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Journal of Immunology (Baltimore, Md. :... Nov 2016Elimination of virus-infected cells by cytotoxic lymphocytes is triggered by activating receptors, among which NKG2D and DNAM-1/CD226 play an important role. Their...
Elimination of virus-infected cells by cytotoxic lymphocytes is triggered by activating receptors, among which NKG2D and DNAM-1/CD226 play an important role. Their ligands, that is, MHC class I-related chain (MIC) A/B and UL16-binding proteins (ULBP)1-6 (NKG2D ligand), Nectin-2/CD112, and poliovirus receptor (PVR)/CD155 (DNAM-1 ligand), are often induced on virus-infected cells, although some viruses, including human CMV (HCMV), can block their expression. In this study, we report that infection of different cell types with laboratory or low-passage HCMV strains upregulated MICA, ULBP3, and PVR, with NKG2D and DNAM-1 playing a role in NK cell-mediated lysis of infected cells. Inhibition of viral DNA replication with phosphonoformic acid did not prevent ligand upregulation, thus indicating that early phases of HCMV infection are involved in ligand increase. Indeed, the major immediate early (IE) proteins IE1 and IE2 stimulated the expression of MICA and PVR, but not ULBP3. IE2 directly activated MICA promoter via its binding to an IE2-responsive element that we identified within the promoter and that is conserved among different alleles of MICA. Both IE proteins were instead required for PVR upregulation via a mechanism independent of IE DNA binding activity. Finally, inhibiting IE protein expression during HCMV infection confirmed their involvement in ligand increase. We also investigated the contribution of the DNA damage response, a pathway activated by HCMV and implicated in ligand regulation. However, silencing of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related protein, and DNA-dependent protein kinase did not influence ligand expression. Overall, these data reveal that MICA and PVR are directly regulated by HCMV IE proteins, and this may be crucial for the onset of an early host antiviral response.
Topics: Antigens, Differentiation, T-Lymphocyte; Cell Line; Cytotoxicity, Immunologic; DNA Replication; Fibroblasts; Foscarnet; GPI-Linked Proteins; Gene Expression Regulation; HEK293 Cells; Histocompatibility Antigens Class I; Host-Pathogen Interactions; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Killer Cells, Natural; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Virus; Trans-Activators; Transcriptional Activation; Up-Regulation; Viral Proteins; Virus Replication
PubMed: 27733551
DOI: 10.4049/jimmunol.1502527 -
Journal of Virology Jul 2015We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long noncoding RNAs (vlncRNAs) that are expressed during reactivation. Here...
New Noncoding Lytic Transcripts Derived from the Epstein-Barr Virus Latency Origin of Replication, oriP, Are Hyperedited, Bind the Paraspeckle Protein, NONO/p54nrb, and Support Viral Lytic Transcription.
UNLABELLED
We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long noncoding RNAs (vlncRNAs) that are expressed during reactivation. Here we show that the EBV latency origin of replication (oriP) is transcribed bi-directionally during reactivation and that both leftward (oriPtLs) and rightward (oriPtRs) transcripts are largely localized in the nucleus. While the oriPtLs are most likely noncoding, at least some of the oriPtRs contain the BCRF1/vIL10 open reading frame. Nonetheless, oriPtR transcripts with long 5' untranslated regions may partially serve noncoding functions. Both oriPtL and oriPtR transcripts are expressed with late kinetics, and their expression is inhibited by phosphonoacetic acid. RNA sequencing (RNA-seq) analysis showed that oriPtLs and oriPtRs exhibited extensive "hyperediting" at their Family of Repeat (FR) regions. RNA secondary structure prediction revealed that the FR region of both oriPtLs and oriPtRs may form large evolutionarily conserved and thermodynamically stable hairpins. The double-stranded RNA-binding protein and RNA-editing enzyme ADAR was found to bind to oriPtLs, likely facilitating editing of the FR hairpin. Further, the multifunctional paraspeckle protein, NONO, was found to bind to oriPt transcripts, suggesting that oriPts interact with the paraspeckle-based innate antiviral immune pathway. Knockdown and ectopic expression of oriPtLs showed that it contributes to global viral lytic gene expression and viral DNA replication. Together, these results show that these new vlncRNAs interact with cellular innate immune pathways and that they help facilitate progression of the viral lytic cascade.
IMPORTANCE
Recent studies have revealed that the complexity of lytic herpesviral transcriptomes is significantly greater than previously appreciated with hundreds of viral long noncoding RNAs (vlncRNAs) being recently discovered. Work on cellular lncRNAs over the past several years has just begun to give us an initial appreciation for the array of functions they play in complex formation and regulatory processes in the cell. The newly identified herpesvirus lncRNAs are similarly likely to play a variety of different functions, although these functions are likely tailored to specific needs of the viral infection cycles. Here we describe novel transcripts derived from the EBV latency origin of replication. We show that they are hyperedited, that they interact with a relatively newly appreciated antiviral pathway, and that they play a role in facilitating viral lytic gene expression. These investigations are a starting point to unraveling the complex arena of vlncRNA function in herpesvirus lytic replication.
Topics: Cell Line; DNA-Binding Proteins; Gene Expression Regulation, Viral; Herpesvirus 4, Human; Host-Pathogen Interactions; Humans; Immunity, Innate; Nuclear Matrix-Associated Proteins; Octamer Transcription Factors; Protein Binding; RNA, Untranslated; RNA-Binding Proteins; Replication Origin; Transcription, Genetic; Virus Replication
PubMed: 25926645
DOI: 10.1128/JVI.00608-15 -
Journal of Virology Nov 2015Viruses modulate cellular processes and metabolism in diverse ways, but these are almost universally studied in the infected cell itself. Here, we study spatial...
UNLABELLED
Viruses modulate cellular processes and metabolism in diverse ways, but these are almost universally studied in the infected cell itself. Here, we study spatial organization of DNA synthesis during multiround transmission of herpes simplex virus (HSV) using pulse-labeling with ethynyl nucleotides and cycloaddition of azide fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with the current understanding of the single-step growth cycle, HSV suppresses host DNA synthesis and promotes viral DNA synthesis in spatially segregated compartments within the cell. In striking contrast, during progressive rounds of infection initiated at a single cell, we observe that infection induces a clear and pronounced stimulation of cellular DNA replication in remote uninfected cells. This induced DNA synthesis was observed in hundreds of uninfected cells at the extended border, outside the perimeter of the progressing infection. Moreover, using pulse-chase analysis, we show that this activation is maintained, resulting in a propagating wave of host DNA synthesis continually in advance of infection. As the virus reaches and infects these activated cells, host DNA synthesis is then shut off and replaced with virus DNA synthesis. Using nonpropagating viruses or conditioned medium, we demonstrate a paracrine effector of uninfected cell DNA synthesis in remote cells continually in advance of infection. These findings have significant implications, likely with broad applicability, for our understanding of the ways in which virus infection manipulates cell processes not only in the infected cell itself but also now in remote uninfected cells, as well as of mechanisms governing host DNA synthesis.
IMPORTANCE
We show that during infection initiated by a single particle with progressive cell-cell virus transmission (i.e., the normal situation), HSV induces host DNA synthesis in uninfected cells, mediated by a virus-induced paracrine effector. The field has had no conception that this process occurs, and the work changes our interpretation of virus-host interaction during advancing infection and has implications for understanding controls of host DNA synthesis. Our findings demonstrate the utility of chemical biology techniques in analysis of infection processes, reveal distinct processes when infection is examined in multiround transmission versus single-step growth curves, and reveal a hitherto-unknown process in virus infection, likely relevant for other viruses (and other infectious agents) and for remote signaling of other processes, including transcription and protein synthesis.
Topics: Animals; Azides; Bacterial Proteins; Cell Line; Chlorocebus aethiops; DNA Replication; Herpes Simplex; Host-Pathogen Interactions; Humans; Luminescent Proteins; Paracrine Communication; Phosphonoacetic Acid; Simplexvirus; Vero Cells; Viral Plaque Assay; Virus Internalization
PubMed: 26311877
DOI: 10.1128/JVI.01950-15 -
Emerging Infectious Diseases Sep 2017FosA, a glutathione S-transferase that inactivates fosfomycin, has been reported as the cause of enzymatic resistance to fosfomycin. We show that multiple lineages of...
FosA, a glutathione S-transferase that inactivates fosfomycin, has been reported as the cause of enzymatic resistance to fosfomycin. We show that multiple lineages of FosA-producing extended spectrum β-lactamase Escherichia coli have circulated in France since 2012, potentially reducing the efficacy of fosfomycin in treating infections with antimicrobial drug-resistant gram-negative bacilli.
Topics: Anti-Bacterial Agents; Drug Resistance, Multiple, Bacterial; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Foscarnet; Fosfomycin; France; Gene Expression; Humans; Isoenzymes; Microbial Sensitivity Tests; Plasmids; Prevalence; beta-Lactamases
PubMed: 28820368
DOI: 10.3201/eid2309.170560 -
Antimicrobial Agents and Chemotherapy Aug 2014In immunosuppressed hosts, the development of multidrug resistance complicates the treatment of cytomegalovirus (CMV) infection. Improved genotypic detection of...
In immunosuppressed hosts, the development of multidrug resistance complicates the treatment of cytomegalovirus (CMV) infection. Improved genotypic detection of impending drug resistance may follow from recent technical advances. A severely T-cell-depleted patient with chronic lymphocytic leukemia developed CMV pneumonia and high plasma viral loads that were poorly responsive to antiviral therapy. Serial plasma specimens were analyzed for mutant viral populations by conventional and high-throughput deep-sequencing methods. Uncharacterized mutations were phenotyped for drug resistance using recombinant viruses. Conventional genotyping detected viruses with the UL97 kinase substitution C607Y after ganciclovir treatment, a transient subpopulation of UL54 polymerase L773V mutants first detected 8 weeks after foscarnet was started, and a subpopulation of a mutant with deletion of UL54 codons 981 and 982 2 months after the addition of cidofovir. Deep sequencing of the same serial specimens revealed the same UL54 mutants sooner, along with a more complex evolution of known and newly recognized mutant subpopulations missed by conventional sequencing. The UL54 exonuclease substitutions D413N, K513R, and C539G were newly shown to confer ganciclovir-cidofovir resistance, while L773V was shown to confer foscarnet resistance and add to the ganciclovir resistance conferred by UL97 C607Y. Increased sequencing depth provided a more timely and detailed diagnosis of mutant viral subpopulations that evolved with changing anti-CMV therapy.
Topics: Antiviral Agents; Cidofovir; Cytomegalovirus; Cytomegalovirus Infections; Cytosine; DNA-Directed DNA Polymerase; Drug Resistance, Viral; Foscarnet; Ganciclovir; High-Throughput Nucleotide Sequencing; Humans; Immunocompromised Host; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Mutation; Organophosphonates; Pneumonia, Viral; Time Factors; Viral Proteins; Virus Replication
PubMed: 24890586
DOI: 10.1128/AAC.03214-14 -
Biology of Blood and Marrow... Sep 2020Despite a well-established risk of chronic kidney disease (CKD) after allogeneic hematopoietic cell transplant (allo-HCT), the benefits of using nephrotoxic...
Despite a well-established risk of chronic kidney disease (CKD) after allogeneic hematopoietic cell transplant (allo-HCT), the benefits of using nephrotoxic anti-infective agents to treat serious peritransplant infections often outweigh this risk. While there is no consensus on the optimal management of post-allo-HCT human herpes virus 6 (HHV6) reactivation, the nephrotoxic drug foscarnet is often used, although its long-term impact on renal function has not been established. We retrospectively reviewed 987 adult patients who underwent transplantation between 2002 and 2016, of whom 45.3% (n = 447) were exposed to foscarnet. The most frequent indications for foscarnet treatment were cytomegalovirus (n = 257, 57.5%) and HHV6 (n = 139, 31.1%). In the first 3 months post-transplant, patients exposed versus unexposed had similar rates of acute kidney injury and acute kidney failure (defined as 3 times baseline creatinine or <75% baseline estimated glomerular filtration rate [eGFR], 61.6% versus 58.7%, P = .42 and 28.1% versus 26.6%, P = .64, respectively). There was no difference in the eGFR at 3 months (P = .36), but patients treated with foscarnet had significantly lower median eGFRs (mL/min/1.73 m) at 6 months (69.3, interquartile range [IQR] 51.4 to 92.8 versus 77.4, IQR 57.3 to 99.3; P = .009) and 12 months (67.8, IQR 52.7 to 85.0 versus 80.7, IQR 63.1 to 102.0; P < .001), respectively. There was also a significant difference in the decline in eGFR from baseline to 12 months (median 32.8, IQR 14.6 to 53.2 versus 21.9, IQR 6.4 to 37.4; P < .001), irrespective of the duration of foscarnet treatment. Multivariate analysis revealed that patients treated with foscarnet were more likely to experience a >30% decrease in eGFR from baseline to 12 months compared to those who were not (odds ratio, 2.30; 95% CI, 1.40 to 3.78; P = .001). We conclude that foscarnet use following allo-HCT had a profound impact on long-term renal function independent of other transplant-related factors.
Topics: Adult; Foscarnet; Glomerular Filtration Rate; Hematopoietic Stem Cell Transplantation; Humans; Retrospective Studies; Transplantation, Homologous
PubMed: 32450288
DOI: 10.1016/j.bbmt.2020.05.007 -
Journal of Virology Mar 2015Kaposi's sarcoma-associated herpesvirus (KSHV) infects many target cells (e.g., endothelial, epithelial, and B cells, keratinocytes, and monocytes) to establish lifelong...
UNLABELLED
Kaposi's sarcoma-associated herpesvirus (KSHV) infects many target cells (e.g., endothelial, epithelial, and B cells, keratinocytes, and monocytes) to establish lifelong latent infections. Viral latent-protein expression is critical in inducing and maintaining KSHV latency. Infected cells are programmed to retain the incoming viral genomes during primary infection. Immediately after infection, KSHV transcribes many lytic genes that modulate various cellular pathways to establish successful infection. Analysis of the virion particle showed that the virions contain viral mRNAs, microRNAs, and other noncoding RNAs that are transduced into the target cells during infection, but their biological functions are largely unknown. We performed a comprehensive analysis of the KSHV virion packaged transcripts and the profiles of viral genes transcribed after de novo infections of various cell types (human peripheral blood mononuclear cells [PBMCs], CD14(+) monocytes, and telomerase-immortalized vascular endothelial [TIVE] cells), from viral entry until latency establishment. A next-generation sequence analysis of the total transcriptome showed that several viral RNAs (polyadenylated nuclear RNA, open reading frame 58 [ORF58], ORF59, T0.7, and ORF17) were abundantly present in the KSHV virions and effectively transduced into the target cells. Analysis of the transcription profiles of each viral gene showed specific expression patterns in different cell lines, with the majority of the genes, other than latent genes, silencing after 24 h postinfection. We differentiated the actively transcribing genes from the virion-transduced transcripts using a nascent RNA capture approach (Click-iT chemistry), which identified transcription of a number of viral genes during primary infection. Treating the infected cells with phosphonoacetic acid (PAA) to block the activity of viral DNA polymerase confirmed the involvement of lytic DNA replication during primary infection. To further understand the role of DNA replication during primary infection, we performed de novo PBMC infections with a recombinant ORF59-deleted KSHV virus, which showed significantly reduced numbers of viral copies in the latently infected cells. In summary, the transduced KSHV RNAs as well as the actively transcribed genes control critical processes of early infection to establish KSHV latency.
IMPORTANCE
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple human malignancies in immunocompromised individuals. KSHV establishes a lifelong latency in the infected host, during which only a limited number of viral genes are expressed. However, a fraction of latently infected cells undergo spontaneous reactivation to produce virions that infect the surrounding cells. These newly infected cells are primed early to retain the incoming viral genome and induce cell growth. KSHV transcribes a variety of lytic proteins during de novo infections that modulate various cellular pathways to establish the latent infection. Interestingly, a large number of viral proteins and RNA are encapsidated in the infectious virions and transduced into the infected cells during a de novo infection. This study determined the kinetics of the viral gene expression during de novo KSHV infections and the functional role of the incoming viral transcripts in establishing latency.
Topics: B-Lymphocytes; Endothelial Cells; Gene Expression Profiling; Gene Expression Regulation, Viral; Herpesviridae Infections; Herpesvirus 8, Human; Humans; Transcriptome; Viral Proteins; Virus Latency
PubMed: 25552714
DOI: 10.1128/JVI.02507-14 -
BMC Neurology May 2023Pseudorabies virus (PRV) was thought to only infect animals. Recent studies have shown that it can also infect human.
BACKGROUND
Pseudorabies virus (PRV) was thought to only infect animals. Recent studies have shown that it can also infect human.
CASE PRESENTATION
We report a case of pseudorabies virus encephalitis and endophthalmitis, diagnosed 89 days after onset, confirmed with intraocular fluid metagenomic next generation sequencing (mNGS) after the result of two cerebrospinal fluid (CSF) mNGS tests were negative. Although treatment with intravenous acyclovir, foscarnet sodium, and methylprednisolone improved the symptoms of encephalitis, significant diagnostic delay resulted in permanent visual loss.
CONCLUSIONS
This case suggests that pseudorabies virus (PRV) DNA in the intraocular fluid may have a higher positivity than that in the CSF. PRV may persist in the intraocular fluid for an extended period and may thus require extended antiviral therapy. Patients with severe encephalitis and PRV should be examined with the focus on pupil reactivity and light reflex. A fundus examination should be performed in patients with a central nervous system infection, specifically, those in a comatose state, to help reduce eye disability.
Topics: Pseudorabies; Encephalitis, Viral; Endophthalmitis; Herpesvirus 1, Suid; Metagenomics; High-Throughput Nucleotide Sequencing; Delayed Diagnosis; Humans; Male; Middle Aged; Aqueous Humor; Acyclovir; Foscarnet; Methylprednisolone; Antiviral Agents; Blindness; DNA, Viral
PubMed: 37194001
DOI: 10.1186/s12883-023-03227-1 -
Kidney & Blood Pressure Research 2016Renal reabsorption of inorganic phosphate (Pi) is mediated by SLC34 and SLC20 Na+/Pi-cotransporters the abundance of which is under hormonal control. Extracellular Pi...
BACKGROUND/AIMS
Renal reabsorption of inorganic phosphate (Pi) is mediated by SLC34 and SLC20 Na+/Pi-cotransporters the abundance of which is under hormonal control. Extracellular Pi itself also regulates the expression of cotransporters and the concentration of Pi-regulating hormones, though the signaling pathways are largely unknown. Here, we explored the mechanisms that allow renal proximal cells to adapt to changes in the concentration of Pi.
METHODS
opossum kidney (OK) cells, a model of proximal epithelia, were incubated with different concentrations of Pi in the absence/presence of phosphonoformic acid (PFA), a Pi-analogue and SLC34-inhibitor, and of inhibitors of kinases involved in hormonal control of Pi-homeostasis; cells cultured in normal media were treated with uncouplers of oxidative phosphorylation. Then, the intracellular concentration of ATP and/or the Pi-transport capacity of the cultures were analyzed.
RESULTS
luminal Pi regulates the Pi-transport and the intracellular ATP levels. Changes in ATP seem secondary to alterations in Pi-transport, rather than ATP acting as a signal. Adaptation of Pi-transport to high Pi was not mimicked by PFA. Transport adaptation was blocked by PFA but not by kinase inhibitors.
CONCLUSIONS
in OK cells, adaptation of Pi-transport to luminal Pi does not depend on the same signaling pathways involved in hormonal regulation.
Topics: Adenosine Triphosphate; Animals; Biological Transport; Cells, Cultured; Foscarnet; Kidney; Kidney Tubules, Proximal; Opossums; Phosphates; Protein Kinase Inhibitors; Signal Transduction; Sodium-Phosphate Cotransporter Proteins, Type II
PubMed: 27165344
DOI: 10.1159/000443432 -
The Journal of Organic Chemistry Oct 2015A versatile and general catalytic strategy has been developed for the α-arylation of phosphonoacetates utilizing parallel microscale experimentation. These...
A versatile and general catalytic strategy has been developed for the α-arylation of phosphonoacetates utilizing parallel microscale experimentation. These α-substituted phosphonoacetates are widely useful, notably as substrates in the Horner-Wadsworth-Emmons-type olefinations. However, the current routes to these products involve harsh conditions, limiting the variety of functionality. The reported method can be used with a variety of aryl chlorides and aryl bromides, including several heterocyclic examples.
Topics: Alkenes; Catalysis; Heterocyclic Compounds; Molecular Structure; Phosphonoacetic Acid; Stereoisomerism
PubMed: 26405824
DOI: 10.1021/acs.joc.5b01887