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

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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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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Origin and evolution of the Zinc Finger Antiviral Protein

10.1101/2020.12.04.412510 ◽

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.

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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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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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Identification of a Dominant Negative Inhibitor of Human Zinc Finger Antiviral Protein Reveals a Functional Endogenous Pool and Critical Homotypic Interactions

Journal of Virology ◽

10.1128/jvi.02018-09 ◽

2010 ◽

Vol 84 (9) ◽

pp. 4504-4512 ◽

Cited By ~ 18

Author(s):

Lok Man J. Law ◽

Owen R. Albin ◽

John-William N. Carroll ◽

Christopher T. Jones ◽

Charles M. Rice ◽

...

Keyword(s):

Antiviral Activity ◽

Zinc Finger ◽

Viral Genome ◽

Sindbis Virus ◽

Mutational Analysis ◽

Dominant Negative ◽

Antiviral Protein ◽

Antiviral Function ◽

Dominant Negative Inhibitor ◽

In Cells

ABSTRACT The zinc finger antiviral protein (ZAP) is a host factor with potent antiviral activity when overexpressed in cells. ZAP blocks replication of the prototype alphavirus Sindbis virus (SINV) at a step at or before translation of the incoming viral genome. The mechanism of ZAP anti-SINV activity and the determinants of its antiviral function, however, have not been defined. Here, we have identified a dominant negative inhibitor of human ZAP. Rat ZAP with a cysteine-to-arginine mutation at position 88 (rZAPC88R), previously reported as a nonfunctional form of ZAP, increases SINV growth in cells. These results led us to discover a previously undetectable pool of endogenous functional ZAP within human cells. Investigation of the mechanism of dominant negative inhibition, combined with a comprehensive mutational analysis of the antiviral factor, revealed that homotypic associations are required for ZAP function in limiting SINV propagation.

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Zinc finger antiviral protein (ZAP) activity in mammalian and avian hosts in CpG and UpA-mediated restriction of RNA viruses and investigation of ZAP-mediated shaping of host transcriptome compositions

10.1101/2021.11.04.467232 ◽

2021 ◽

Author(s):

Valerie Odon ◽

Steven fiddaman ◽

Adrian Smith ◽

Peter Simmonds

Keyword(s):

Cell Lines ◽

Zinc Finger ◽

Influenza A ◽

Rna Viruses ◽

Rna Virus ◽

Virus Genome ◽

Antiviral Protein ◽

Cpg Dinucleotides ◽

Antiviral State ◽

Mammalian Cell Lines

The ability of zinc finger antiviral protein (ZAP) to recognise and respond to RNA virus sequences with elevated frequencies of CpG dinucleotides has been proposed as a functional part of the vertebrate innate immune antiviral response. It has been further proposed that ZAP activity shapes compositions of cytoplasmic mRNA sequences to avoid self-recognition, particularly mRNAs for interferons (IFNs) and IFN-stimulated genes highly expressed when ZAP is upregulated during the antiviral state. We investigated the ZAP functional activity in different species of mammals and birds, and potential downstream effects of differences in CpG and UpA dinucleotide representations in host transcriptomes and in RNA viruses that infect them. Cell lines from different bird orders showed variability in restriction of influenza A virus and echovirus 7 replicons with elevated CpG frequencies and none restricted UpA-high mutants, in marked contrast to mammalian cell lines. Given this variability, we compared CpG and UpA representation in coding regions of ISGs and IFNs with the total cellular transcriptome to determine whether differences in ZAP activity shaped dinucleotide compositions of highly expressed genes during the antiviral state. While type 1 IFN genes typically showed often profound suppression of CpG and UpA frequencies, there was no over-suppression of CpGs or UpAs in ISGs in any species, irrespective of underlying ZAP activity. Similarly, mammalian and avian RNA virus genome sequences were compositionally equivalent as were IAV serotypes recovered from ducks, chickens and humans. Overall, we found no evidence for host variability in ZAP function impacting compositions of antiviral genes.

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CpG Frequency in the 5′ Third of the env Gene Determines Sensitivity of Primary HIV-1 Strains to the Zinc-Finger Antiviral Protein

mBio ◽

10.1128/mbio.02903-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 10

Author(s):

Dorota Kmiec ◽

Rayhane Nchioua ◽

Scott Sherrill-Mix ◽

Christina M. Stürzel ◽

Elena Heusinger ◽

...

Keyword(s):

Zinc Finger ◽

Structural Features ◽

Clinical Progression ◽

Antiviral Protein ◽

Primate Lentiviruses ◽

Cpg Dinucleotide ◽

Specific Part ◽

Hiv 1 ◽

Cpg Suppression

ABSTRACT CpG dinucleotide suppression has been reported to allow HIV-1 to evade inhibition by the zinc-finger antiviral protein (ZAP). Here, we show that primate lentiviruses display marked differences in CpG frequencies across their genome, ranging from 0.44% in simian immunodeficiency virus SIVwrc from Western red colobus to 2.3% in SIVmon infecting mona monkeys. Moreover, functional analyses of a large panel of human and simian immunodeficiency viruses revealed that the magnitude of CpG suppression does not correlate with their susceptibility to ZAP. However, we found that the number of CpG dinucleotides within a region of ∼700 bases at the 5′ end of the env gene determines ZAP sensitivity of primary HIV-1 strains but not of HIV-2. Increased numbers of CpGs in this region were associated with reduced env mRNA expression and viral protein production. ZAP sensitivity profiles of chimeric simian-human immunodeficiency viruses (SHIVs) expressing different HIV-1 env genes were highly similar to those of the corresponding HIV-1 strains. The frequency of CpGs in the identified env region correlated with differences in clinical progression rates. Thus, the CpG frequency in a specific part of env, rather than the overall genomic CpG content, governs the susceptibility of HIV-1 to ZAP and might affect viral pathogenicity in vivo. IMPORTANCE Evasion of the zinc-finger antiviral protein (ZAP) may drive CpG dinucleotide suppression in HIV-1 and many other viral pathogens but the viral determinants of ZAP sensitivity are poorly defined. Here, we examined CpG suppression and ZAP sensitivity in a large number of primate lentiviruses and demonstrate that their genomic frequency of CpGs varies substantially and does not correlate with ZAP sensitivity. We further show that the number of CpG residues in a defined region at the 5′ end of the env gene together with structural features plays a key role in HIV-1 susceptibility to ZAP and correlates with differences in clinical progression rates in HIV-1-infected individuals. Our identification of a specific part of env as a major determinant of HIV-1 susceptibility to ZAP restriction provides a basis for future studies of the underlying inhibitory mechanisms and their potential relevance in the pathogenesis of AIDS.

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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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