Author response: KHNYN is essential for the zinc finger antiviral protein (ZAP) to restrict HIV-1 containing clustered CpG dinucleotides

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.

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Poly(ADP-ribose) potentiates ZAP antiviral activity

10.1101/2020.12.17.423219 ◽

2020 ◽

Author(s):

Guangai Xue ◽

Klaudia Braczyk ◽

Daniel Gonçalves-Carneiro ◽

Daria M. Dawidziak ◽

Katarzyna Zawada ◽

...

Keyword(s):

Zinc Finger ◽

Rna Binding ◽

Stress Granules ◽

Human Pathogens ◽

Antiviral Protein ◽

Cpg Dinucleotides ◽

Dna And Rna ◽

Cellular Compartments ◽

Hiv 1

AbstractZinc-finger antiviral protein (ZAP), also known as poly(ADP-ribose) polymerase 13 (PARP13), is an antiviral factor that selectively targets viral RNA for degradation. ZAP is active against both DNA and RNA viruses, including important human pathogens such as hepatitis B virus and type 1 human immunodeficiency virus (HIV-1). ZAP selectively binds CpG dinucleotides through its N-terminal RNA-binding domain, which consists of four zinc fingers. ZAP also contains a central region that consists of a fifth zinc finger and two WWE domains. Through structural and biochemical studies, we found that the fifth zinc finger and tandem WWEs of ZAP combine into a single integrated domain that binds to poly(ADP-ribose) (PAR), a cellular polynucleotide. PAR binding is mediated by the second WWE module of ZAP and likely involves specific recognition of iso(ADP-ribose), a repeating structural unit of PAR. Mutation of the putative iso(ADP-ribose) binding site in ZAP abrogates the interaction in vitro and diminishes ZAP activity against a CpG-rich HIV-1 reporter virus. In cells, PAR facilitates formation of non-membranous sub-cellular compartments such as DNA repair foci, spindle poles and cytosolic RNA stress granules. Our results suggest that ZAP-mediated viral mRNA degradation is facilitated by PAR, and provides a biophysical rationale for the reported association of ZAP with RNA stress granules.

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Decision letter: KHNYN is essential for the zinc finger antiviral protein (ZAP) to restrict HIV-1 containing clustered CpG dinucleotides

10.7554/elife.46767.021 ◽

2019 ◽

Author(s):

Michael Emerman ◽

Eric Freed

Keyword(s):

Zinc Finger ◽

Antiviral Protein ◽

Cpg Dinucleotides ◽

Hiv 1

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KHNYN is essential for the zinc finger antiviral protein (ZAP) to restrict HIV-1 containing clustered CpG dinucleotides

eLife ◽

10.7554/elife.46767 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 26

Author(s):

Mattia Ficarelli ◽

Harry Wilson ◽

Rui Pedro Galão ◽

Michela Mazzon ◽

Irati Antzin-Anduetza ◽

...

Keyword(s):

Zinc Finger ◽

Infectious Virus ◽

Rna Virus ◽

Leukemia Virus ◽

Virus Production ◽

Antiviral Protein ◽

Cpg Dinucleotides ◽

Endonuclease Domain ◽

Virus Genomes ◽

Hiv 1

CpG 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. We found 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 and also enhanced the production of murine leukemia virus. Overall, we have identified KHNYN as a novel cofactor for ZAP to target CpG-containing retroviral RNA for degradation.

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

mBio ◽

10.1128/mbio.01930-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 3

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.

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Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1101676108 ◽

2011 ◽

Vol 108 (38) ◽

pp. 15834-15839 ◽

Cited By ~ 143

Author(s):

Y. Zhu ◽

G. Chen ◽

F. Lv ◽

X. Wang ◽

X. Ji ◽

...

Keyword(s):

Zinc Finger ◽

Antiviral Protein ◽

Viral Mrnas ◽

Hiv 1

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

Viruses ◽

10.3390/v13091857 ◽

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.

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Origin and evolution of the zinc finger antiviral protein

PLoS Pathogens ◽

10.1371/journal.ppat.1009545 ◽

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.

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

10.1101/581785 ◽

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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Association of Zinc Finger Antiviral Protein Binding to Viral Genomic RNA with Attenuation of Replication of Echovirus 7

mSphere ◽

10.1128/msphere.01138-20 ◽

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.

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