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The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides.

ABSTRACT: Zinc finger antiviral protein (ZAP) is a powerful restriction factor for viruses with elevated CpG dinucleotide frequencies. We report that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides. Attenuation of both CpG- and UpA-high viruses and replicon mutants was reversed in ZAP k/o cell lines, and restored by plasmid-derived reconstitution of expression in k/o cells. In pull-down assays, ZAP bound to viral RNA transcripts with either CpG- and UpA-high sequences inserted in the R2 region. We found no evidence that attenuation of CpG- or UpA-high mutants was mediated through either translation inhibition or accelerated RNA degradation. Reversal of the attenuation of CpG-high, and UpA-high E7 viruses and replicons was also achieved through knockout of RNAseL and oligodenylate synthetase 3 (OAS3), but not OAS1. WT levels of replication of CpG- and UpA-high mutants were observed in OAS3 k/o cells despite abundant expression of ZAP, indicative of synergy or complementation of these hitherto unconnected pathways. The dependence on expression of ZAP, OAS3 and RNAseL for CpG/UpA-mediated attenuation and the variable and often low level expression of these pathway proteins in certain cell types, such as those of the central nervous system, has implications for the use of CpG-elevated mutants as attenuated live vaccines against neurotropic viruses.

SUBMITTER: Odon V

PROVIDER: S-EPMC6735852 | biostudies-other | 2019 Sep

REPOSITORIES: biostudies-other

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The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides.

Odon Valerie V Fros Jelke J JJ Goonawardane Niluka N Dietrich Isabelle I Ibrahim Ahmad A Alshaikhahmed Kinda K Nguyen Dung D Simmonds Peter P

Nucleic acids research 20190901 15

Zinc finger antiviral protein (ZAP) is a powerful restriction factor for viruses with elevated CpG dinucleotide frequencies. We report that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides. Attenuation of both CpG- and UpA-high viruses and replicon mutants was reversed in ZAP k/o cell lines, and restored by plasmid-derived reconstitution of expression in k/o cells. In pull-down assays, ZAP bound to viral RNA transcripts ...[more]

PMID: 31276592

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Project description:BackgroundLoss of CpG dinucleotides in genomic DNA through methylation-induced mutation is characteristic of vertebrates and plants. However, these and other eukaryotic phyla show a range of other dinucleotide frequency biases with currently uncharacterized underlying mutational or selection mechanisms. We developed a parameterized Markov process to identify what neighbour context-dependent mutations best accounted for patterns of dinucleotide frequency biases in genomic and cytoplasmically expressed mRNA sequences of different vertebrates, other eukaryotic groups and RNA viruses that infect them.ResultsConsistently, 11- to 14-fold greater frequencies of the methylation-associated mutation of C to T upstream of G (depicted as C?T,G) than other transitions best modelled dinucleotide frequencies in mammalian genomic DNA. However, further mutations such as G?T,T (5-fold greater than the default transversion rate) were required to account for the full spectrum of dinucleotide frequencies in mammalian sequence datasets. Consistent with modeling predictions for these two mutations, instability of both CpG and CpT dinucleotides was identified through SNP frequency analysis of human DNA sequences. Different sets of context-dependent mutations were modelled in other eukaryotes with non-methylated genomic DNA. In contrast to genomic DNA, best-fit models of dinucleotide frequencies in transcribed RNA sequences expressed in the cytoplasm from all organisms were dominated by mutations that eliminated UpA dinucleotides, observations consistent with cytoplasmically driven selection for mRNA stability. Surprisingly, mRNA sequences from organisms with methylated genomes showed evidence for additional selection against CpG through further context-dependent mutations (eg. C?A,G). Similar mutation or selection processes were identified among single-stranded mammalian RNA viruses; these potentially account for their previously described but unexplained under-representations of CpG and UpA dinucleotides.ConclusionsMethods we have developed identify mutational processes and selection pressures in organisms that provide new insights into nucleotide compositional constraints and a wealth of biochemical and evolutionarily testable predictions for the future.

Project description:The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with specific suppression of CpG motifs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. We show that acute virus replication is mutually exclusive with high levels of cellular ZAP, potentially explaining the higher levels of CpG in viral genes expressed subsequent to IE1 due to the loss of pressure from ZAP in infected cells. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early 1 transcript. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.

Project description:CpG dinucleotides are suppressed in the genomes of many vertebrate RNA viruses, including HIV-1. The cellular antiviral protein ZAP (zinc finger antiviral protein) binds CpGs and inhibits HIV-1 replication when CpGs are introduced into the viral genome. However, it is not known if ZAP-mediated restriction is the only mechanism driving CpG suppression. To determine how CpG dinucleotides affect HIV-1 replication, we increased their abundance in multiple regions of the viral genome and analyzed the effect on RNA expression, protein abundance, and infectious-virus production. We found that the antiviral effect of CpGs was not correlated with their abundance. Interestingly, CpGs inserted into some regions of the genome sensitize the virus to ZAP antiviral activity more efficiently than insertions into other regions, and this sensitivity can be modulated by interferon treatment or ZAP overexpression. Furthermore, the sensitivity of the virus to endogenous ZAP was correlated with its sensitivity to the ZAP cofactor KHNYN. Finally, we show that CpGs in some contexts can also inhibit HIV-1 replication by ZAP-independent mechanisms, and one of these is the activation of a cryptic splice site at the expense of a canonical splice site. Overall, we show that the location and sequence context of the CpG in the viral genome determines its antiviral activity.IMPORTANCE Some RNA virus genomes are suppressed in the nucleotide combination of a cytosine followed by a guanosine (CpG), indicating that they are detrimental to the virus. The antiviral protein ZAP binds viral RNA containing CpGs and prevents the virus from multiplying. However, it remains unknown how the number and position of CpGs in viral genomes affect restriction by ZAP and whether CpGs have other antiviral mechanisms. Importantly, manipulating the CpG content in viral genomes could help create new vaccines. HIV-1 shows marked CpG suppression, and by introducing CpGs into its genome, we show that ZAP efficiently targets a specific region of the viral genome, that the number of CpGs does not predict the magnitude of antiviral activity, and that CpGs can inhibit HIV-1 gene expression through a ZAP-independent mechanism. Overall, the position of CpGs in the HIV-1 genome determines the magnitude and mechanism through which they inhibit the virus.

Dataset Information

The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides.

Publications

The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides.

Similar Datasets

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