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 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 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.

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 Regulation of KHNYN antiviral activity by the extended di-KH domain and nucleo-cytoplasmic trafficking

2021 ◽  
Author(s):  
María José Lista ◽  
Rui Pedro Galão ◽  
Mattia Ficarelli ◽  
Dorota Kmiec ◽  
Harry Wilson ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Nuclear Export ◽  
Rna Binding ◽  
Nuclear Export Signal ◽  
Antiviral Protein ◽  
Similar Time ◽  
Cpg Dinucleotides ◽  
C Terminus ◽  
Kh Domain ◽  
Retroviral Replication

The zinc finger antiviral protein (ZAP) restricts a broad range of viruses by binding CpG dinucleotides in viral RNA to target it for degradation and inhibit its translation. KHNYN was recently identified as an antiviral protein required for ZAP to inhibit retroviral replication, though little is known about its functional determinants. KHNYN contains an N-terminal extended di-KH-like domain, a PIN endoribonuclease domain and a C-terminal CUBAN domain that binds NEDD8 and ubiquitin. We show that deletion of the extended di-KH domain reduces its antiviral activity. However, despite its similarity to RNA binding KH domains, the extended di-KH domain in KHNYN does not appear to bind RNA. Mutation of residues in the CUBAN domain that bind NEDD8 increase KHNYN abundance but do not alter its antiviral activity, suggesting that this interaction regulates KHNYN homeostatic turnover. In contrast, a CRM1-dependent nuclear export signal (NES) at the C-terminus of the CUBAN domain is required for antiviral activity. Deletion of this signal retains KHNYN in the nucleus and inhibits its interaction with ZAP. Interestingly, this NES appeared in the KHNYN lineage at a similar time as when ZAP evolved in tetrapods, indicating that these proteins may have co-evolved to restrict viral replication.

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.

Sign in / Sign up

Export Citation Format

Share Document