scholarly journals Structure of the zinc-finger antiviral protein in complex with RNA reveals a mechanism for selective targeting of CG-rich viral sequences

2019 ◽  
Vol 116 (48) ◽  
pp. 24303-24309 ◽  
Author(s):  
Jennifer L. Meagher ◽  
Matthew Takata ◽  
Daniel Gonçalves-Carneiro ◽  
Sarah C. Keane ◽  
Antoine Rebendenne ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Rna Structure ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Specific Binding ◽  
Binding Pocket ◽  
Animal Cells ◽  
Antiviral Protein ◽  
Cytoplasmic Rna ◽  
Cg Dinucleotide

Infection of animal cells by numerous viruses is detected and countered by a variety of means, including recognition of nonself nucleic acids. The zinc finger antiviral protein (ZAP) depletes cytoplasmic RNA that is recognized as foreign in mammalian cells by virtue of its elevated CG dinucleotide content compared with endogenous mRNAs. Here, we determined a crystal structure of a protein-RNA complex containing the N-terminal, 4-zinc finger human (h) ZAP RNA-binding domain (RBD) and a CG dinucleotide-containing RNA target. The structure reveals in molecular detail how hZAP is able to bind selectively to CG-rich RNA. Specifically, the 4 zinc fingers create a basic patch on the hZAP RBD surface. The highly basic second zinc finger contains a pocket that selectively accommodates CG dinucleotide bases. Structure guided mutagenesis, cross-linking immunoprecipitation sequencing assays, and RNA affinity assays show that the structurally defined CG-binding pocket is not required for RNA binding per se in human cells. However, the pocket is a crucial determinant of high-affinity, specific binding to CG dinucleotide-containing RNA. Moreover, variations in RNA-binding specificity among a panel of CG-binding pocket mutants quantitatively predict their selective antiviral activity against a CG-enriched HIV-1 strain. Overall, the hZAP RBD RNA structure provides an atomic-level explanation for how ZAP selectively targets foreign, CG-rich RNA.

scholarly journals TRIM25 Is Required for the Antiviral Activity of Zinc Finger Antiviral Protein

2017 ◽  
Vol 91 (9) ◽  
Author(s):  
Xiaojiao Zheng ◽  
Xinlu Wang ◽  
Fan Tu ◽  
Qin Wang ◽  
Zusen Fan ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Zinc Finger ◽  
Ebola Virus ◽  
Rna Binding ◽  
E3 Ligase ◽  
Antiviral Protein ◽  
Viral Mrnas ◽  
Target Mrna ◽  
Ubiquitin E3 Ligase ◽  
Target Rna

ABSTRACTZinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to viral mRNAs and repressing the translation and/or promoting the degradation of target mRNA. In addition, ZAP regulates the expression of certain cellular genes. Here, we report that tripartite motif-containing protein 25 (TRIM25), a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 abolished ZAP's antiviral activity. The E3 ligase activity of TRIM25 is required for this regulation. TRIM25 mediated ZAP ubiquitination, but the ubiquitination of ZAP itself did not seem to be required for its antiviral activity. Downregulation of endogenous ubiquitin or overexpression of the deubiquitinase OTUB1 impaired ZAP's activity. We provide evidence indicating that TRIM25 modulates the target RNA binding activity of ZAP. These results uncover a mechanism by which the antiviral activity of ZAP is regulated.IMPORTANCEZAP is a host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1, Sindbis virus, and Ebola virus. ZAP binds directly to target mRNA, and it represses the translation and promotes the degradation of target mRNA. While the mechanisms by which ZAP posttranscriptionally inhibits target RNA expression have been extensively studied, how its antiviral activity is regulated is not very clear. Here, we report that TRIM25, a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 remarkably abolished ZAP's activity. TRIM25 is required for ZAP optimal binding to target mRNA. These results help us to better understand how the antiviral activity of ZAP is regulated.

scholarly journals Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity

2021 ◽  
Author(s):  
Omer Ziv ◽  
Svetlana Farberov ◽  
Jian You Lau ◽  
Eric A Miska ◽  
Grzegorz Kudla ◽  
...  
Keyword(s):  
Rna Structure ◽  
Mammalian Cells ◽  
Noncoding Rna ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Features ◽  
Structural Domain ◽  
Mrna Targets ◽  
Structural Principles ◽  
Pumilio Proteins

It is increasingly appreciated that long non-coding RNAs (lncRNAs) carry out important functions in mammalian cells, but how these are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA binding proteins, but the structural principles that underlie this mode of regulation are underexplored. Here, we focused on the NORAD lncRNA, which binds Pumilio proteins and modulates their ability to repress hundreds of mRNA targets. We probed the RNA structure and long-range RNA-RNA interactions formed by NORAD inside cells, under different stressful conditions. We discovered that NORAD structure is highly modular, and consists of well-defined domains that contribute independently to NORAD function. We discovered that NORAD structure spatially clusters the Pumilio binding sites along NORAD in a manner that contributes to the de-repression of Pumilio target proteins. Following arsenite stress, the majority of NORAD structure undergoes relaxation and forms inter-molecular interactions with RNAs that are targeted to stress granules. NORAD sequence thus dictates elaborated structural domain organization that facilitates its function on multiple levels, and which helps explain the extensive evolutionary sequence conservation of NORAD regions that are not predicted to directly bind Pumilio proteins.

scholarly journals Аffinity properties of plant-made anti-her2 antibodies

2018 ◽  
Vol 17 (1) ◽  
pp. 95-100
Author(s):  
A. I. Scherbakov ◽  
E. N. Kosobokova ◽  
M. V. Pinyugina ◽  
E. V. Sheshukova ◽  
V. S. Kosorukov
Keyword(s):  
Monoclonal Antibodies ◽  
Mammalian Cells ◽  
Plant Biomass ◽  
Specific Binding ◽  
High Yield ◽  
Immunocytochemical Staining ◽  
Animal Cells ◽  
Her2 Receptor ◽  
Her2 Antigen ◽  
Culture Technology

The Her2 receptor is an important target for antitumor therapy in the treatment of breast cancer. Trastuzumab, based on anti-Her2 monoclonal antibodies, is used in clinical practice. Trastuzumab is produced by animal cells culture technology and is quite expensive. We use the technology of production of recombinant antibodies in the plants Nicotiana benthamiana with a high yield of final purified protein. Objective. The aim of following study is a comparison of monoclonal antibodies received via classic cell culture technology and produced in plant biomass. Materials and methods. Recombinant plant-made antibodies were isolated by affinity chromatography from the biomass of N. benthamiana plants agroinfiltrated by vector constructs. Comparison of affinity properties was carried out by immunocytochemical staining of cells and competitive binding using flow cytometry analysis. Results and conclusion. We show that the antibodies expressed in N. benthamiana are equal to those obtained from mammalian cells in binding to Her2 antigen localized on the surface of the SK-BR-3 cells. In the present work it was shown that the plant-made anti-Her2 antibodies do not differ in specific binding with the Her2 antigen, as well as with IV subdomain of the Her2 receptor.

scholarly journals Association of Zinc Finger Antiviral Protein Binding to Viral Genomic RNA with Attenuation of Replication of Echovirus 7

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Niluka Goonawardane ◽  
Dung Nguyen ◽  
Peter Simmonds
Keyword(s):  
Zinc Finger ◽  
Specific Binding ◽  
Stress Granules ◽  
Rna Degradation ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Antiviral Protein ◽  
Rna Sequences ◽  
Cpg Dinucleotides ◽  
Cellular Compartments

ABSTRACT Previous studies have implicated both zinc finger antiviral protein (ZAP) and oligoadenylate synthetase 3 (OAS3)/RNase L in the attenuation of RNA viruses with elevated CpG and UpA dinucleotides. Mechanisms and interrelationships between these two pathways were investigated using an echovirus 7 (E7) replicon with compositionally modified sequences inserted into the 3′ untranslated region. ZAP and OAS3 immunoprecipitation (IP) assays provided complementary data on dinucleotide composition effects on binding. Elevated frequencies of alternative pyrimidine/purine (CpA and UpG) and reversed (GpC and ApU) dinucleotides showed no attenuating effect on replication or specific binding to ZAP by IP. However, the bases 3′ and 5′ of CpG motifs influenced replication and ZAP binding; UCGU enhanced CpG-mediated attenuation and ZAP binding, while A residues shielded CpGs from ZAP recognition. Attenuating effects of elevated frequencies of UpA on replication occurred independently of CpG dinucleotides and bound noncompetitively with CpG-enriched RNA, consistent with a separate recognition site from CpG. Remarkably, immunoprecipitation with OAS3 antibody reproduced the specific binding to CpG- and UpA-enriched RNA sequences. However, OAS3 and ZAP were coimmunoprecipitated in both ZAP and OAS3 IP and colocalized with E7 and stress granules (SGs) by confocal microscopy analysis of infected cells. ZAP’s association with larger cellular complexes may mediate the recruitment of OAS3/RNase L, KHNYN, and other RNA degradation pathways. IMPORTANCE We recently discovered that the OAS3/RNase L antiviral pathway is essential for restriction of CpG- and UpA-enriched viruses, in addition to the requirement for zinc finger antiviral protein (ZAP). The current study provides evidence for the specific dinucleotide and wider recognition contexts associated with virus recognition and attenuation. It further documents the association of ZAP and OAS3 and association with stress granules and a wider protein interactome that may mediate antiviral effects in different cellular compartments. The study provides a striking reconceptualization of the pathways associated with this aspect of antiviral defense.

Expression and RNA-binding of human zinc-finger antiviral protein

2010 ◽  
Vol 396 (3) ◽  
pp. 696-702 ◽  
Author(s):  
Mi Suk Jeong ◽  
Eun Jung Kim ◽  
Se Bok Jang
Keyword(s):  
Zinc Finger ◽  
Rna Binding ◽  
Antiviral Protein

scholarly journals Insect-Specific Flavivirus Replication in Mammalian Cells Is Inhibited by Physiological Temperature and the Zinc-Finger Antiviral Protein

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 573
Author(s):  
Agathe M.G. Colmant ◽  
Jody Hobson-Peters ◽  
Teun A.P. Slijkerman ◽  
Jessica J. Harrison ◽  
Gorben P. Pijlman ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Interferon Response ◽  
Cell Culture Supernatant ◽  
Progeny Virus ◽  
Infection Cycle ◽  
Antiviral Protein ◽  
Response Factors ◽  
Cpg Dinucleotides

The genus Flavivirus contains pathogenic vertebrate-infecting flaviviruses (VIFs) and insect-specific flaviviruses (ISF). ISF transmission to vertebrates is inhibited at multiple stages of the cellular infection cycle, via yet to be elucidated specific antiviral responses. The zinc-finger antiviral protein (ZAP) in vertebrate cells can bind CpG dinucleotides in viral RNA, limiting virus replication. Interestingly, the genomes of ISFs contain more CpG dinucleotides compared to VIFs. In this study, we investigated whether ZAP prevents two recently discovered lineage II ISFs, Binjari (BinJV) and Hidden Valley viruses (HVV) from replicating in vertebrate cells. BinJV protein and dsRNA replication intermediates were readily observed in human ZAP knockout cells when cultured at 34 °C. In ZAP-expressing cells, inhibition of the interferon response via interferon response factors 3/7 did not improve BinJV protein expression, whereas treatment with kinase inhibitor C16, known to reduce ZAP’s antiviral function, did. Importantly, at 34 °C, both BinJV and HVV successfully completed the infection cycle in human ZAP knockout cells evident from infectious progeny virus in the cell culture supernatant. Therefore, we identify vertebrate ZAP as an important barrier that protects vertebrate cells from ISF infection. This provides new insights into flavivirus evolution and the mechanisms associated with host switching.

scholarly journals The Zinc Finger Antiviral Protein Directly Binds to Specific Viral mRNAs through the CCCH Zinc Finger Motifs

2004 ◽  
Vol 78 (23) ◽  
pp. 12781-12787 ◽  
Author(s):  
Xuemin Guo ◽  
John-William N. Carroll ◽  
Margaret R. MacDonald ◽  
Stephen P. Goff ◽  
Guangxia Gao
Keyword(s):  
Zinc Finger ◽  
Sindbis Virus ◽  
Rna Binding ◽  
Leukemia Virus ◽  
Luciferase Reporter ◽  
Antiviral Protein ◽  
Viral Mrnas ◽  
Ccch Zinc Finger ◽  
Zinc Finger Motifs ◽  
Viral Sequences

ABSTRACT The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. For this report, we mapped the viral sequences that are sensitive to ZAP inhibition. The viral sequences were cloned into a luciferase reporter and analyzed for the ability to mediate ZAP-dependent destabilization of the reporter. The sensitive sequence in MLV was mapped to the 3′ long terminal repeat; the sensitive sequences in SIN were mapped to multiple fragments. The fragment of SIN that displayed the highest destabilizing activity was further analyzed by deletion mutagenesis for the minimal sequence that retained the activity. This led to the identification of a fragment of 653 nucleotides. Any further deletion of this fragment resulted in significantly lower activity. We provide evidence that ZAP directly binds to the active but not the inactive fragments. The CCCH zinc finger motifs of ZAP play important roles in RNA binding and antiviral activity. Disruption of the second and fourth zinc fingers abolished ZAP's activity, whereas disruption of the first and third fingers just slightly lowered its activity.

scholarly journals Rec (Formerly Corf) Function Requires Interaction with a Complex, Folded RNA Structure within Its Responsive Element rather than Binding to a Discrete Specific Binding Site

2001 ◽  
Vol 75 (21) ◽  
pp. 10359-10371 ◽  
Author(s):  
Christine Magin-Lachmann ◽  
Silvia Hahn ◽  
Heike Strobel ◽  
Ulrike Held ◽  
Johannes Löwer ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Nuclear Export ◽  
Rna Structure ◽  
Rna Binding ◽  
Specific Binding ◽  
Regulatory Protein ◽  
Human Endogenous Retrovirus ◽  
Responsive Element ◽  
Folded Rna

ABSTRACT It was recently reported that the human endogenous retrovirus HTDV/HERV-K encodes the regulatory protein Rec (formerly designated Corf), which is functionally equivalent to the nuclear export adapter proteins Rev of human immunodeficiency virus and Rex of human T-cell leukemia virus. We have demonstrated that the Rec protein interacts with a characteristic 429-nucleotide RNA element, the Rec-responsive element (RcRE), present in the 3′ long terminal repeat of HTDV/HERV-K transcripts. In analogy to the Rev and Rex proteins, which have distinct RNA binding sites in their responsive elements, we have proposed that Rec may also have a defined binding site in the RcRE. In this report, we demonstrate that not every HTDV/HERV-K copy present in the human genome contains an active RcRE, and we characterize mutations that abrogate Rec function. In addition, we demonstrate that Rec function requires binding to a complex, folded RNA structure rather than binding to a discrete specific binding site, in contrast to Rev and Rex and their homologous responsive elements. We define four stem-loop structures in the RcRE that are essential for Rec function. Finally, we demonstrate that both Rev and Rex can mediate nuclear export through the RcRE but that their binding sites are different from each other and from that of Rec.

scholarly journals Association of zinc-finger antiviral protein (ZAP) binding to viral genomic RNA with attenuation of replication of echovirus 7

2020 ◽  
Author(s):  
Niluka Goonawardane ◽  
Dung Nguyen ◽  
Peter Simmonds
Keyword(s):  
Zinc Finger ◽  
Specific Binding ◽  
Stress Granules ◽  
Rna Degradation ◽  
Oligoadenylate Synthetase ◽  
Antiviral Protein ◽  
Rna Sequences ◽  
Cpg Dinucleotides ◽  
Infected Cells ◽  
Cellular Compartments

AbstractPrevious studies have implicated both zinc finger antiviral protein (ZAP) and oligoadenylate synthetase 3 (OAS3)/RNASeL in the attenuation of RNA viruses with elevated CpG and UpA dinucleotides. Mechanisms and inter-relationships between these two pathways were investigated using an echovirus 7 (E7) replicon with compositionally modified sequences inserted into the 3’untranslated region. ZAP and OAS3 immunoprecipitation (IP) assays provided complementary data on dinucleotide composition effects on binding. Elevated frequencies of alternative pyrimidine/purine (CpA and UpG) and reversed (GpC and ApU) dinucleotides showed no attenuating effect nor specific binding to ZAP by IP. However, the bases 3’ and 5’ to CpG motifs influenced replication and ZAP binding; UCGU enhanced CpG-mediated attenuation and ZAP-binding while A residues shielded CpGs from ZAP recognition. Attenuating effects of elevated frequencies of UpA on replication occurred independently of CpG dinucleotides and bound non-competitively with CpG-enriched RNA consistent with a separate recognition site from CpG. Remarkably, immunoprecipitation with OAS3 antibody reproduced the specific binding to CpG- and UpA-enriched RNA sequences. However, OAS3 and ZAP were coimmunoprecipitated in both ZAP and OAS3 IP, and colocalised with E7 and stress granules (SGs) by confocal microscopy analysis of infected cells. ZAP’s association with larger cellular complexes may mediate the recruitment of OAS3/RNAseL, KHNYN and other RNA degradation pathways.ImportanceWe have recently discovered that the OAS3/RNAseL antiviral pathway is essential for restriction of CpG- and UpA-enriched viruses, in addition to the requirement for zinc finger antiviral protein (ZAP). The current study provides evidence for the specific dinucleotide and wider recognition contexts associated with virus recognition and attenuation. It further documents the association of ZAP and OAS3 and association with stress granules and a wider protein interactome that may mediate antiviral effects in different cellular compartments. The study provides a striking re-conceptualisation of the pathways associated with this aspect of antiviral defence.

scholarly journals Dynamic Regulation of RNA Structure in Mammalian Cells

2018 ◽  
Author(s):  
Lei Sun ◽  
Furqan Fazal ◽  
Pan Li ◽  
James P. Broughton ◽  
Byron Lee ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Structure ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Structural Information ◽  
Rna Structures ◽  
Rna Modifications ◽  
Dynamic Regulation ◽  
Comparative Structure

RNA structure is intimately connected to each step of gene expression. Recent advances have enabled transcriptome-wide maps of RNA secondary structure, termed RNA structuromes. However, previous whole-cell analyses lacked the resolution to unravel the dynamic regulation of RNA structure across subcellular states. Here we reveal the RNA structuromes in three compartments — chromatin, nucleoplasm and cytoplasm. The cytotopic structuromes substantially expand RNA structural information, and enable detailed investigation of the central role of RNA structure in linking transcription, translation, and RNA decay. Through comparative structure analysis, we develop a resource to visualize the interplay of RNA-protein interactions, RNA chemical modifications, and RNA structure, and predict both direct and indirect reader proteins of RNA modifications. We validate the novel role of the RNA binding protein LIN28A as an N6-methyladenosine (m6A) modification “anti-reader”. Our results highlight the dynamic nature of RNA structures and its functional significance in gene regulation.

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