Homo sapiens
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Project description:Infection of animal cells by many viruses is detected and countered by a variety of means, including recognition of non-self 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 to endogenous mRNAs. Here, we determined a crystal structure of a protein-RNA complex containing the N-terminal, four-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 four 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, crosslinking-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 the RNA binding specificity 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.
2019-11-01 | GSE139667 | GEO
Project description:Vertebrate genomes exhibit marked CG-suppression, that is lower than expected numbers of 5’-CG-3’ dinucleotides1. This feature is likely due to C-to-T mutations that have accumulated over hundreds of millions of years, driven by CG-specific DNA methyl transferases and spontaneous methyl-cytosine deamination. Remarkably, many RNA viruses of vertebrates that are not substrates for DNA methyl transferases mimic the CG-suppression of their hosts2-4. This striking property of viral genomes is unexplained4-6. In a synonymous mutagenesis experiment, we found that CG-suppression is essential for HIV-1 replication. The deleterious effect of CG dinucleotides on HIV-1 replication was cumulative, evident as cytoplasmic RNA depletion, and exerted by CG dinucleotides in both translated and non-translated exonic RNA sequences. A focused siRNA screen revealed that zinc finger antiviral protein (ZAP)7 inhibited virion production by cells infected with CG-enriched HIV-1. Crucially, HIV-1 mutants containing segments whose CG-content mimicked random sequence were defective in unmanipulated cells, but replicated normally in ZAP-deficient cells. Crosslinking-immunoprecipitation-sequencing assays demonstrated that ZAP binds directly and selectively to RNA sequences containing CG dinucleotides. These findings suggest that ZAP exploits host CG-suppression to discriminate non-self RNA. The dinucleotide composition of HIV-1, and perhaps other RNA viruses, appears to have adapted to evade this host defense.
2017-08-22 | GSE102843 | GEO
Project description:Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonises several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is largely unknown. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a β-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, the long (ZAP-L) and short (ZAP-S), is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP decelerates the progression of HCMV infection. SLAM-sequencing revealed that ZAP restricts HCMV at early stages of infection by destabilising a distinct subset of viral transcripts with low CG content. In summary, this report provides evidence of an important antiviral role for ZAP in host defense against HCMV infection and highlights its differentiated function during DNA virus infection.
2021-04-22 | PXD023559 | Pride
Project description:CG-dinucleotide suppression enables antiviral defense targeting non-self RNA
| PRJNA399117 | ENA
Project description:The Epstein-Barr virus (EBV) B-ZIP transcription factor (TF) Zta binds to many DNA sequences containing methylated CG dinucleotides. Using protein binding microarrays (PBMs), we analyzed the binding of Zta to four kinds of double-stranded DNA: 1) DNA containing cytosine on both strands, 2) DNA with 5-methylcytosine (5mC) on one strand and cytosine on the second strand, 3) DNA with 5-hydroxymethylcytosine (5hmC) on one strand and cytosine on the second strand, and 4) DNA where both cytosines in all CG dinucleotides contain 5mC. We compared the resulting data to PBM data for three other B-ZIP proteins (CREB1 and CEBPB homodimers, and cFos-cJun heterodimers). With cytosine, Zta binds the TRE motif TGAC/GTCA as previously reported. With CG dinucleotides containing 5mC on both strands, many TRE motif variants containing a methylated CG dinucleotide at two positions in the motif, such as MGAGTCA and TGAGMGA (where M=5mC) were preferentially bound. 5mC inhibits Zta binding to both TRE motif half sites GTCA and CTCA. Like the CREB1 homodimer, the Zta homodimer and the cJun|cFos heterodimer bind the C/EBP half site tetranucleotide GCAA stronger when it contains 5mC. Our results identify new DNA sequences that are well-bound by the viral B-ZIP protein Zta only when they contain 5mC or 5hmC, opening the potential for discovery of new viral and host regulatory programs controlled by EBV.
2017-10-26 | GSE100870 | GEO
Project description:The Epstein-Barr virus (EBV) B-ZIP transcription factor (TF) Zta binds to many DNA sequences containing methylated CG dinucleotides. Using protein binding microarrays (PBMs), we analyzed the binding of Zta to four kinds of double-stranded DNA: 1) DNA containing cytosine on both strands, 2) DNA with 5-methylcytosine (5mC) on one strand and cytosine on the second strand, 3) DNA with 5-hydroxymethylcytosine (5hmC) on one strand and cytosine on the second strand, and 4) DNA where both cytosines in all CG dinucleotides contain 5mC. We compared the resulting data to PBM data for three other B-ZIP proteins (CREB1 and CEBPB homodimers, and cFos-cJun heterodimers). With cytosine, Zta binds the TRE motif TGAC/GTCA as previously reported. With CG dinucleotides containing 5mC on both strands, many TRE motif variants containing a methylated CG dinucleotide at two positions in the motif, such as MGAGTCA and TGAGMGA (where M=5mC) were preferentially bound. 5mC inhibits Zta binding to both TRE motif half sites GTCA and CTCA. Like the CREB1 homodimer, the Zta homodimer and the cJun|cFos heterodimer bind the C/EBP half site tetranucleotide GCAA stronger when it contains 5mC. Our results identify new DNA sequences that are well-bound by the viral B-ZIP protein Zta only when they contain 5mC or 5hmC, opening the potential for discovery of new viral and host regulatory programs controlled by EBV.
2017-10-26 | GSE100869 | GEO
Project description:Synonymous recoding of viral genome can attenuate their replication, but can have pleiotropic effects, with multiple mechanisms contributing to attenuation. We set out to design recoded viral genomes whose attenuation was specific and conditional. The zinc finger antiviral protein (ZAP) recognizes CpG dinucleotides and targets CpG-rich RNAs for depletion, but RNA features such as CpG numbers, spacing and surrounding nucleotide composition that enable specific modulation by ZAP are undescribed. Using synonymously mutated HIV-1 genomes, we define several sequence features that govern ZAP sensitivity and stable attenuation. Using features defined using HIV-1, we then designed a mutant enterovirus A71 genome whose attenuation was also stable and strictly ZAP-dependent, both in cell culture and in mice. This conditionally attenuated enterovirus A71 elicited neutralizing antibodies that were protective against wild-type enterovirus 71 infection and disease. Elucidation of the determinants of ZAP sensitivity can thus enable the rational design of conditionally attenuated viral vaccines.
2022-07-26 | GSE208611 | GEO
Project description:DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpC and CpT dinucleotides. Here we report a comprehensive analysis of non-CG methylation in 72 genome-scale DNA methylation maps across human pluripotent and differentiated cell types. We confirm non-CG methylation to be predominant in pluripotent cell types and observe an expected decrease upon differentiation and near complete absence in various differentiated cells. Our data highlight that non-CG methylation is highly variable and shows little conservation between different pluripotent cell lines. While we show a strong correlation of non-CG methylation and DNMT3 expression levels we find a statistical independence of non-CG methylation from pluripotency associated gene expression. Finally, non-CG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of DNA methylation in human cells and help clarify previous observations using a large representative sample set. Examination of nonCG DNA methylation patterns in pluripotent and differentiated cells
2011-08-31 | E-GEOD-27432 | biostudies-arrayexpress
Project description:Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.
2021-11-25 | PXD029656 | Pride
Project description:Background: Interferon-alpha (IFN-a) contributes extensively to the host immune response upon viral infection through antiviral, pro-apoptotic, antiproliferative and immunomodulatory activities. Although extensively documented in various types of human cancers and viral infections, controversy exists in the exact mechanism of action of IFN-a in human immunodeficiency virus type 1 (HIV-1) and human T lymphotropic virus type 1 (HTLV-1) retroviral infections. Principal Findings: IFN-a displayed robust anti-HIV-1 effects in HTLV-1/HIV-1 co-infected MT-4 cells in vitro, demonstrated by the dose-dependent inhibition of the HIV-1-induced cytopathic effect (IC50 = 83.5IU/ml, p < 0.0001) and p24 secretion (IC50 = 1.2 IU/ml, p < 0.0001). In contrast, IFN-a treatment did not affect cell viability nor HTLV-1 viral RNA levels in HTLV-1 mono-infected cell lines, based on flow cytometry and nCounter analysis. However, we were able to confirm the previously described posttranscriptional inhibition of HTLV-1 p19 secretion by IFN-a, both in cell lines (p = 0.0045) as well as in adult T cell leukemia patient samples (p = 0.031). In addition, through microarray and nCounter analysis, we demonstrated significant transcriptional activation of interferon-stimulated genes and intact IFN-a signaling in HTLV-1-infected cell lines. Conclusions: Taken together, our results indicate that both the absence of in vitro antiproliferative and pro-apoptotic activity, as well as the modest posttranscriptional antiviral activity of IFN-a against HTLV-1, was not due to a cell intrinsic defect in IFN-a signalisation, but rather represents a retrovirus-specific phenomenon, considering the robust HIV-1 inhibition in co-infected cells.
2015-01-01 | GSE34870 | GEO