hepatitis Delta/fisiología , Hepatitis B/virología , Hepatitis B/inmunología , Biología Molecular , Japón , Hepatitis D/virología , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genéticaRESUMEN
IMPORTANCE: The biogenesis and clinical application of serum HBV pgRNA have been a research hotspot in recent years. This study further characterized the heterogeneity of the 3' terminus of capsid RNA by utilizing a variety of experimental systems conditionally supporting HBV genome replication and secretion, and reveal that the 3' truncation of capsid pgRNA is catalyzed by cellular ribonuclease(s) and viral RNaseH at positions after and before 3' DR1, respectively, indicating the 3' DR1 as a boundary between the encapsidated portion of pgRNA for reverse transcription and the 3' unprotected terminus, which is independent of pgRNA length and the 3' terminal sequence. Thus, our study provides new insights into the mechanism of pgRNA encapsidation and reverse transcription, as well as the optimization of serum HBV RNA diagnostics.
Asunto(s)
Cápside , Genoma Viral , Virus de la Hepatitis B , ARN Viral , Replicación Viral , Cápside/metabolismo , Genoma Viral/genética , Hepatitis B/diagnóstico , Hepatitis B/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/crecimiento & desarrollo , Virus de la Hepatitis B/metabolismo , Transcripción Reversa , Ribonucleasa H/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Replicación Viral/genéticaRESUMEN
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), serving as the viral persistence form and transcription template of HBV infection, hijacks host histone and non-histone proteins to form a minichromosome and utilizes posttranslational modifications (PTMs) "histone code" for its transcriptional regulation. HBV X protein (HBx) is known as a cccDNA transcription activator. In this study we established a dual system of the inducible reporter cell lines modelling infection with wildtype (wt) and HBx-null HBV, both secreting HA-tagged HBeAg as a semi-quantitative marker for cccDNA transcription. The cccDNA-bound histone PTM profiling of wt and HBx-null systems, using chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR), confirmed that HBx is essential for maintenance of cccDNA at transcriptionally active state, characterized by active histone PTM markers. Differential proteomics analysis of cccDNA minichromosome established in wt and HBx-null HBV cell lines revealed group-specific hits. One of the hits in HBx-deficient condition was a non-histone host DNA-binding protein high mobility group box 1 (HMGB1). Its elevated association to HBx-null cccDNA was validated by ChIP-qPCR assay in both the HBV stable cell lines and infection systems in vitro. Furthermore, experimental downregulation of HMGB1 in HBx-null HBV inducible and infection models resulted in transcriptional re-activation of the cccDNA minichromosome, accompanied by a switch of the cccDNA-associated histones to euchromatic state with activating histone PTMs landscape and subsequent upregulation of cccDNA transcription. Mechanistically, HBx interacts with HMGB1 and prevents its binding to cccDNA without affecting the steady state level of HMGB1. Taken together, our results suggest that HMGB1 is a novel host restriction factor of HBV cccDNA with epigenetic silencing mechanism, which can be counteracted by viral transcription activator HBx.
Asunto(s)
Proteína HMGB1 , Hepatitis B , ADN Circular/genética , ADN Circular/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Epigénesis Genética , Proteína HMGB1/genética , Células Hep G2 , Virus de la Hepatitis B/metabolismo , Histonas/metabolismo , Humanos , Transactivadores , Factores de Transcripción/metabolismo , Proteínas Reguladoras y Accesorias Virales , Replicación Viral/genéticaRESUMEN
We have previously developed a bacterial artificial chromosome (BAC)-vectored SARS-CoV-2 replicon, namely BAC-CoV2-Rep, which, upon transfection into host cells, serves as a transcription template for SARS-CoV-2 replicon mRNA to initiate replicon replication and produce nanoluciferase (Nluc) reporter from the subgenomic viral mRNA. However, an inherent issue of such DNA-launched replicon system is that the nascent full-length replicon transcript undergoes process by host RNA splicing machinery, which reduces replicon replication and generates spliced mRNA species expressing NLuc reporter independent of replicon replication. To mitigate this problem, we employed Isoginkgetin, a universal eukaryotic host splicing inhibitor, to treat cells transfected with BAC-CoV2-Rep. Isoginkgetin effectively increased the level of full-length replicon transcripts while concurrently reducing the level of Nluc signal derived from spliced replicon mRNA, making the Nluc reporter signal more correlated with replicon replication, as evidenced by treatment with known SARS-CoV-2 replication inhibitors including Remdesivir, GC376, and EIDD-1931. Thus, our study emphasizes that host RNA splicing is a confounding factor for DNA-launched SARS-CoV-2 replicon systems, which can be mitigated by Isoginkgetin treatment.
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Biflavonoides , COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Replicón , ARN Mensajero , Replicación ViralRESUMEN
Emerging evidence supports a high prevalence of cancer type-specific microbiota residing within tumor tissues. The intratumoral microbiome in hepatocellular carcinoma (HCC), especially in viral (hepatitis B virus [HBV]/hepatitis C virus [HCV]) HCC, has not been well characterized for their existence, composition, distribution, and biological functions. We report herein a finding of specific microbial signature in viral HCC as compared to non-HBV/non-HCV (NBNC) HCC. However, the significantly diverse tumor microbiome was only observed in HBV-related HCC, and Cutibacterium was identified as the representative taxa biomarker. Biological function of the unique tumor microbiota in modulating tumor microenvironment (TME) was characterized by using formalin-fixed paraffin-embedded (FFPE) tissue-based multiplex immunofluorescence histochemistry (mIFH) allowing simultaneous in situ detection of the liver cancer cells surrounded with high/low density of microbiota, and the infiltrating immune cells. In HBV_HCC, the intratumoral microbiota are positively associated with increased tumor-infiltrating CD8+ T lymphocytes, but not the CD56+ NK cells. Two subtypes of myeloid-derived suppressor cells (MDSCs): monocytic MDSCs and polymorphonuclear MDSCs, were also found to be positively correlated with the intratumoral microbiota in HBV_HCC, indicating an inhibitory role of these microbial species in antitumor immunity and the contribution to the liver TME in combination of chronic viral hepatitis during HCC development.
Asunto(s)
Carcinoma Hepatocelular , Hepatitis B , Hepatitis C , Neoplasias Hepáticas , Humanos , Virus de la Hepatitis B , Hepatitis C/complicaciones , Hepatitis B/complicaciones , Hepatitis B/patología , Microambiente TumoralRESUMEN
The hollow core anti-resonant fibers (HC-ARFs) based on soft glass are in high demand for 3-6 µm laser delivery. A HC-ARF based on tellurite glass with 6 touching capillaries as cladding was designed and fabricated for the first time, to the best of our knowledge. A relatively low loss of 3.75â dB/m at 4.45 µm was realized in it. The effects of capillary number, core diameter, wall thickness of capillary, and material absorption loss on the loss of the HC-ARF were analyzed by the numerically simulation. The output beam quality was measured and the influence of bending on the fiber loss was discussed. The results of numerical simulation suggested that the theoretical loss of the prepared fiber can be reduced to 0.1â dB/m, indicating that tellurite HC-ARFs have great potential for mid-infrared laser applications.
RESUMEN
In this work, the concentration of rare-earth ions in doped silica whispering gallery lasers (WGLs) is controlled by evaporation. The fabrication of WGLs is used to experimentally evaluate the evaporation rate (mol/µm) and ratio (mol/mol) of erbium and silica lost from a doped fiber during heating. Fixed lengths of doped silica fiber are spliced to different lengths of undoped fiber and then evaporated by feeding into the focus of a CO2 laser. During evaporation, erbium ions are precipitated in the doped silica fiber to control the erbium concentration in the remaining SiO2, which is melted into a microsphere. By increasing the length of the undoped section, a critical point is reached where effectively no ions remain in the glass microsphere. The critical point is found using the spectra of the whispering gallery modes in microspheres with equal sizes. From the critical point, it is estimated that, for a given CO2 laser power, 6.36 × 10-21 mol of Er3+ is lost during the evaporation process for every cubic micron of silica fiber. This is equivalent to 1.74 × 10-7 mol of Er3+ lost per mol of SiO2 evaporated. This result facilitates the control of the doping concentration in WGLs and provides insight into the kinetics of laser-induced evaporation of doped silica.
RESUMEN
OBJECTIVES: A major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA quantification is essential in preclinical and clinical studies. Southern blot (SB) permits cccDNA visualisation but lacks sensitivity and is very laborious. Quantitative PCR (qPCR) has no such limitations but inaccurate quantification due to codetection of viral replicative intermediates (RI) can occur. The use of different samples, preservation conditions, DNA extraction, nuclease digestion methods and qPCR strategies has hindered standardisation. Within the ICE-HBV consortium, available and novel protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures were compared in six laboratories to develop evidence-based guidance for best practices. DESIGN: Reference material (HBV-infected humanised mouse livers and HepG2-NTCP cells) was exchanged for cross-validation. Each group compared different DNA extraction methods (Hirt extraction, total DNA extraction with or without proteinase K treatment (+PK/-PK)) and nuclease digestion protocols (plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, exonucleases I/III). Samples were analysed by qPCR and SB. RESULTS: Hirt and -PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by qPCR. T5 and Exo I/III nucleases efficiently removed all RI forms. In contrast, PSD did not digest pf-rcDNA, but was less prone to induce cccDNA overdigestion. In stabilised tissues (eg, Allprotect), nucleases had detrimental effects on cccDNA. CONCLUSIONS: We present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.
Asunto(s)
ADN Circular , Virus de la Hepatitis B , Animales , Ratones , Humanos , Virus de la Hepatitis B/genética , ADN Circular/genética , Hígado , Reacción en Cadena de la Polimerasa/métodos , Células Hep G2 , ADN Viral/genéticaRESUMEN
Hepatitis B virus (HBV) utilizes host DNA repair mechanisms to convert viral relaxed circular DNA (rcDNA) into a persistent viral genome, the covalently closed circular DNA (cccDNA). To identify host factors involved in cccDNA formation, we developed an unbiased approach to discover proteins involved in cccDNA formation by precipitating nuclear rcDNA from induced HepAD38 cells and identifying the coprecipitated proteins by mass spectrometry. DNA damage binding protein 1 (DDB1) surfaced as a hit, coinciding with our previously reported short hairpin RNA (shRNA) screen in which shRNA-DDB1 in HepDES19 cells reduced cccDNA production. DDB1 binding to nuclear rcDNA was confirmed in HepAD38 cells via ChIP-qPCR. DDB1 and DNA damage binding protein 2 (DDB2) form the UV-DDB complex, and the latter senses DNA damage to initiate the global genome nucleotide excision repair (GG-NER) pathway. To investigate the role of the DDB complex in cccDNA formation, DDB2 was knocked out in HepAD38 and HepG2-NTCP cells. In both knockout cell lines, cccDNA formation was stunted significantly, and in HepG2-NTCP-DDB2 knockout cells, downstream indicators of cccDNA such as HBV RNA, HBcAg, and HBeAg were similarly reduced. Knockdown of DDB2 in HBV-infected HepG2-NTCP cells and primary human hepatocytes (PHH) also resulted in cccDNA reduction. Transcomplementation of wild-type DDB2 in HepG2-NTCP-DDB2 knockout cells rescued cccDNA formation and its downstream indicators. However, ectopic expression of DDB2 mutants deficient in DNA binding, DDB1 binding, or ubiquitination failed to rescue cccDNA formation. Our study thus suggests an integral role of UV-DDB, specifically DDB2, in the formation of HBV cccDNA. IMPORTANCE Serving as a key viral factor for chronic hepatitis B virus (HBV) infection, HBV covalently closed circular DNA (cccDNA) is formed in the cell nucleus from viral relaxed circular DNA (rcDNA) by hijacking host DNA repair machinery. Previous studies have identified several host DNA repair factors involved in cccDNA formation through hypothesis-driven research with some help from RNA interference (RNAi) screening and/or biochemistry approaches. To enrich the landscape of tools for discovering host factors responsible for rcDNA-to-cccDNA conversion, we developed an rcDNA immunoprecipitation paired mass spectrometry assay, which allowed us to pull down nuclear rcDNA in its transitional state to cccDNA and observe the associated host factors. From this assay, we discovered a novel relationship between the UV-DDB complex and cccDNA formation, providing a proof of concept for a more direct discovery of novel HBV DNA-host interactions that can be exploited to develop new cccDNA-targeting antivirals.
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ADN Circular/metabolismo , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Virus de la Hepatitis B/fisiología , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/virología , Replicación del ADN , Proteínas de Unión al ADN/genética , Antígenos de la Hepatitis B/metabolismo , Virus de la Hepatitis B/metabolismo , Humanos , Unión Proteica , Proteómica , ARN Viral/metabolismo , Ubiquitinación , Replicación ViralRESUMEN
Tegument, which occupies the space between the nucleocapsid and the envelope, is a unique structure of a herpesvirion. Tegument proteins are major components of tegument and play critical roles in virus life cycle. Murine gammaherpesvirus 68 (MHV-68), a member of the gammaherpesvirus subfamily, is closely related to two human herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). We have previously shown that MHV-68 ORF33, conserved among all herpesviruses, encodes a tegument protein that is associated with intranuclear capsids and is essential for virion morphogenesis and egress. Another tegument protein ORF45, which is conserved only among gammaherpesviruses, also plays an essential role in virion morphogenesis of MHV-68. In this study, we investigated the underlying mechanism and showed that these two proteins colocalize and interact with each other during virus infection. We mapped the ORF33-interacting domain to the conserved carboxyl-terminal 23 amino acids (C23) of ORF45. Deletion of the C23 coding sequence in the context of viral genome abolished the production of infectious virions. Transmission electron microscopy results demonstrated that C23 of ORF45 are essential for virion tegumentation in the cytoplasm. We further mapped the ORF45-interacting domain to the N-terminal 17 amino acids (N17) of ORF33. Deletion of the N17 coding sequence in the context of viral genome also abolished production of infectious virions, and N17 of ORF33 are also essential for virion tegumentation in the cytoplasm. Taken together, our data strongly indicate that the interaction between ORF45 and ORF33 plays an essential role in cytoplasmic maturation of MHV-68 virions. IMPORTANCE A critical step in viral lytic replication is the assembly of progeny viral particles. Herpesviruses are important pathogens. A herpesvirus particle comprises, from inside to outside, four layers: DNA core, capsid, tegument, and envelope. The tegument layer contains dozens of virally encoded tegument proteins, which play critical roles in virus assembly. Murine gammaherpesvirus 68 (MHV-68) is a tumor-associated herpesvirus and is closely related to Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. We previously found that the absence of either tegument protein ORF33 or ORF45 inhibits the translocation of nucleocapsids to the cytoplasm and blocks virion maturation, but the underlying mechanism remained unclear. Here, we showed that ORF33 interacts with ORF45. We mapped their interaction domains and constructed viral mutants with defects in ORF33-ORF45 interaction. Transmission electron microscopy data demonstrated that the assembly of these viral mutants in the cytoplasm is blocked. Our results indicate that ORF33-ORF45 interaction is essential for gammaherpesvirus replication.
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Proteínas de la Cápside , Proteínas Inmediatas-Precoces , Rhadinovirus , Ensamble de Virus , Animales , Ratones , Citoplasma/metabolismo , Herpesvirus Humano 4 , Herpesvirus Humano 8 , Rhadinovirus/genética , Rhadinovirus/fisiología , Virión/genética , Virión/fisiología , Replicación Viral , Proteínas de la Cápside/metabolismo , Proteínas Inmediatas-Precoces/metabolismoRESUMEN
Chronic HBV infection can hardly be cured due to the persistence of an intrahepatic pool of viral covalently closed circular DNA (cccDNA) transcription template, which is refractory to current antivirals. The direct analyses of cccDNA quantity and transcriptional activity require an invasive biopsy. Recently, circulating HBV RNA has been identified as a promising noninvasive surrogate marker of cccDNA and can be used for monitoring disease progression and predicting prognosis of patients with chronic HBV infection. To better understand this surrogate biomarker of cccDNA, we reviewed the current knowledge about the molecular characteristics and potential clinical applications of circulating HBV RNA. Specifically, we summarized the reported species and existing forms of circulating HBV RNA and discussed their biogenesis and the capacity of de novo infection by RNA virions. Moreover, we described the potential applications of circulating HBV RNA in different clinical scenarios, such as classifying the phases of chronic HBV infection, analyzing sustained on-treatment and off-treatment outcomes of treated patients, as well as predicting HCC development. Perspectives on future research of circulating HBV RNA were also proposed in this review.
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Carcinoma Hepatocelular , Ácidos Nucleicos Libres de Células , Neoplasias Hepáticas , Humanos , Virus de la Hepatitis B/genética , ADN Viral/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , ADN Circular , Antivirales/uso terapéutico , ARN , Biomarcadores , Biología , Replicación Viral/genéticaRESUMEN
Since the report of the first COVID-19 case in 2019, SARS-CoV-2 variants of concern (VOCs) have continued to emerge, manifesting diverse infectivity, evasion of host immunity and pathology. While ACE2 is the predominant receptor of SARS-CoV-2, TMPRSS2, Kim-1, NRP-1, CD147, furin, CD209L, and CD26 have also been implicated as viral entry-related cofactors. To understand the variations in infectivity and pathogenesis of VOCs, we conducted infection analysis in human cells from different organ systems using pseudoviruses of VOCs including Alpha, Beta, Gamma, and Delta. Recombinant spike S1, RBD, ACE2, Kim-1, and NRP-1 proteins were tested for their ability to block infection to dissect their roles in SARS-CoV-2 entry into cells. Compared with wild type SARS-CoV-2 (WT), numerous VOCs had significant increases of infectivity across a wide spectrum of cell types. Recombinant ACE2 protein more effectively inhibited the infection of VOCs including Delta and Omicron (BA.1 and BA.2) than that of WT. Interestingly, recombinant S1, RBD, Kim-1, and NRP-1 proteins inhibited the infection of all pseudoviruses in a manner dependent on the levels of ACE2 expression in different cell types. These results provide insights into the diverse infectivity of SARS-CoV-2 VOCs, which might be helpful for managing the emergence of new VOCs.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Hepatitis B Virus (HBV) replication has been reported to be restricted by the intrahepatic host restriction factors and antiviral signaling pathways. The intracellular mechanisms underlying the significant viremia difference among different phases of the natural history chronic HBV infection remain elusive. We herein report that the hypoxia-induced gene domain protein-1a (HIGD1A) was highly expressed in the liver of inactive HBV carriers with low viremia. Ectopic expression of HIGD1A in hepatocyte-derived cells significantly inhibited HBV transcription and replication in a dose-dependent manner, while silence of HIGD1A promoted HBV gene expression and replication. Similar results were also observed in both de novo HBV-infected cell culture model and HBV persistence mouse model. Mechanistically, HIGD1A is located on the mitochondrial inner membrane and activates nuclear factor kappa B (NF-κB) signaling pathway through binding to paroxysmal nonkinesigenic dyskinesia (PNKD), which further enhances the expression of a transcription factor NR2F1 to inhibit HBV transcription and replication. Consistently, knockdown of PNKD or NR2F1 and blockage of NF-κB signaling pathway abrogated the inhibitory effect of HIGD1A on HBV replication. Mitochondrial HIGD1A exploits the PNKD-NF-κB-NR2F1 nexus to act as a host restriction factor of HBV infection. Our study thus shed new lights on the regulation of HBV by hypoxia-related genes and related antiviral strategies.
Asunto(s)
Virus de la Hepatitis B , Hepatitis B , Animales , Ratones , Antivirales/farmacología , Virus de la Hepatitis B/fisiología , FN-kappa B/genética , FN-kappa B/metabolismo , Transcripción Viral , Viremia , Replicación Viral , HumanosRESUMEN
BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests diverse clinical pathologies involving multiple organs. While the respiratory tract is the primary SARS-CoV-2 target, acute kidney injury is common in COVID-19 patients, displaying as acute tubular necrosis (ATN) resulting from focal epithelial necrosis and eosinophilia, glomerulosclerosis, and autolysis of renal tubular cells. However, whether any renal cells are infected by SARS-CoV-2 and the mechanism involved in the COVID-19 kidney pathology remain unclear. METHODS: Kidney tissues obtained at autopsy from four severe COVID-19 patients and one healthy subject were examined by hematoxylin and eosin staining. Indirect immunofluorescent antibody assay was performed to detect SARS-CoV-2 spike protein S1 and nonstructural protein 8 (NSP8) together with markers of different kidney cell types and immune cells to identify the infected cells. RESULTS: Renal parenchyma showed tissue injury comprised of ATN and glomerulosclerosis. Positive staining of S1 protein was observed in renal parenchymal and tubular epithelial cells. Evidence of viral infection was also observed in innate monocytes/macrophages and NK cells. Positive staining of NSP8, which is essential for viral RNA synthesis and replication, was confirmed in renal parenchymal cells, indicating the presence of active viral replication in the kidney. CONCLUSIONS: In fatal COVID-19 kidneys, there are SARS-CoV-2 infection, minimally infiltrated innate immune cells, and evidence of viral replication, which could contribute to tissue damage in the form of ATN and glomerulosclerosis.
Asunto(s)
Lesión Renal Aguda , COVID-19 , Humanos , COVID-19/patología , SARS-CoV-2 , Riñón/patología , Lesión Renal Aguda/patología , Necrosis/patologíaRESUMEN
SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Animales , Ratas , SARS-CoV-2/metabolismo , Anticuerpos Monoclonales , Células Endoteliales , ARN Polimerasa Dependiente del ARN/genética , Antivirales/metabolismoRESUMEN
The presence of hepatitis B virus (HBV) covalently closed circular (ccc) DNA (cccDNA), which serves as a template for viral replication and integration of HBV DNA into the host cell genome, sustains liver pathogenesis and constitutes an intractable barrier to the eradication of chronic HBV infection. The current antiviral therapy for HBV infection, using nucleos(t)ide analogues (NAs), can suppress HBV replication but cannot eliminate integrated HBV DNA and episomal cccDNA. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a powerful genetic tool that can edit integrated HBV DNA and minichromosomal cccDNA for gene therapy, but its expression and delivery require a viral vector, which poses safety concerns for therapeutic applications in humans. In the present study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a nonviral formulation to develop a novel CRISPR/Cas9-mediated gene therapy for eradicating HBV infection. We designed a series of gRNAs targeting multiple specific HBV genes and tested their antiviral efficacy and cytotoxicity in different HBV cellular models. Transfection of stably HBV-infected human hepatoma cell line HepG2.2.15 with HBV-specific gRNA/Cas9 RNPs resulted in a substantial reduction in HBV transcripts. Specifically, gRNA5 and/or gRNA9 RNPs significantly reduced HBV cccDNA, total HBV DNA, pregenomic RNA, and HBV antigen (HBsAg, HBeAg) levels. T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing confirmed specific HBV gene cleavage and mutations at or around the gRNA target sites. Notably, this gene-editing system did not alter cellular viability or proliferation in the treated cells. Because of their rapid DNA cleavage capability, low off-target effects, low risk of insertional mutagenesis, and readiness for use in clinical application, these results suggest that synthetic gRNA/Cas9 RNP-based gene-editing can be utilized as a promising therapeutic drug for eradicating chronic HBV infection.