scholarly journals Origin and evolution of the Zinc Finger Antiviral Protein

2020 ◽  
Author(s):  
Daniel Gonçalves-Carneiro ◽  
Matthew A. Takata ◽  
Heley Ong ◽  
Amanda Shilton ◽  
Paul D. Bieniasz
Keyword(s):  
Antiviral Activity ◽  
Zinc Finger ◽  
Phylogenetic Analyses ◽  
Viral Rna ◽  
Interspecies Transmission ◽  
Antiviral Protein ◽  
Ancestral Gene ◽  
Cpg Dinucleotides ◽  
Species Specific ◽  
Cpg Suppression

AbstractThe human zinc finger antiviral protein (ZAP) recognizes RNA by binding to CpG dinucleotides. Mammalian transcriptomes are CpG-poor, and ZAP may have evolved to exploit this feature to specifically target non-self viral RNA. Phylogenetic analyses reveal that ZAP and its paralogue PARP12 share an ancestral gene that arose prior to extensive eukaryote divergence, and the ZAP lineage diverged from the PARP12 lineage in tetrapods. Notably, The CpG content of modern eukaryote genomes varies widely, and ZAP-like genes arose subsequent to the emergence of CpG-suppression in vertebrates. Human PARP12 exhibited no antiviral activity against wild type and CpG-enriched HIV-1, but ZAP proteins from several tetrapods had antiviral activity when expressed human cells. In some cases, ZAP antiviral activity required a TRIM25 protein from the same or a related species, suggesting functional co-evolution of these genes. Indeed, a hypervariable sequence in the N-terminal domain of ZAP contributed to species-specific TRIM25 dependence in antiviral activity assays. Crosslinking immunoprecipitation coupled with RNA sequencing revealed that ZAP proteins from human, mouse, bat and alligator exhibit a high degree of CpG-specificity, while some avian ZAP proteins appear more promiscuous. Together, these data suggest that the CpG-rich RNA directed antiviral activity of ZAP-related proteins arose in tetrapods, subsequent to the onset of CpG suppression in certain eukaryote lineages, with subsequent species-specific adaptation of cofactor requirements and RNA target specificity.Author SummaryTo control viral infections, cells have evolved a variety of mechanisms that detect, modify and sometimes eliminate viral components. One of such mechanism is the Zinc Finger Antiviral Protein (ZAP) which binds RNA sequences that are rich in elements composed of a cytosine followed by a guanine. Selection of viral RNA can only be achieved because such elements are sparse in RNAs encoded by human genes. Here, we traced the molecular evolution of ZAP. We found that ZAP and a closely related gene, PARP12, originated from the same ancestral gene that existed in a predecessor of vertebrates and invertebrates. We found that ZAP proteins from mammals, birds and reptiles have antiviral activity but only in the presence of a co-factor, TRIM25, from the same species. ZAP proteins from birds were particularly interesting since they demonstrated a broader antiviral activity, primarily driven by relaxed requirement for cytosine-guanine. Our findings suggest that viruses that infect birds – which are important vectors for human diseases – are under differential selective pressures and this property may influence the outcome of interspecies transmission.

scholarly journals Origin and evolution of the zinc finger antiviral protein

2021 ◽  
Vol 17 (4) ◽  
pp. e1009545
Author(s):  
Daniel Gonçalves-Carneiro ◽  
Matthew A. Takata ◽  
Heley Ong ◽  
Amanda Shilton ◽  
Paul D. Bieniasz
Keyword(s):  
Antiviral Activity ◽  
Zinc Finger ◽  
Phylogenetic Analyses ◽  
Antiviral Protein ◽  
Ancestral Gene ◽  
Cpg Dinucleotides ◽  
Origin And Evolution ◽  
Species Specific ◽  
High Degree ◽  
Cpg Suppression

The human zinc finger antiviral protein (ZAP) recognizes RNA by binding to CpG dinucleotides. Mammalian transcriptomes are CpG-poor, and ZAP may have evolved to exploit this feature to specifically target non-self viral RNA. Phylogenetic analyses reveal that ZAP and its paralogue PARP12 share an ancestral gene that arose prior to extensive eukaryote divergence, and the ZAP lineage diverged from the PARP12 lineage in tetrapods. Notably, the CpG content of modern eukaryote genomes varies widely, and ZAP-like genes arose subsequent to the emergence of CpG-suppression in vertebrates. Human PARP12 exhibited no antiviral activity against wild type and CpG-enriched HIV-1, but ZAP proteins from several tetrapods had antiviral activity when expressed in human cells. In some cases, ZAP antiviral activity required a TRIM25 protein from the same or related species, suggesting functional co-evolution of these genes. Indeed, a hypervariable sequence in the N-terminal domain of ZAP contributed to species-specific TRIM25 dependence in antiviral activity assays. Crosslinking immunoprecipitation coupled with RNA sequencing revealed that ZAP proteins from human, mouse, bat and alligator exhibit a high degree of CpG-specificity, while some avian ZAP proteins appear more promiscuous. Together, these data suggest that the CpG- rich RNA directed antiviral activity of ZAP-related proteins arose in tetrapods, subsequent to the onset of CpG suppression in certain eukaryote lineages, with subsequent species-specific adaptation of cofactor requirements and RNA target specificity.

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

2019 ◽  
Vol 94 (6) ◽  
Author(s):  
Mattia Ficarelli ◽  
Irati Antzin-Anduetza ◽  
Rupert Hugh-White ◽  
Andrew E. Firth ◽  
Helin Sertkaya ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Zinc Finger ◽  
Splice Site ◽  
Viral Genome ◽  
Rna Virus ◽  
Antiviral Protein ◽  
Cpg Dinucleotides ◽  
Viral Genomes ◽  
Hiv 1 ◽  
Cpg Suppression

ABSTRACT 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.

scholarly journals The Zinc Finger Antiviral Protein restricts SARS-CoV-2

2020 ◽  
Author(s):  
Rayhane Nchioua ◽  
Dorota Kmiec ◽  
Janis Müller ◽  
Carina Conzelmann ◽  
Rüdiger Groß ◽  
...  
Keyword(s):  
Zinc Finger ◽  
High Efficiency ◽  
Antiviral Effect ◽  
Lung Cells ◽  
Type I ◽  
List Type ◽  
Antiviral Protein ◽  
Cpg Dinucleotides ◽  
Human Lung Cells ◽  
Cpg Suppression

SUMMARYRecent evidence shows that the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly sensitive to interferons (IFNs). However, the underlying antiviral effectors remain to be defined. Here, we show that Zinc finger antiviral protein (ZAP) that specifically targets CpG dinucleotides in viral RNA sequences restricts SARS-CoV-2. We demonstrate that ZAP and its cofactors KHNYN and TRIM25 are expressed in human lung cells. Type I, II and III IFNs all strongly inhibited SARS-CoV-2 and further induced ZAP expression. Strikingly, SARS-CoV-2 and its closest relatives from bats show the strongest CpG suppression among all known human and bat coronaviruses, respectively. Nevertheless, knock-down of ZAP significantly increased SARS-CoV-2 production in lung cells, particularly upon treatment with IFN-α or IFN-γ. Thus, our results identify ZAP as an effector of the IFN response against SARS-CoV-2, although this pandemic pathogen may be preadapted to the low CpG environment in humans.HighlightsSARS-CoV-2 and its closest bat relatives show strong CpG suppressionIFN-β, -γ and -λ inhibit SARS-CoV-2 with high efficiencyZAP restricts SARS-CoV-2 and contributes to the antiviral effect of IFNs

scholarly journals Use of a small DNA virus model to investigate mechanisms of CpG dinucleotide-induced attenuation of virus replication

2020 ◽  
Vol 101 (11) ◽  
pp. 1202-1218
Author(s):  
Lisa Loew ◽  
Niluka Goonawardane ◽  
Jeremy Ratcliff ◽  
Dung Nguyen ◽  
Peter Simmonds
Keyword(s):  
Mutational Analysis ◽  
A549 Cells ◽  
Translation Efficiency ◽  
Antiviral Protein ◽  
Dna Virus ◽  
Dna Viruses ◽  
Cpg Dinucleotides ◽  
Functional Basis ◽  
Cpg Dinucleotide ◽  
Cpg Suppression

Suppression of the CpG dinucleotide is widespread in RNA viruses infecting vertebrates and plants, and in the genomes of retroviruses and small mammalian DNA viruses. The functional basis for CpG suppression in the latter was investigated through the construction of mutants of the parvovirus, minute virus of mice (MVM) with increased CpG or TpA dinucleotides in the VP gene. CpG-high mutants displayed extraordinary attenuation in A9 cells compared to wild-type MVM (>six logs), while TpA elevation showed no replication effect. Attenuation was independent of Toll-like receptor 9 and STING-mediated DNA recognition pathways and unrelated to effects on translation efficiency. While translation from codon-optimized VP RNA was enhanced in a cell-free assay, MVM containing this sequence was highly attenuated. Further mutational analysis indicated that this arose through its increased numbers of CpG dinucleotides (7→70) and separately from its increased G+C content (42.3→57.4 %), which independently attenuated replication. CpG-high viruses showed impaired NS mRNA expression by qPCR and reduced NS and particularly VP protein expression detected by immunofluorescence and replication in A549 cells, effects reversed in zinc antiviral protein (ZAP) knockout cells, even though nuclear relocalization of VP remained defective. The demonstrated functional basis for CpG suppression in MVM and potentially other small DNA viruses and the observed intolerance of CpGs in coding sequences, even after codon optimization, has implications for the use of small DNA virus vectors in gene therapy and immunization.

Zinc finger protein ZFP36L1 inhibits flavivirus infection by both 5′-3′ XRN1 and 3′-5′ RNA-exosome RNA decay pathways.

2021 ◽  
Author(s):  
Han Chiu ◽  
Hsin-Ping Chiu ◽  
Han-Pang Yu ◽  
Li-Hsiung Lin ◽  
Zih-Ping Chen ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Dengue Virus ◽  
Zinc Finger ◽  
Rna Binding ◽  
Zinc Finger Protein ◽  
Viral Rna ◽  
Rna Decay ◽  
Finger Protein ◽  
Zinc Finger Motifs ◽  
Rna Exosome

Zinc-finger protein 36, CCCH type-like 1 (ZFP36L1), containing tandem CCCH-type zinc-finger motifs with an RNA-binding property, plays an important role in cellular RNA metabolism mainly via RNA decay pathways. Recently, we demonstrated that human ZFP36L1 has potent antiviral activity against influenza A virus infection. However, its role in the host defense response against flaviviruses has not been addressed. Here, we demonstrate that ZFP36L1 functions as a host innate defender against flaviviruses, including Japanese encephalitis virus (JEV) and dengue virus (DENV). Overexpression of ZFP36L1 reduced JEV and DENV infection, and ZFP36L1 knockdown enhanced viral replication. ZFP36L1 destabilized the JEV genome by targeting and degrading viral RNA mediated by both 5′-3′ XRN1 and 3′-5′ RNA-exosome RNA decay pathways. Mutation in both zinc-finger motifs of ZFP36L1 disrupted RNA-binding and antiviral activity. Furthermore, the viral RNA sequences specifically recognized by ZFP36L1 were mapped to the 3'-untranslated region of the JEV genome with the AU-rich element (AUUUA) motif. We extend the function of ZFP36L1 to host antiviral defense by directly binding and destabilizing the viral genome via recruiting cellular mRNA decay machineries. Importance Cellular RNA-binding proteins are among the first lines of defense against various viruses, particularly RNA viruses. ZFP36L1 belongs to the CCCH-type zinc-finger protein family and has RNA-binding activity; it has been reported to directly bind to the AU-rich elements (AREs) of a subset of cellular mRNAs and then lead to mRNA decay by recruiting mRNA degrading enzymes. However, the antiviral potential of ZFP36L1 against flaviviruses has not yet been fully demonstrated. Here, we reveal the antiviral potential of human ZFP36L1 against Japanese encephalitis virus (JEV) and dengue virus (DENV). ZFP36L1 specifically targeted the ARE motif within viral RNA and triggered the degradation of viral RNA transcripts via cellular degrading enzymes, 5′-3′ XRN1 and 3′-5′ RNA exosome. These findings provide mechanistic insights into how human ZFP36L1 serves as a host antiviral factor to restrict flavivirus replication.

scholarly journals SARS-CoV-2 Is Restricted by Zinc Finger Antiviral Protein despite Preadaptation to the Low-CpG Environment in Humans

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Rayhane Nchioua ◽  
Dorota Kmiec ◽  
Janis A. Müller ◽  
Carina Conzelmann ◽  
Rüdiger Groß ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Human Lung ◽  
Lung Cells ◽  
Type I ◽  
Antiviral Protein ◽  
Viral Pathogen ◽  
Cpg Dinucleotides ◽  
Human Lung Cells ◽  
Ifn Γ ◽  
Horseshoe Bat

ABSTRACT Recent evidence shows that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is sensitive to interferons (IFNs). However, the most effective types of IFNs and the underlying antiviral effectors remain to be defined. Here, we show that zinc finger antiviral protein (ZAP), which preferentially targets CpG dinucleotides in viral RNA sequences, restricts SARS-CoV-2. We further demonstrate that ZAP and its cofactors KHNYN and TRIM25 are expressed in human lung cells. Type I, II, and III IFNs all strongly inhibited SARS-CoV-2 and further induced ZAP expression. Comprehensive sequence analyses revealed that SARS-CoV-2 and its closest relatives from horseshoe bats showed the strongest CpG suppression among all known human and bat coronaviruses, respectively. Nevertheless, endogenous ZAP expression restricted SARS-CoV-2 replication in human lung cells, particularly upon treatment with IFN-α or IFN-γ. Both the long and the short isoforms of human ZAP reduced SARS-CoV-2 RNA expression levels, but the former did so with greater efficiency. Finally, we show that the ability to restrict SARS-CoV-2 is conserved in ZAP orthologues of the reservoir bat and potential intermediate pangolin hosts of human coronaviruses. Altogether, our results show that ZAP is an important effector of the innate response against SARS-CoV-2, although this pandemic pathogen emerged from zoonosis of a coronavirus that was preadapted to the low-CpG environment in humans. IMPORTANCE Although interferons inhibit SARS-CoV-2 and have been evaluated for treatment of coronavirus disease 2019 (COVID-19), the most effective types and antiviral effectors remain to be defined. Here, we show that IFN-γ is particularly potent in restricting SARS-CoV-2 and in inducing expression of the antiviral factor ZAP in human lung cells. Knockdown experiments revealed that endogenous ZAP significantly restricts SARS-CoV-2. We further show that CpG dinucleotides which are specifically targeted by ZAP are strongly suppressed in the SARS-CoV-2 genome and that the two closest horseshoe bat relatives of SARS-CoV-2 show the lowest genomic CpG content of all coronavirus sequences available from this reservoir host. Nonetheless, both the short and long isoforms of human ZAP reduced SARS-CoV-2 RNA levels, and this activity was conserved in horseshoe bat and pangolin ZAP orthologues. Our findings indicating that type II interferon is particularly efficient against SARS-CoV-2 and that ZAP restricts this pandemic viral pathogen might promote the development of effective immune therapies against COVID-19.

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 Does the Zinc Finger Antiviral Protein (ZAP) Shape the Evolution of Herpesvirus Genomes?

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1857
Author(s):  
Yao-Tang Lin ◽  
Long-Fung Chau ◽  
Hannah Coutts ◽  
Matin Mahmoudi ◽  
Vayalena Drampa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Zinc Finger ◽  
Gc Content ◽  
Nucleotide Composition ◽  
Antiviral Protein ◽  
Dna Viruses ◽  
Cpg Dinucleotides ◽  
Host Restriction ◽  
Counter Measures ◽  
Cpg Dinucleotide

An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses, reducing their fitness, and ultimately minimising pathogenic effects. In turn, viruses have evolved sophisticated counter-measures that mediate evasion of host defence mechanisms. A key aspect of host defences is the ability to differentiate between self and non-self. Previous studies have demonstrated significant suppression of CpG and UpA dinucleotide frequencies in the coding regions of RNA and small DNA viruses. Artificially increasing these dinucleotide frequencies results in a substantial attenuation of virus replication, suggesting dinucleotide bias could facilitate recognition of non-self RNA. The interferon-inducible gene, zinc finger antiviral protein (ZAP) is the host factor responsible for sensing CpG dinucleotides in viral RNA and restricting RNA viruses through direct binding and degradation of the target RNA. Herpesviruses are large DNA viruses that comprise three subfamilies, alpha, beta and gamma, which display divergent CpG dinucleotide patterns within their genomes. ZAP has recently been shown to act as a host restriction factor against human cytomegalovirus (HCMV), a beta-herpesvirus, which in turn evades ZAP detection by suppressing CpG levels in the major immediate-early transcript IE1, one of the first genes expressed by the virus. While suppression of CpG dinucleotides allows evasion of ZAP targeting, synonymous changes in nucleotide composition that cause genome biases, such as low GC content, can cause inefficient gene expression, especially in unspliced transcripts. To maintain compact genomes, the majority of herpesvirus transcripts are unspliced. Here we discuss how the conflicting pressures of ZAP evasion, the need to maintain compact genomes through the use of unspliced transcripts and maintaining efficient gene expression may have shaped the evolution of herpesvirus genomes, leading to characteristic CpG dinucleotide patterns.

scholarly journals KHNYN is essential for ZAP-mediated restriction of HIV-1 containing clustered CpG dinucleotides

2019 ◽  
Author(s):  
Mattia Ficarelli ◽  
Harry Wilson ◽  
Rui Pedro Galão ◽  
Stuart J D Neil ◽  
Chad M Swanson
Keyword(s):  
Infectious Virus ◽  
Rna Virus ◽  
Virus Production ◽  
Innate Immune ◽  
Viral Rna ◽  
Antiviral Protein ◽  
Cpg Dinucleotides ◽  
Endonuclease Domain ◽  
Virus Genomes ◽  
Hiv 1

AbstractCpG dinucleotides are suppressed in most vertebrate RNA viruses, including HIV-1, and introducing CpGs into RNA virus genomes inhibits their replication. The zinc-finger antiviral protein (ZAP) binds regions of viral RNA containing CpGs and targets them for degradation. ZAP does not have enzymatic activity and recruits other cellular proteins to inhibit viral replication. Here we show that KHNYN, a protein with no previously known function, interacts with ZAP. KHNYN overexpression selectively inhibits HIV-1 containing clustered CpG dinucleotides and this requires ZAP and its cofactor TRIM25. KHNYN requires both its KH-like domain and NYN endonuclease domain for antiviral activity. Crucially, depletion of KHNYN eliminated the deleterious effect of CpG dinucleotides on HIV-1 RNA abundance and infectious virus production indicating that KHNYN is required for this antiviral pathway. Overall, we have identified KHNYN as a novel ZAP cofactor that is essential for innate immune destruction of CpG containing viral RNA.

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