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Article ; Online: Effects of donor source on transcriptomic profiles of human kidney tissue.

Arat, Seda / Huynh, Renee / Kumpf, Steven / Qian, Jessie / Shoieb, Ahmed / Virgen-Slane, Richard / Voigt, Frank / Xie, Zhiyong / Jakubczak, John L

FASEB journal : official publication of the Federation of American Societies for Experimental Biology

2023 Volume 37, Issue 3, Page(s) e22804

Abstract: Normal human tissue is a critical reference control in biomedical research. However, the type of tissue donor can significantly affect the underlying biology of the samples. We investigated the impact of tissue donor source type by performing ... ...

Abstract	Normal human tissue is a critical reference control in biomedical research. However, the type of tissue donor can significantly affect the underlying biology of the samples. We investigated the impact of tissue donor source type by performing transcriptomic analysis on healthy kidney tissue from three donor source types: cadavers, organ donors, and normal-adjacent tissue from surgical resections of clear cell renal cell carcinomas, and we compared the gene expression profiles to those of clear cell renal cell carcinoma samples. Comparisons among the normal samples revealed general similarity, with notable differences in gene expression pathways involving immune system and inflammatory processes, response to extracellular stimuli, ion transport, and metabolism. When compared to tumors, the transcriptomic profiles of the normal adjacent tissue were highly similar to the profiles from cadaveric and organ donor tissue samples, arguing against the presence of a field cancerization effect in clear cell renal cell carcinoma. We conclude that all three normal source types are suitable for reference kidney control samples, but important differences must be noted for particular research areas and tissue banking strategies.
MeSH term(s)	Humans ; Carcinoma, Renal Cell/genetics ; Transcriptome ; Kidney ; Kidney Neoplasms/genetics ; Tissue Donors
Language	English
Publishing date	2023-02-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	639186-2
ISSN	1530-6860 ; 0892-6638
ISSN (online)	1530-6860
ISSN	0892-6638
DOI	10.1096/fj.202201754R
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Zs.A 2266: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Btla signaling in conventional and regulatory lymphocytes coordinately tempers humoral immunity in the intestinal mucosa.

Stienne, Caroline / Virgen-Slane, Richard / Elmén, Lisa / Veny, Marisol / Huang, Sarah / Nguyen, Jennifer / Chappell, Elizabeth / Balmert, Mary Olivia / Shui, Jr-Wen / Hurchla, Michelle A / Kronenberg, Mitchell / Peterson, Scott N / Murphy, Kenneth M / Ware, Carl F / Šedý, John R

Cell reports

2022 Volume 38, Issue 12, Page(s) 110553

Abstract: The Btla inhibitory receptor limits innate and adaptive immune responses, both preventing the development of autoimmune disease and restraining anti-viral and anti-tumor responses. It remains unclear how the functions of Btla in diverse lymphocytes ... ...

Abstract	The Btla inhibitory receptor limits innate and adaptive immune responses, both preventing the development of autoimmune disease and restraining anti-viral and anti-tumor responses. It remains unclear how the functions of Btla in diverse lymphocytes contribute to immunoregulation. Here, we show that Btla inhibits activation of genes regulating metabolism and cytokine signaling, including Il6 and Hif1a, indicating a regulatory role in humoral immunity. Within mucosal Peyer's patches, we find T-cell-expressed Btla-regulated Tfh cells, while Btla in T or B cells regulates GC B cell numbers. Treg-expressed Btla is required for cell-intrinsic Treg homeostasis that subsequently controls GC B cells. Loss of Btla in lymphocytes results in increased IgA bound to intestinal bacteria, correlating with altered microbial homeostasis and elevations in commensal and pathogenic bacteria. Together our studies provide important insights into how Btla functions as a checkpoint in diverse conventional and regulatory lymphocyte subsets to influence systemic immune responses.
MeSH term(s)	B-Lymphocytes ; Immunity, Humoral ; Intestinal Mucosa ; Signal Transduction ; T-Lymphocytes, Regulatory
Language	English
Publishing date	2022-03-23
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2022.110553
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Article ; Online: Lymphotoxin β Receptor: a Crucial Role in Innate and Adaptive Immune Responses against Toxoplasma gondii.

Tersteegen, Anne / Sorg, Ursula R / Virgen-Slane, Richard / Helle, Marcel / Petzsch, Patrick / Dunay, Ildiko R / Köhrer, Karl / Degrandi, Daniel / Ware, Carl F / Pfeffer, Klaus

Infection and immunity

2021 Volume 89, Issue 6

Abstract: The lymphotoxin β receptor (LTβR) plays an essential role in the initiation of immune responses to intracellular pathogens. In mice, the LTβR is crucial for surviving acute toxoplasmosis; however, until now, a functional analysis was largely incomplete. ... ...

Abstract	The lymphotoxin β receptor (LTβR) plays an essential role in the initiation of immune responses to intracellular pathogens. In mice, the LTβR is crucial for surviving acute toxoplasmosis; however, until now, a functional analysis was largely incomplete. Here, we demonstrate that the LTβR is a key regulator required for the intricate balance of adaptive immune responses.
MeSH term(s)	Adaptive Immunity ; Animals ; Disease Models, Animal ; Host-Parasite Interactions/immunology ; Immunity, Innate ; Lymphotoxin beta Receptor/genetics ; Lymphotoxin beta Receptor/metabolism ; Mice ; Mice, Knockout ; Toxoplasma/immunology ; Toxoplasmosis/immunology ; Toxoplasmosis/metabolism ; Toxoplasmosis/parasitology
Chemical Substances	LTBR protein, human ; Lymphotoxin beta Receptor
Language	English
Publishing date	2021-05-17
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	218698-6
ISSN	1098-5522 ; 0019-9567
ISSN (online)	1098-5522
ISSN	0019-9567
DOI	10.1128/IAI.00026-21
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Article ; Online: Regnase-1 is essential for B cell homeostasis to prevent immunopathology.

Bhat, Numana / Virgen-Slane, Richard / Ramezani-Rad, Parham / Leung, Charlotte R / Chen, Cindi / Balsells, Daniel / Shukla, Ashima / Kao, Elaine / Apgar, John R / Fu, Mingui / Ware, Carl F / Rickert, Robert C

The Journal of experimental medicine

2021 Volume 218, Issue 5

Abstract: Regnase-1 is an emerging regulator of immune responses with essential roles in the posttranscriptional control of immune cell activation. Regnase-1 is expressed in B cells; however, its B cell-specific functions remain unknown. Here, we demonstrate that ... ...

Abstract	Regnase-1 is an emerging regulator of immune responses with essential roles in the posttranscriptional control of immune cell activation. Regnase-1 is expressed in B cells; however, its B cell-specific functions remain unknown. Here, we demonstrate that Regnase-1 prevents severe autoimmune pathology and show its essential role in maintaining B cell homeostasis. Using Cre driver mice for ablation of Regnase-1 at various stages of B cell development, we demonstrate that loss of Regnase-1 leads to aberrant B cell activation and differentiation, resulting in systemic autoimmunity and early morbidity. The basis of these findings was informed by gene expression data revealing a regulatory role for Regnase-1 in the suppression of a transcriptional program that promotes B cell activation, survival, and differentiation. Overall, our study shows that Regnase-1 exerts critical control of B cell activation, which is required for prevention of immunopathology.
MeSH term(s)	Animals ; Autoimmunity/genetics ; B-Lymphocytes/metabolism ; Cell Differentiation/genetics ; Gene Expression Profiling/methods ; Homeostasis/genetics ; Lymphocyte Activation/genetics ; Mice, Knockout ; Mice, Transgenic ; RNA-Seq/methods ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Ribonucleases/genetics ; Ribonucleases/metabolism ; Mice
Chemical Substances	Ribonucleases (EC 3.1.-) ; Zc3h12a protein, mouse (EC 3.1.-)
Language	English
Publishing date	2021-04-06
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	218343-2
ISSN	1540-9538 ; 0022-1007
ISSN (online)	1540-9538
ISSN	0022-1007
DOI	10.1084/jem.20200971
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Article ; Online: T cells and monocyte-derived myeloid cells mediate immunotherapy-related hepatitis in a mouse model.

Llewellyn, Heather P / Arat, Seda / Gao, Jingjin / Wen, Ji / Xia, Shuhua / Kalabat, Dalia / Oziolor, Elias / Virgen-Slane, Richard / Affolter, Timothy / Ji, Changhua

Journal of hepatology

2021 Volume 75, Issue 5, Page(s) 1083–1095

Abstract: Background & aims: Immune checkpoint inhibitors (ICIs) are associated with immune-related adverse events (irAEs) which are more severe when ICIs are used in combination. We aimed to use a mouse model to elucidate the molecular mechanisms of immune- ... ...

Abstract	Background & aims: Immune checkpoint inhibitors (ICIs) are associated with immune-related adverse events (irAEs) which are more severe when ICIs are used in combination. We aimed to use a mouse model to elucidate the molecular mechanisms of immune-related hepatitis, one of the common irAEs associated with ICIs. Methods: Immune phenotyping and molecular profiling were performed on Pdcd1 Results: ICI combination-induced hepatitis and 4-1BB agonist-mediated hepatitis share similar features yet maintain distinct immune signatures. Both were characterized by an expansion of periportal infiltrates and pan-zonal inflammation albeit with different morphologic characteristics. In both cases, infiltrates were predominantly CD4+ and CD8+ T cells with upregulated T-cell activation markers, ICOS and CD44. Depletion of CD8+ T cells abolished ICI-mediated hepatitis. Single-cell transcriptomics revealed that the hepatitis induced by combination ICIs is associated with a robust immune activation signature in all subtypes of T cells and T helper 1 skewing. Expression profiling revealed a central role for IFNγ and liver monocyte-derived macrophages in promoting a pro-inflammatory T-cell response to ICI combination and 4-1BB agonism. Conclusion: We developed a novel mouse model which offers significant value in yielding deeper mechanistic insight into immune-mediated liver toxicity associated with various immunotherapies. Lay summary: Hepatitis is one of the common immune-related adverse events in cancer patients receiving immune checkpoint inhibitor (ICI) therapy. The mechanisms of ICI-induced hepatitis are not well understood. In this paper, we identify key molecular mechanisms mediating immune intracellular crosstalk between liver T cells and macrophages in response to ICI in a mouse model.
MeSH term(s)	Animals ; Disease Models, Animal ; Hepatitis/immunology ; Immunotherapy/methods ; Immunotherapy/statistics & numerical data ; Mice ; Monocytes/immunology ; Myeloid Cells/metabolism ; T-Lymphocytes/immunology
Language	English
Publishing date	2021-07-07
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	605953-3
ISSN	1600-0641 ; 0168-8278
ISSN (online)	1600-0641
ISSN	0168-8278
DOI	10.1016/j.jhep.2021.06.037
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Article ; Online: Engineered picornavirus VPg-RNA substrates: analysis of a tyrosyl-RNA phosphodiesterase activity.

Rozovics, Janet M / Virgen-Slane, Richard / Semler, Bert L

PloS one

2011 Volume 6, Issue 3, Page(s) e16559

Abstract: Using poliovirus, the prototypic member of Picornaviridae, we have further characterized a host cell enzymatic activity found in uninfected cells, termed "unlinkase," that recognizes and cleaves the unique 5' tyrosyl-RNA phosphodiester bond found at the ... ...

Abstract	Using poliovirus, the prototypic member of Picornaviridae, we have further characterized a host cell enzymatic activity found in uninfected cells, termed "unlinkase," that recognizes and cleaves the unique 5' tyrosyl-RNA phosphodiester bond found at the 5' end of picornavirus virion RNAs. This bond connects VPg, a viral-encoded protein primer essential for RNA replication, to the viral RNA; it is cleaved from virion RNA prior to its engaging in protein synthesis as mRNA. Due to VPg retention on nascent RNA strands and replication templates, but not on viral mRNA, we hypothesize that picornaviruses utilize unlinkase activity as a means of controlling the ratio of viral RNAs that are translated versus those that either serve as RNA replication templates or are encapsidated. To test our hypothesis and further characterize this enzyme, we have developed a novel assay to detect unlinkase activity. We demonstrate that unlinkase activity can be detected using this assay, that this unique activity remains unchanged over the course of a poliovirus infection in HeLa cells, and that unlinkase activity is unaffected by the presence of exogenous VPg or anti-VPg antibodies. Furthermore, we have determined that unlinkase recognizes and cleaves a human rhinovirus-poliovirus chimeric substrate with the same efficiency as the poliovirus substrate.
MeSH term(s)	Amino Acid Sequence ; Cell Extracts ; Enzyme Assays ; Genetic Engineering ; Genome, Viral/genetics ; HeLa Cells ; Humans ; Isotope Labeling ; Molecular Sequence Data ; Mutant Proteins/chemistry ; Mutant Proteins/metabolism ; Phosphoric Diester Hydrolases/metabolism ; Poliovirus/genetics ; Poliovirus/metabolism ; RNA, Viral/metabolism ; Rhinovirus/metabolism ; Ribonuclease T1/metabolism ; Substrate Specificity ; Tyrosine/metabolism ; Viral Proteins/chemistry ; Viral Proteins/metabolism ; Virion/metabolism
Chemical Substances	Cell Extracts ; Mutant Proteins ; RNA, Viral ; VPg protein, poliovirus ; Viral Proteins ; Tyrosine (42HK56048U) ; Ribonuclease T1 (EC 3.1.27.3) ; Phosphoric Diester Hydrolases (EC 3.1.4.-)
Language	English
Publishing date	2011-03-07
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0016559
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Article ; Online: Cutting Edge: The RNA-Binding Protein Ewing Sarcoma Is a Novel Modulator of Lymphotoxin β Receptor Signaling.

Virgen-Slane, Richard / Correa, Ricardo G / Ramezani-Rad, Parham / Steen-Fuentes, Seth / Detanico, Thiago / DiCandido, Michael J / Li, Jun / Ware, Carl F

Journal of immunology (Baltimore, Md. : 1950)

2020 Volume 204, Issue 5, Page(s) 1085–1090

Abstract: Lymphotoxin β receptor (LTβR) signaling is crucial for lymphoid tissue organogenesis and immune homeostasis. To identify novel regulatory mechanisms for signaling, we implemented a two-step screen that uses coexpression analysis of human fibroblasts ... ...

Abstract	Lymphotoxin β receptor (LTβR) signaling is crucial for lymphoid tissue organogenesis and immune homeostasis. To identify novel regulatory mechanisms for signaling, we implemented a two-step screen that uses coexpression analysis of human fibroblasts undergoing LTβR stimulation and affinity-purification mass spectrometry for the LTβR signaling protein TNFR-associated factor 3 (TRAF3). We identify Ewing sarcoma (EWS) protein as a novel LTβR signaling component that associates with TRAF3 but not with TNFR-associated factor 2 (TRAF2). The EWS:TRAF3 complex forms under unligated conditions that are disrupted following activation of the LTβR. We conclude that EWS limits expression of proinflammatory molecules, GM-CSF, and ERK-2, promoting immune homeostasis.
MeSH term(s)	Granulocyte-Macrophage Colony-Stimulating Factor/genetics ; Granulocyte-Macrophage Colony-Stimulating Factor/immunology ; HEK293 Cells ; Humans ; Lymphotoxin beta Receptor/genetics ; Lymphotoxin beta Receptor/immunology ; MAP Kinase Signaling System/genetics ; MAP Kinase Signaling System/immunology ; Mitogen-Activated Protein Kinase 1/genetics ; Mitogen-Activated Protein Kinase 1/immunology ; Multiprotein Complexes/genetics ; Multiprotein Complexes/immunology ; RNA-Binding Protein EWS/genetics ; RNA-Binding Protein EWS/immunology ; TNF Receptor-Associated Factor 2/genetics ; TNF Receptor-Associated Factor 2/immunology ; TNF Receptor-Associated Factor 3/genetics ; TNF Receptor-Associated Factor 3/immunology
Chemical Substances	EWSR1 protein, human ; LTBR protein, human ; Lymphotoxin beta Receptor ; Multiprotein Complexes ; PSMD2 protein, human ; RNA-Binding Protein EWS ; TNF Receptor-Associated Factor 2 ; TNF Receptor-Associated Factor 3 ; TRAF3 protein, human ; Granulocyte-Macrophage Colony-Stimulating Factor (83869-56-1) ; MAPK1 protein, human (EC 2.7.11.24) ; Mitogen-Activated Protein Kinase 1 (EC 2.7.11.24)
Language	English
Publishing date	2020-01-22
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	3056-9
ISSN	1550-6606 ; 0022-1767 ; 1048-3233 ; 1047-7381
ISSN (online)	1550-6606
ISSN	0022-1767 ; 1048-3233 ; 1047-7381
DOI	10.4049/jimmunol.1901260
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Ud II Zs.37: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 2021: Bestellungen von Artikeln über das Online-Bestellformular ab Jg. 2022: Lesesaal (EG)
Zs.MG 28: Show issues

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Article ; Online: Tandem mass tag-based quantitative proteomic profiling identifies candidate serum biomarkers of drug-induced liver injury in humans.

Ravindra, Kodihalli C / Vaidya, Vishal S / Wang, Zhenyu / Federspiel, Joel D / Virgen-Slane, Richard / Everley, Robert A / Grove, Jane I / Stephens, Camilla / Ocana, Mireia F / Robles-Díaz, Mercedes / Isabel Lucena, M / Andrade, Raul J / Atallah, Edmond / Gerbes, Alexander L / Weber, Sabine / Cortez-Pinto, Helena / Fowell, Andrew J / Hussaini, Hyder / Bjornsson, Einar S /

Patel, Janisha / Stirnimann, Guido / Verma, Sumita / Elsharkawy, Ahmed M / Griffiths, William J H / Hyde, Craig / Dear, James W / Aithal, Guruprasad P / Ramaiah, Shashi K

Nature communications

2023 Volume 14, Issue 1, Page(s) 1215

Abstract: Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. Here, we identify, confirm, and replicate the biomarker performance characteristics of ... ...

Abstract	Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. Here, we identify, confirm, and replicate the biomarker performance characteristics of candidate proteins in patients with DILI at onset (DO; n = 133) and follow-up (n = 120), acute non-DILI at onset (NDO; n = 63) and follow-up (n = 42), and healthy volunteers (HV; n = 104). Area under the receiver operating characteristic curve (AUC) for cytoplasmic aconitate hydratase, argininosuccinate synthase, carbamoylphosphate synthase, fumarylacetoacetase, fructose-1,6-bisphosphatase 1 (FBP1) across cohorts achieved near complete separation (range: 0.94-0.99) of DO and HV. In addition, we show that FBP1, alone or in combination with glutathione S-transferase A1 and leukocyte cell-derived chemotaxin 2, could potentially assist in clinical diagnosis by distinguishing NDO from DO (AUC range: 0.65-0.78), but further technical and clinical validation of these candidate biomarkers is needed.
MeSH term(s)	Humans ; Proteomics ; Chemical and Drug Induced Liver Injury ; Argininosuccinate Synthase ; Biomarkers ; CD8 Antigens ; Fructose
Chemical Substances	Argininosuccinate Synthase (EC 6.3.4.5) ; Biomarkers ; CD8 Antigens ; Fructose (30237-26-4)
Language	English
Publishing date	2023-03-03
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-36858-6
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Article ; Online: Co-expression Networks Identify DHX15 RNA Helicase as a B Cell Regulatory Factor.

Detanico, Thiago / Virgen-Slane, Richard / Steen-Fuentes, Seth / Lin, Wai W / Rhode-Kurnow, Antje / Chappell, Elizabeth / Correa, Ricardo G / DiCandido, Michael J / Mbow, M Lamine / Li, Jun / Ware, Carl F

Frontiers in immunology

2019 Volume 10, Page(s) 2903

Abstract: Genome-wide co-expression analysis is often used for annotating novel gene functions from high-dimensional data. Here, we developed an R package with a Shiny visualization app that creates immuno-networks from RNAseq data using a combination of Weighted ... ...

Abstract	Genome-wide co-expression analysis is often used for annotating novel gene functions from high-dimensional data. Here, we developed an R package with a Shiny visualization app that creates immuno-networks from RNAseq data using a combination of Weighted Gene Co-expression Network Analysis (WGCNA), xCell immune cell signatures, and Bayesian Network Learning. Using a large publicly available RNAseq dataset we generated a Gene Module-Immune Cell (GMIC) network that predicted causal relationships between DEAH-box RNA helicase (DHX)15 and genes associated with humoral immunity, suggesting that DHX15 may regulate B cell fate. Deletion of DHX15 in mouse B cells led to impaired lymphocyte development, reduced peripheral B cell numbers, and dysregulated expression of genes linked to antibody-mediated immune responses similar to the genes predicted by the GMIC network. Moreover, antigen immunization of mice demonstrated that optimal primary IgG1 responses required DHX15. Intrinsic expression of DHX15 was necessary for proliferation and survival of activated of B cells. Altogether, these results support the use of co-expression networks to elucidate fundamental biological processes.
MeSH term(s)	Animals ; B-Lymphocytes/immunology ; B-Lymphocytes/metabolism ; Biomarkers ; Biopsy ; Female ; Gene Expression Profiling ; Gene Expression Regulation ; Gene Regulatory Networks ; Immunomodulation/genetics ; Mice ; RNA Helicases/genetics ; RNA Helicases/metabolism
Chemical Substances	Biomarkers ; Dhx15 protein, mouse (EC 3.6.4.13) ; RNA Helicases (EC 3.6.4.13)
Language	English
Publishing date	2019-12-10
Publishing country	Switzerland
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2019.02903
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Article ; Online: Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA.

Langereis, Martijn A / Feng, Qian / Nelissen, Frank H T / Virgen-Slane, Richard / van der Heden van Noort, Gerbrand J / Maciejewski, Sonia / Filippov, Dmitri V / Semler, Bert L / van Delft, Floris L / van Kuppeveld, Frank J M

Nucleic acids research

2013 Volume 42, Issue 4, Page(s) 2473–2482

Abstract: Picornaviruses constitute a large group of viruses comprising medically and economically important pathogens such as poliovirus, coxsackievirus, rhinovirus, enterovirus 71 and foot-and-mouth disease virus. A unique characteristic of these viruses is the ... ...

Abstract	Picornaviruses constitute a large group of viruses comprising medically and economically important pathogens such as poliovirus, coxsackievirus, rhinovirus, enterovirus 71 and foot-and-mouth disease virus. A unique characteristic of these viruses is the use of a viral peptide (VPg) as primer for viral RNA synthesis. As a consequence, all newly formed viral RNA molecules possess a covalently linked VPg peptide. It is known that VPg is enzymatically released from the incoming viral RNA by a host protein, called TDP2, but it is still unclear whether the release of VPg is necessary to initiate RNA translation. To study the possible requirement of VPg release for RNA translation, we developed a novel method to modify the genomic viral RNA with VPg linked via a 'non-cleavable' bond. We coupled an azide-modified VPg peptide to an RNA primer harboring a cyclooctyne [bicyclo[6.1.0]nonyne (BCN)] by a copper-free 'click' reaction, leading to a VPg-triazole-RNA construct that was 'non-cleavable' by TDP2. We successfully ligated the VPg-RNA complex to the viral genomic RNA, directed by base pairing. We show that the lack of VPg unlinkase does not influence RNA translation or replication. Thus, the release of the VPg from the incoming viral RNA is not a prerequisite for RNA translation or replication.
MeSH term(s)	Click Chemistry ; Enterovirus/genetics ; Genome, Viral ; HeLa Cells ; Humans ; Peptides/chemistry ; Picornaviridae/genetics ; Picornaviridae/physiology ; Protein Biosynthesis ; RNA/chemistry ; RNA, Viral/biosynthesis ; RNA, Viral/chemistry ; Viral Proteins/chemistry ; Virus Replication
Chemical Substances	Peptides ; RNA primers ; RNA, Viral ; VPg protein, poliovirus ; Viral Proteins ; RNA (63231-63-0)
Language	English
Publishing date	2013-11-15
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkt1162
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