Publications by authors named "Brad R Rosenberg"

25 Publications

  • Page 1 of 1

Decoupling expression and editing preferences of ADAR1 p150 and p110 isoforms.

Proc Natl Acad Sci U S A 2021 Mar;118(12)

Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065;

Human adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine deamination reactions on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Defective ADAR1 editing leads to disorders such as Aicardi-Goutières syndrome, an autoinflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. Two ADAR1 protein isoforms, p150 (150 kDa) and p110 (110 kDa), are expressed and can edit RNA, but the contribution of each isoform to the editing landscape remains unclear, largely because of the challenges in expressing p150 without p110. In this study, we demonstrate that p110 is coexpressed with p150 from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. The presence of a strong Kozak consensus context surrounding the p110 start codon suggests the p150 mRNA is optimized to leak p110 alongside expression of p150. To reduce leaky scanning and translation initiation at the p110 start codon, we introduced synonymous mutations in the coding region between the p150 and p110 start codons. Cells expressing p150 constructs with these mutations produced significantly reduced levels of p110. Editing analysis of total RNA from ADAR1 knockout cells reconstituted separately with modified p150 and p110 revealed that more than half of the A-to-I edit sites are selectively edited by p150, and the other half are edited by either p150 or p110. This method of isoform-selective editing analysis, making use of the modified p150, has the potential to be adapted for other cellular contexts.
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http://dx.doi.org/10.1073/pnas.2021757118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000508PMC
March 2021

Pathogenesis, miR-122 gene-regulation, and protective immune responses after acute equine hepacivirus infection.

Hepatology 2021 Mar 13. Epub 2021 Mar 13.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.

Background & Aims: Equine hepacivirus (EqHV) is phylogenetically the closest relative of hepatitis C virus (HCV) and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease, and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses.

Approach & Results: Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same, and subsequently a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred non-sterilizing immunity resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV specific T cells were identified. Additionally, an interferon stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver-specific microRNA, miR-122. Interestingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology.

Conclusions: EqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.
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http://dx.doi.org/10.1002/hep.31802DOI Listing
March 2021

Single-cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse cell types including T-bet B cells.

BMC Biol 2021 Jan 22;19(1):13. Epub 2021 Jan 22.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, 1 Gustave L. Levy Place, New York, NY, 10029, USA.

Background: Traditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species. Immunology research in non-traditional model organisms can be advantageous or even necessary, such as when studying host-pathogen interactions. However, such research presents multiple challenges, many stemming from an incomplete understanding of potentially species-specific immune cell types, frequencies, and phenotypes. Identifying and characterizing immune cells in such organisms is frequently limited by the availability of species-reactive immunophenotyping reagents for flow cytometry, and insufficient prior knowledge of cell type-defining markers.

Results: Here, we demonstrate the utility of single-cell RNA sequencing (scRNA-Seq) to characterize immune cells for which traditional experimental tools are limited. Specifically, we used scRNA-Seq to comprehensively define the cellular diversity of equine peripheral blood mononuclear cells (PBMC) from healthy horses across different breeds, ages, and sexes. We identified 30 cell type clusters partitioned into five major populations: monocytes/dendritic cells, B cells, CD3PRF1 lymphocytes, CD3PRF1 lymphocytes, and basophils. Comparative analyses revealed many cell populations analogous to human PBMC, including transcriptionally heterogeneous monocytes and distinct dendritic cell subsets (cDC1, cDC2, plasmacytoid DC). Remarkably, we found that a majority of the equine peripheral B cell compartment is comprised of T-bet B cells, an immune cell subpopulation typically associated with chronic infection and inflammation in human and mouse.

Conclusions: Taken together, our results demonstrate the potential of scRNA-Seq for cellular analyses in non-traditional model organisms and form the basis for an immune cell atlas of horse peripheral blood.
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http://dx.doi.org/10.1186/s12915-020-00947-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820527PMC
January 2021

Single-cell RNA sequencing of equine mesenchymal stromal cells from primary donor-matched tissue sources reveals functional heterogeneity in immune modulation and cell motility.

Stem Cell Res Ther 2020 12 4;11(1):524. Epub 2020 Dec 4.

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA.

Background: The efficacy of mesenchymal stromal cell (MSC) therapy is thought to depend on the intrinsic heterogeneity of MSC cultures isolated from different tissue sources as well as individual MSCs isolated from the same tissue source, neither of which is well understood. To study this, we used MSC cultures isolated from horses. The horse is recognized as a physiologically relevant large animal model appropriate for translational MSC studies. Moreover, due to its large size the horse allows for the simultaneous collection of adequate samples from multiple tissues of the same animal, and thus, for the unique collection of donor matched MSC cultures from different sources. The latter is much more challenging in mice and humans due to body size and ethical constraints, respectively.

Methods: In the present study, we performed single-cell RNA sequencing (scRNA-seq) on primary equine MSCs that were collected from three donor-matched tissue sources; adipose tissue (AT), bone marrow (BM), and peripheral blood (PB). Based on transcriptional differences detected with scRNA-seq, we performed functional experiments to examine motility and immune regulatory function in distinct MSC populations.

Results: We observed both inter- and intra-source heterogeneity across the three sources of equine MSCs. Functional experiments demonstrated that transcriptional differences correspond with phenotypic variance in cellular motility and immune regulatory function. Specifically, we found that (i) differential expression of junctional adhesion molecule 2 (JAM2) between MSC cultures from the three donor-matched tissue sources translated into altered cell motility of BM-derived MSCs when RNA interference was used to knock down this gene, and (ii) differences in C-X-C motif chemokine ligand 6 (CXCL6) expression in clonal MSC lines derived from the same tissue source correlated with the chemoattractive capacity of PB-derived MSCs.

Conclusions: Ultimately, these findings will enhance our understanding of MSC heterogeneity and will lead to improvements in the therapeutic potential of MSCs, accelerating the transition from bench to bedside.
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http://dx.doi.org/10.1186/s13287-020-02043-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716481PMC
December 2020

Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.

Cell 2021 01 24;184(1):92-105.e16. Epub 2020 Oct 24.

New York Genome Center, New York, NY, USA; Department of Biology, New York University, New York, NY, USA. Electronic address:

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.
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http://dx.doi.org/10.1016/j.cell.2020.10.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584921PMC
January 2021

SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling.

Proc Natl Acad Sci U S A 2020 11 23;117(45):28344-28354. Epub 2020 Oct 23.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029;

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.
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http://dx.doi.org/10.1073/pnas.2016650117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668094PMC
November 2020

Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function.

Immunity 2020 09 3;53(3):672-684.e11. Epub 2020 Aug 3.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Autoinflammatory disease can result from monogenic errors of immunity. We describe a patient with early-onset multi-organ immune dysregulation resulting from a mosaic, gain-of-function mutation (S703I) in JAK1, encoding a kinase essential for signaling downstream of >25 cytokines. By custom single-cell RNA sequencing, we examine mosaicism with single-cell resolution. We find that JAK1 transcription was predominantly restricted to a single allele across different cells, introducing the concept of a mutational "transcriptotype" that differs from the genotype. Functionally, the mutation increases JAK1 activity and transactivates partnering JAKs, independent of its catalytic domain. S703I JAK1 is not only hypermorphic for cytokine signaling but also neomorphic, as it enables signaling cascades not canonically mediated by JAK1. Given these results, the patient was treated with tofacitinib, a JAK inhibitor, leading to the rapid resolution of clinical disease. These findings offer a platform for personalized medicine with the concurrent discovery of fundamental biological principles.
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http://dx.doi.org/10.1016/j.immuni.2020.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398039PMC
September 2020

Equine pegiviruses cause persistent infection of bone marrow and are not associated with hepatitis.

PLoS Pathog 2020 07 10;16(7):e1008677. Epub 2020 Jul 10.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.

Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1-4% of healthy individuals and another 5-13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler's disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler's disease) in horses. Although no subsequent reports link TDAV to disease, any association with hepatitis has not been formally examined. Here, we characterized EPgV-1 and TDAV tropism, sequence diversity, persistence and association with liver disease in horses. Among more than 20 tissue types, we consistently detected high viral loads only in serum, bone marrow and spleen, and viral RNA replication was consistently identified in bone marrow. PBMCs and lymph nodes, but not liver, were sporadically positive. To exclude potential effects of co-infecting agents in experimental infections, we constructed full-length consensus cDNA clones; this was enabled by determination of the complete viral genomes, including a novel TDAV 3' terminus. Clone derived RNA transcripts were used for direct intrasplenic inoculation of healthy horses. This led to productive infection detectable from week 2-3 and persisting beyond the 28 weeks of study. We did not observe any clinical signs of illness or elevation of circulating liver enzymes. The polyprotein consensus sequences did not change, suggesting that both clones were fully functional. To our knowledge, this is the first successful extrahepatic viral RNA launch and the first robust reverse genetics system for a pegivirus. In conclusion, equine pegiviruses are bone marrow tropic, cause persistent infection in horses, and are not associated with hepatitis. Based on these findings, it may be appropriate to rename the group of TDAV and related viruses as EPgV-2.
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http://dx.doi.org/10.1371/journal.ppat.1008677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375656PMC
July 2020

The ETS transcription factor ELF1 regulates a broadly antiviral program distinct from the type I interferon response.

PLoS Pathog 2019 11 4;15(11):e1007634. Epub 2019 Nov 4.

Department of Microbiology, New York University School of Medicine, New York, New York, United States of America.

Induction of vast transcriptional programs is a central event of innate host responses to viral infections. Here we report a transcriptional program with potent antiviral activity, driven by E74-like ETS transcription factor 1 (ELF1). Using microscopy to quantify viral infection over time, we found that ELF1 inhibits eight diverse RNA and DNA viruses after multi-cycle replication. Elf1 deficiency results in enhanced susceptibility to influenza A virus infections in mice. ELF1 does not feed-forward to induce interferons, and ELF1's antiviral effect is not abolished by the absence of STAT1 or by inhibition of JAK phosphorylation. Accordingly, comparative expression analyses by RNA-seq revealed that the ELF1 transcriptional program is distinct from interferon signatures. Thus, ELF1 provides an additional layer of the innate host response, independent from the action of type I interferons.
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http://dx.doi.org/10.1371/journal.ppat.1007634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932815PMC
November 2019

Cyclin F Controls Cell-Cycle Transcriptional Outputs by Directing the Degradation of the Three Activator E2Fs.

Mol Cell 2019 06 23;74(6):1264-1277.e7. Epub 2019 May 23.

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA; Perlmutter NYU Cancer Center, New York University School of Medicine, New York, NY 10016, USA; Howard Hughes Medical Institute, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

E2F1, E2F2, and E2F3A, the three activators of the E2F family of transcription factors, are key regulators of the G1/S transition, promoting transcription of hundreds of genes critical for cell-cycle progression. We found that during late S and in G2, the degradation of all three activator E2Fs is controlled by cyclin F, the substrate receptor of 1 of 69 human SCF ubiquitin ligase complexes. E2F1, E2F2, and E2F3A interact with the cyclin box of cyclin F via their conserved N-terminal cyclin binding motifs. In the short term, E2F mutants unable to bind cyclin F remain stable throughout the cell cycle, induce unscheduled transcription in G2 and mitosis, and promote faster entry into the next S phase. However, in the long term, they impair cell fitness. We propose that by restricting E2F activity to the S phase, cyclin F controls one of the main and most critical transcriptional engines of the cell cycle.
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http://dx.doi.org/10.1016/j.molcel.2019.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588466PMC
June 2019

Longitudinal transcriptomic characterization of the immune response to acute hepatitis C virus infection in patients with spontaneous viral clearance.

PLoS Pathog 2018 09 17;14(9):e1007290. Epub 2018 Sep 17.

Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, QC, Canada.

Most individuals exposed to hepatitis C virus (HCV) become persistently infected while a minority spontaneously eliminate the virus. Although early immune events influence infection outcome, the cellular composition, molecular effectors, and timeframe of the host response active shortly after viral exposure remain incompletely understood. Employing specimens collected from people who inject drugs (PWID) with high risk of HCV exposure, we utilized RNA-Seq and blood transcriptome module (BTM) analysis to characterize immune function in peripheral blood mononuclear cells (PBMC) before, during, and after acute HCV infection resulting in spontaneous resolution. Our results provide a detailed description of innate immune programs active in peripheral blood during acute HCV infection, which include prominent type I interferon and inflammatory signatures. Innate immune gene expression rapidly returns to pre-infection levels upon viral clearance. Comparative analyses using peripheral blood gene expression profiles from other viral and vaccine studies demonstrate similarities in the immune responses to acute HCV and flaviviruses. Of note, both acute dengue virus (DENV) infection and acute HCV infection elicit similar innate antiviral signatures. However, while transient in DENV infection, this signature was sustained for many weeks in the response to HCV. These results represent the first longitudinal transcriptomic characterization of human immune function in PBMC during acute HCV infection and identify several dynamically regulated features of the complex response to natural HCV exposure.
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http://dx.doi.org/10.1371/journal.ppat.1007290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160227PMC
September 2018

Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection.

Cell 2018 02;172(5):952-965.e18

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065, USA; Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Paris 75015, France; Paris Descartes University, Imagine Institute, Paris 75015, France; Howard Hughes Medical Institute, New York, NY 10065, USA; Pediatric Immunology-Hematology Unit, Necker Hospital for Sick Children, Paris 75015, France.

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.
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http://dx.doi.org/10.1016/j.cell.2018.02.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886375PMC
February 2018

Human ADAR1 Prevents Endogenous RNA from Triggering Translational Shutdown.

Cell 2018 02 25;172(4):811-824.e14. Epub 2018 Jan 25.

Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Type I interferon (IFN) is produced when host sensors detect foreign nucleic acids, but how sensors differentiate self from nonself nucleic acids, such as double-stranded RNA (dsRNA), is incompletely understood. Mutations in ADAR1, an adenosine-to-inosine editing enzyme of dsRNA, cause Aicardi-Goutières syndrome, an autoinflammatory disorder associated with spontaneous interferon production and neurologic sequelae. We generated ADAR1 knockout human cells to explore ADAR1 substrates and function. ADAR1 primarily edited Alu elements in RNA polymerase II (pol II)-transcribed mRNAs, but not putative pol III-transcribed Alus. During the IFN response, ADAR1 blocked translational shutdown by inhibiting hyperactivation of PKR, a dsRNA sensor. ADAR1 dsRNA binding and catalytic activities were required to fully prevent endogenous RNA from activating PKR. Remarkably, ADAR1 knockout neuronal progenitor cells exhibited MDA5 (dsRNA sensor)-dependent spontaneous interferon production, PKR activation, and cell death. Thus, human ADAR1 regulates sensing of self versus nonself RNA, allowing pathogen detection while avoiding autoinflammation.
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http://dx.doi.org/10.1016/j.cell.2017.12.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831367PMC
February 2018

Intrinsic Immunity Shapes Viral Resistance of Stem Cells.

Cell 2018 01 14;172(3):423-438.e25. Epub 2017 Dec 14.

Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.
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http://dx.doi.org/10.1016/j.cell.2017.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5786493PMC
January 2018

Genetic Variation at IFNL4 Influences Extrahepatic Interferon-Stimulated Gene Expression in Chronic HCV Patients.

J Infect Dis 2018 01;217(4):650-655

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas.

Polymorphisms at IFNL4 strongly influence spontaneous resolution and interferon therapeutic response in hepatitis C virus (HCV) infection. In chronic HCV, unfavorable alleles are associated with elevated interferon (IFN)-stimulated gene (ISG) expression in the liver, but extrahepatic effects are less well characterized. We used RNA sequencing (RNA-Seq) to examine whether IFNL4 genetic variation (rs368234815) modulates ISG expression in peripheral blood mononuclear cells (PBMC) during chronic HCV infection. ISG expression was elevated in unstimulated PBMC homozygous for the unfavorable ΔG IFNL4 variant; expression following IFN-α stimulation was comparable across genotypes. These findings suggest that lambda interferons may have broader systemic effects during HCV infection.
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http://dx.doi.org/10.1093/infdis/jix593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853921PMC
January 2018

Human Genetic Determinants of Viral Diseases.

Annu Rev Genet 2017 11 30;51:241-263. Epub 2017 Aug 30.

Department of Microbial Infection and Immunity, The Ohio State University, Columbus, Ohio 43210, USA; email: , , ,

Much progress has been made in the identification of specific human gene variants that contribute to enhanced susceptibility or resistance to viral diseases. Herein we review multiple discoveries made with genome-wide or candidate gene approaches that have revealed significant insights into virus-host interactions. Genetic factors that have been identified include genes encoding virus receptors, receptor-modifying enzymes, and a wide variety of innate and adaptive immunity-related proteins. We discuss a range of pathogenic viruses, including influenza virus, respiratory syncytial virus, human immunodeficiency virus, human T cell leukemia virus, human papilloma virus, hepatitis B and C viruses, herpes simplex virus, norovirus, rotavirus, parvovirus, and Epstein-Barr virus. Understanding the genetic underpinnings that affect infectious disease outcomes should allow tailored treatment and prevention approaches in the future.
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http://dx.doi.org/10.1146/annurev-genet-120116-023425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038703PMC
November 2017

Transcriptomic characterization of fibrolamellar hepatocellular carcinoma.

Proc Natl Acad Sci U S A 2015 Nov 21;112(44):E5916-25. Epub 2015 Oct 21.

Laboratory of Cellular Biophysics, The Rockefeller University, New York, NY 10065;

Fibrolamellar hepatocellular carcinoma (FLHCC) tumors all carry a deletion of ∼ 400 kb in chromosome 19, resulting in a fusion of the genes for the heat shock protein, DNAJ (Hsp40) homolog, subfamily B, member 1, DNAJB1, and the catalytic subunit of protein kinase A, PRKACA. The resulting chimeric transcript produces a fusion protein that retains kinase activity. No other recurrent genomic alterations have been identified. Here we characterize the molecular pathogenesis of FLHCC with transcriptome sequencing (RNA sequencing). Differential expression (tumor vs. adjacent normal tissue) was detected for more than 3,500 genes (log2 fold change ≥ 1, false discovery rate ≤ 0.01), many of which were distinct from those found in hepatocellular carcinoma. Expression of several known oncogenes, such as ErbB2 and Aurora Kinase A, was increased in tumor samples. These and other dysregulated genes may serve as potential targets for therapeutic intervention.
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http://dx.doi.org/10.1073/pnas.1424894112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4640752PMC
November 2015

Characterizing immune repertoires by high throughput sequencing: strategies and applications.

Trends Immunol 2014 12 8;35(12):581-590. Epub 2014 Oct 8.

The Rockefeller University, New York, NY, USA; John C. Whitehead Presidential Fellows Program, The Rockefeller University, New York, NY, USA. Electronic address:

As the key cellular effectors of adaptive immunity, T and B lymphocytes utilize specialized receptors to recognize, respond to, and neutralize a diverse array of extrinsic threats. These receptors (immunoglobulins in B lymphocytes, T cell receptors in T lymphocytes) are incredibly variable, the products of specialized genetic diversification mechanisms that generate complex lymphocyte repertoires with extensive collections of antigen specificities. Recent advances in high throughput sequencing (HTS) technologies have transformed our ability to examine antigen receptor repertoires at single nucleotide, and more recently, single cell, resolution. Here we review current approaches to examining antigen receptor repertoires by HTS, and discuss inherent biological and technical challenges. We further describe emerging applications of this powerful methodology for exploring the adaptive immune system.
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http://dx.doi.org/10.1016/j.it.2014.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390416PMC
December 2014

Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma.

Science 2014 Feb;343(6174):1010-4

Laboratory of Cellular Biophysics, Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare liver tumor affecting adolescents and young adults with no history of primary liver disease or cirrhosis. We identified a chimeric transcript that is expressed in FL-HCC but not in adjacent normal liver and that arises as the result of a ~400-kilobase deletion on chromosome 19. The chimeric RNA is predicted to code for a protein containing the amino-terminal domain of DNAJB1, a homolog of the molecular chaperone DNAJ, fused in frame with PRKACA, the catalytic domain of protein kinase A. Immunoprecipitation and Western blot analyses confirmed that the chimeric protein is expressed in tumor tissue, and a cell culture assay indicated that it retains kinase activity. Evidence supporting the presence of the DNAJB1-PRKACA chimeric transcript in 100% of the FL-HCCs examined (15/15) suggests that this genetic alteration contributes to tumor pathogenesis.
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http://dx.doi.org/10.1126/science.1249484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286414PMC
February 2014

A comprehensive analysis of the effects of the deaminase AID on the transcriptome and methylome of activated B cells.

Nat Immunol 2013 Jul 26;14(7):749-55. Epub 2013 May 26.

Laboratory of Lymphocyte Biology, The Rockefeller University, New York, New York, USA.

Beyond its well-characterized functions in antibody diversification, the cytidine deaminase AID can catalyze off-target DNA damage and has been hypothesized to edit RNA and mediate DNA demethylation. To comprehensively examine the effects of AID on the transcriptome and the pattern of DNA methylation ('methylome'), we analyzed AID-deficient (Aicda(-/-)), wild-type and AID-overexpressing activated B cells by high-throughput RNA sequencing (RNA-Seq) and reduced-representation bisulfite sequencing (RRBS). These analyses confirmed the known role of AID in immunoglobulin isotype switching and also demonstrated few other effects of AID on gene expression. Additionally, we detected no evidence of AID-dependent editing of mRNA or microRNA. Finally, the RRBS data did not support the proposed role for AID in regulating DNA methylation. Thus, despite evidence of its additional activities in other systems, antibody diversification seems to be the sole physiological function of AID in activated B cells.
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http://dx.doi.org/10.1038/ni.2616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3688651PMC
July 2013

Identifying mRNA editing deaminase targets by RNA-Seq.

Methods Mol Biol 2011 ;718:103-19

Laboratory of Lymphocyte Biology, The Rockefeller University, New York, NY, USA.

RNA editing deaminases act on a variety of targets in different organisms. A number of such enzymes have been shown to act on mRNA, with the resultant nucleotide changes modifying a transcript's information content. Though the deaminase activity of mRNA editing enzymes is readily demonstrated in vitro, identifying their physiological targets has proved challenging. Recent advances in ultra high-throughput sequencing technologies have allowed for whole transcriptome sequencing and expression profiling (RNA-Seq). We have developed a system to identify novel mRNA editing deamination targets based on comparative analysis of RNA-Seq data. The efficacy and utility of this approach is demonstrated for APOBEC1, a cytidine deaminase with a known and well-characterized mRNA editing target in the mammalian small intestine.
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http://dx.doi.org/10.1007/978-1-61779-018-8_6DOI Listing
June 2011

Transcriptome-wide sequencing reveals numerous APOBEC1 mRNA-editing targets in transcript 3' UTRs.

Nat Struct Mol Biol 2011 Feb 23;18(2):230-6. Epub 2011 Jan 23.

Laboratory of Lymphocyte Biology, The Rockefeller University, New York, New York, USA.

Apolipoprotein B-editing enzyme, catalytic polypeptide-1 (APOBEC1) is a cytidine deaminase initially identified by its activity in converting a specific cytidine (C) to uridine (U) in apolipoprotein B (apoB) mRNA transcripts in the small intestine. Editing results in the translation of a truncated apoB isoform with distinct functions in lipid transport. To address the possibility that APOBEC1 edits additional mRNAs, we developed a transcriptome-wide comparative RNA sequencing (RNA-Seq) screen. We identified and validated 32 previously undescribed mRNA targets of APOBEC1 editing, all of which are located in AU-rich segments of transcript 3' untranslated regions (3' UTRs). Further analysis established several characteristic sequence features of editing targets, which were predictive for the identification of additional APOBEC1 substrates. The transcriptomics approach to RNA editing presented here dramatically expands the list of APOBEC1 mRNA editing targets and reveals a novel cellular mechanism for the modification of transcript 3' UTRs.
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http://dx.doi.org/10.1038/nsmb.1975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075553PMC
February 2011

Circulating plasmacytoid dendritic cells in acutely infected patients with hepatitis C virus genotype 4 are normal in number and phenotype.

J Infect Dis 2010 Dec 19;202(11):1671-5. Epub 2010 Oct 19.

Liver Disease Research Unit, National Hepatology and Tropical Medicine Research Institute, Aim Shams University, Cairo, Egypt.

The incidence of hepatitis C virus (HCV) genotype 4 infection in Egypt provides a unique opportunity to study the innate immune response to symptomatic acute HCV infection. We investigated whether plasmacytoid dendritic cells (pDCs) are activated as a result of HCV infection. We demonstrate that, even during symptomatic acute infection, circulating pDCs maintained a similar precursor frequency and resting phenotype, compared with pDCs in healthy individuals. Moreover, stimulation with a Toll-like receptor 9 agonist resulted in an intact inflammatory response. These data support the growing consensus that pDCs are not directly activated by HCV and therefore are viable targets for immunotherapy throughout HCV infection.
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http://dx.doi.org/10.1086/656777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281401PMC
December 2010

Diverse functions for DNA and RNA editing in the immune system.

RNA Biol 2010 Mar-Apr;7(2):220-8. Epub 2010 Mar 29.

Laboratory of Lymphocyte Biology, The Rockefeller University, New York, NY, USA.

Polynucleotide DNA and RNA editing enzymes alter nucleic acid sequences and can thereby modify encoded informational content. Two major families of polynucleotide editing enzymes, the AID/APOBEC cytidine deaminases (which catalyze the deamination of cytidine to uridine) and the adenosine deaminases acting on RNA (ADARs, which catalyze the deamination of adenosine to inosine), function in a variety of host defense mechanisms. These enzymes act in innate and adaptive immune pathways, with both host and pathogen targets. DNA editing by the cytidine deaminase AID mediates immunoglobulin somatic hypermutation and class switch recombination, providing the antibody response with the flexibility and diversity to defend against an almost limitless array of varied and rapidly adapting pathogenic challenges. Other cytidine deaminases (APOBEC3) restrict retroviral infection by editing viral retrogenomes. Adenosine deaminases (ADARs) shape innate immune responses by modifying host transcripts that encode immune effectors and their regulators. Here we review current knowledge of polynucleotide DNA and RNA editors with a focus on these and other functions they serve in the immune system.
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http://dx.doi.org/10.4161/rna.7.2.11344DOI Listing
September 2010

Beyond SHM and CSR: AID and related cytidine deaminases in the host response to viral infection.

Adv Immunol 2007 ;94:215-44

Laboratory of Lymphocyte Biology, The Rockefeller University, New York, New York, USA.

As the primary effector of immunoglobulin somatic hypermutation (SHM) and class switch recombination (CSR), activation-induced cytidine deaminase (AID) serves an important function in the adaptive immune response. Recent advances have demonstrated that AID and a group of closely related cytidine deaminases, the APOBEC3 proteins, also act in the innate host response to viral infection. Antiviral activity was first attributed to APOBEC3G as a potent inhibitor of HIV. It is now apparent that the targets of the APOBEC3 proteins extend beyond HIV, with family members acting against a wide variety of viruses as well as host-encoded retrotransposable genetic elements. Although it appears to function through a different mechanism, AID also possesses antiviral properties. Independent of its antibody diversification functions, AID protects against transformation by Abelson murine leukemia virus (Ab-MLV), an oncogenic retrovirus. Additionally, AID has been implicated in the host response to other pathogenic viruses. These emerging roles for the AID/APOBEC cytidine deaminases in viral infection suggest an intriguing evolutionary connection of innate and adaptive immune mechanisms.
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http://dx.doi.org/10.1016/S0065-2776(06)94007-3DOI Listing
September 2007