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; Fros, Jelke J.; Goonawardane, Niluka; Dietrich, Isabelle; Ibrahim, Ahmad; Alshaikhahmed, Kinda; Nguyen, Dung; Simmonds, Peter

doi:10.1093/nar/gkz581

Cited by 77 publications

(168 citation statements)

References 82 publications

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“…CpGs directly or indirectly introduced into coding and noncoding regions of picornaviruses have shown that they can potently attenuate viral replication and create strains that protect animals from challenge with the wild-type virus (6-8, 10, 68). ZAP has recently been shown to be necessary for introduced CpGs to inhibit the picornavirus echovirus 7 (32). CpGs have also been shown to attenuate influenza A virus and to protect animals from lethal challenge by the wild-type virus (9).…”

Section: Discussionmentioning

confidence: 99%

CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms

Ficarelli

Antzin-Anduetza

Hugh-White

et al. 2020

J Virol

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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.

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Section: Discussionmentioning

confidence: 99%

CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms

Ficarelli

Antzin-Anduetza

Hugh-White

et al. 2020

J Virol

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“…Interestingly, both ZAPS and TRIM25 inhibited HCMV virus production in an IRF3 independent manner, indicating they may play a role in directly inhibiting the virus. ZAPS and TRIM25 have recently been identified as host factors responsible for inhibiting HIV-1, Echo-7 and influenza virus constructs with synthetically increased CpG levels 9,11,20 . The two co-factors have also been reported to have antiviral activity against multiple viruses 47–51 .…”

Section: Resultsmentioning

confidence: 99%

“…The fact that CpG dinucleotide patterns are so uniform across the virus sub-families also suggests a central underlying biological relevance. For example, alpha-herpesviruses are mainly associated with latent infections in neuronal cells where the interferon response may be limited due to immune privilege and constitutive expression levels of ZAP are low 20 , whereas beta-herpesviruses and gamma-herpesviruses are mainly associated with latency in haematopoietic cells and are therefore under greater pressure to evade host cell IFN responses 58 . Alternatively, alpha-herpesviruses may have evolved a robust mechanism that directly counteracts ZAP antiviral activity, making it unnecessary to repress CpG dinucleotides.…”

Section: Discussionmentioning

confidence: 99%

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Human cytomegalovirus evades ZAP detection by suppressing CpG dinucleotides in the major immediate early genes

Lee

Chiweshe

McCormick

et al. 2020

Preprint

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20The genomes of RNA and small DNA viruses of vertebrates display significant suppression of 21 CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in 22 substantial attenuation of virus replication, suggesting that these compositional changes may 23 facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein 24 ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct 25 binding and possibly downstream targeting for degradation. Using an arrayed interferon 26 stimulated gene expression library screen, we identified ZAPS, and its associated factor 27 TRIM25, as direct inhibitors of human cytomegalovirus (HCMV) replication. Exogenous 28 expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown 29 resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of 30 CpG representation with a specific suppression of CpGs within the IE1 major immediate early 31 transcript which is absent in subsequently expressed genes. We demonstrated that 32 suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. 33During HCMV infection, expression of ZAP and TRIM25 are rapidly reduced, removing 34 pressure to suppress dinucleotide frequencies in viral genes expressed after the immediate 35 early genes, while acute virus replication and high levels of ZAP are mutually exclusive. 36Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long 37 65 would be expected given their overall base composition. Lower CpG frequencies are thought 66 to have arisen through deamination of methylated cytosines in nuclear DNA, resulting in CpG 67 sequences mutating to TpG 10 . RNA and small DNA viruses of vertebrates have evolved a 68 similar pattern of suppressed CpG dinucleotides and artificially increasing dinucleotide 69 frequencies within their viral genomes through synonymous mutations results in considerable 70 attenuation of virus replication 6-9 . 72A recent study identified the short form of the zinc-finger antiviral protein (ZAP) and its 73 associated factor TRIM25 as responsible for recognition of high CpG frequencies in viral RNA 74 3 11 . Mammalian ZAP is expressed in two major isoforms, ZAPS (short) and ZAPL (long) which 75 are generated by differential splicing, with ZAPL encoding an additional catalytically inactive 76 poly (ADP-ribose) polymerase (PARP) domain 12 . ZAP had previously been identified as a 77 host antiviral factor and is capable of binding to viral RNA through a pocket created by the 78 second of four zinc fingers within the RNA binding domain [13][14][15] . TRIM25 is an E3 ubiquitin 79 ligase and member of the tripartite motif (TRIM) family, many of which have been associated 80 with antiviral functions 16,17 . TRIM25 is required for ZAPS antiviral activity, although the precise 81 mechanism by which TRIM25 contributes to ZAPS antiviral activity is not fully understood 18,19 . 82A focused...

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“…It is increasingly recognised that the nucleotide composition of the genomes of viruses has a profound influence of its subsequent interaction with the cell on infection. Elevated frequencies of the CpG dinucleotide in RNA virus genomes or in mRNAs expressed during replication substantially modify replication kinetics and gene expression in mammalian viruses, including enteroviruses (1) and other RNA viruses (2)(3)(4)(5), HIV-1 (6)(7)(8) and other retroviruses (9) and hepatitis B virus (10) through their interactions with zinc finger antiviral protein (ZAP). ZAP has been shown to specifically bind to high CpG containing RNA sequences (1,8) and activate a range of RNA degradation pathways or induce translational arrest.…”

Section: Introductionmentioning

confidence: 99%

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

Goonawardane

Nguyen

Simmonds

2020

Preprint

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Previous 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 CpGenriched 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.Importance. We 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.

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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

Cited by 77 publications

References 82 publications

CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms

CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms

Human cytomegalovirus evades ZAP detection by suppressing CpG dinucleotides in the major immediate early genes

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

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