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

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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Aedes anphevirus (AeAV): an insect-specific virus distributed worldwide inAedes aegyptimosquitoes that has complex interplays withWolbachiaand dengue virus infection in cells

10.1101/335067 ◽

2018 ◽

Cited By ~ 2

Author(s):

Rhys Parry ◽

Sassan Asgari

Keyword(s):

Dengue Virus ◽

Cell Lines ◽

Rna Viruses ◽

Rna Virus ◽

Asia Pacific ◽

Mammalian Cell Lines ◽

Persistent Virus ◽

Specific Virus ◽

Laboratory Colonies

AbstractInsect specific viruses (ISVs) of the yellow fever mosquitoAedes aegyptihave been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome ofAe. aegypti, however, remains poorly understood. In this study, we characterised Aedes anphevirus (AeAV), a negative-sense RNA virus from the orderMononegavirales. AeAV identified fromAedescell lines were infectious to bothAe. aegyptiandAedes albopictuscells, but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available RNA-Seq data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as theAe. aegyptimosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American and Asia-Pacific lineages. The endosymbiotic bacteriumWolbachia pipientisrestricts positive-sense RNA viruses inAe. aegypti. Re-analysis of a small RNA library ofAe. aegypticells co-infected with AeAV andWolbachiaproduces an abundant RNAi response consistent with persistent virus replication. We foundWolbachiaenhances replication of AeAV when compared to a tetracycline cleared cell line, and AeAV modestly reduces DENV replicationin vitro. The results from our study improve understanding of the diversity and evolution of the virome ofAe. aegyptiand adds to previous evidence that showsWolbachiadoes not restrict a range of negative strand RNA viruses.

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

Journal of Virology ◽

10.1128/jvi.01337-19 ◽

2019 ◽

Vol 94 (6) ◽

Cited By ~ 11

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.

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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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Activation of viral transcription by stepwise largescale folding of an RNA virus genome

Nucleic Acids Research ◽

10.1093/nar/gkaa675 ◽

2020 ◽

Vol 48 (16) ◽

pp. 9285-9300

Author(s):

Tamari Chkuaseli ◽

K Andrew White

Keyword(s):

Rna Structure ◽

Rna Viruses ◽

Rna Virus ◽

Initial Step ◽

Binding Pocket ◽

Regulatory Elements ◽

Virus Genome ◽

Genomic Rna ◽

Stem Loop ◽

Rna Complex

Abstract The genomes of RNA viruses contain regulatory elements of varying complexity. Many plus-strand RNA viruses employ largescale intra-genomic RNA-RNA interactions as a means to control viral processes. Here, we describe an elaborate RNA structure formed by multiple distant regions in a tombusvirus genome that activates transcription of a viral subgenomic mRNA. The initial step in assembly of this intramolecular RNA complex involves the folding of a large viral RNA domain, which generates a discontinuous binding pocket. Next, a distally-located protracted stem-loop RNA structure docks, via base-pairing, into the binding site and acts as a linchpin that stabilizes the RNA complex and activates transcription. A multi-step RNA folding pathway is proposed in which rate-limiting steps contribute to a delay in transcription of the capsid protein-encoding viral subgenomic mRNA. This study provides an exceptional example of the complexity of genome-scale viral regulation and offers new insights into the assembly schemes utilized by large intra-genomic RNA structures.

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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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Plant Virus Genome Is Shaped by Specific Dinucleotide Restrictions That Influence Viral Infection

mBio ◽

10.1128/mbio.02818-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Alfonso González de Prádena ◽

Adrián Sánchez Jimenez ◽

David San León ◽

Peter Simmonds ◽

Juan Antonio García ◽

...

Keyword(s):

Rna Polymerase Ii ◽

High Throughput Sequencing ◽

Rna Viruses ◽

Rna Virus ◽

Virus Genome ◽

Viral Fitness ◽

Plum Pox Virus ◽

Dependent Manner ◽

Viral Restriction ◽

Rna Accumulation

ABSTRACT The presence of CpG and UpA dinucleotides is restricted in the genomes of animal RNA viruses to avoid specific host defenses. We wondered whether a similar phenomenon exists in nonanimal RNA viruses. Here, we show that these two dinucleotides, especially UpA, are underrepresented in the family Potyviridae, the most important group of plant RNA viruses. Using plum pox virus (PPV; Potyviridae family) as a model, we show that an increase in UpA frequency strongly diminishes virus accumulation. Remarkably, unlike previous observations in animal viruses, PPV variants harboring CpG-rich fragments display just faint (or no) attenuation. The anticorrelation between UpA frequency and viral fitness additionally demonstrates the relevance of this particular dinucleotide: UpA-high mutants are attenuated in a dose-dependent manner, whereas a UpA-low variant displays better fitness than its parental control. Using high-throughput sequencing, we also show that UpA-rich PPV variants are genetically stable, without apparent changes in sequence that revert and/or compensate for the dinucleotide modification despite its attenuation. In addition, we also demonstrate here that the PPV restriction of UpA-rich variants works independently of the classical RNA silencing pathway. Finally, we show that the anticorrelation between UpA frequency and RNA accumulation applies to mRNA-like fragments produced by the host RNA polymerase II. Together, our results inform us about a dinucleotide-based system in plant cells that controls diverse RNAs, including RNA viruses. IMPORTANCE Dinucleotides (combinations of two consecutive nucleotides) are not randomly present in RNA viruses; in fact, the presence of CpG and UpA is significantly repressed in their genomes. Although the meaning of this phenomenon remains obscure, recent studies with animal-infecting viruses have revealed that their low CpG/UpA frequency prevents virus restriction via a host antiviral system that recognizes, and promotes the degradation of, CpG/UpA-rich RNAs. Whether similar systems act in organisms from other life kingdoms has been unknown. To fill this gap in our knowledge, we built several synthetic variants of a plant RNA virus with deoptimized dinucleotide frequencies and analyzed their viral fitness and genome adaptation. In brief, our results inform us for the first time about an effective dinucleotide-based system that acts in plants against viruses. Remarkably, this viral restriction in plants is reminiscent of, but not identical to, the equivalent antiviral response in animals.

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Evolutionary origins of epidemic potential among human RNA viruses

10.1101/771394 ◽

2019 ◽

Author(s):

Lu Lu ◽

Liam Brierley ◽

Gail Robertson ◽

Feifei Zhang ◽

Samantha Lycett ◽

...

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Virus Genome ◽

Human Populations ◽

Infective Virus ◽

Genome Sequences ◽

Evolutionary Origins ◽

Virus Diversity ◽

Human Viruses ◽

Do So

AbstractTo have epidemic potential, a pathogen must be able to spread in human populations, but of human-infective RNA viruses only a minority can do so. We investigated the evolution of human transmissibility through parallel analyses of 1755 virus genome sequences from 39 RNA virus genera. We identified 57 lineages containing human-transmissible species and estimated that at least 74% of these lineages have evolved directly from non-human viruses in other mammals or birds, a public health threat recently designated “Disease X”. Human-transmissible viruses rarely evolve from virus lineages that can infect but not transmit between humans. This result cautions against focussing surveillance and mitigation efforts narrowly on currently known human-infective virus lineages and supports calls for a better understanding of RNA virus diversity in non-human hosts.

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Direct RNA Sequencing of the Complete Influenza A Virus Genome

10.1101/300384 ◽

2018 ◽

Cited By ~ 4

Author(s):

Matthew W. Keller ◽

Benjamin L. Rambo-Martin ◽

Malania M. Wilson ◽

Callie A. Ridenour ◽

Samuel S. Shepard ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Splice Variants ◽

Rna Virus ◽

Allantoic Fluid ◽

Virus Genome ◽

Chemical Degradation ◽

Original Form ◽

Sequencing Platform ◽

Oxford Nanopore

ABSTRACTFor the first time, a complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabelled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method and total RNA extracted from the allantoic fluid of infected chicken eggs, we demonstrate successful sequencing of the complete influenza virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza. By utilizing the same methodology we can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle. This has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.

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Quantitative analysis of cellular proteome alterations in human influenza A virus-infected mammalian cell lines

PROTEOMICS ◽

10.1002/pmic.200800893 ◽

2009 ◽

Vol 9 (12) ◽

pp. 3316-3327 ◽

Cited By ~ 72

Author(s):

Diana Vester ◽

Erdmann Rapp ◽

Dörte Gade ◽

Yvonne Genzel ◽

Udo Reichl

Keyword(s):

Quantitative Analysis ◽

Influenza A Virus ◽

Cell Lines ◽

Mammalian Cell ◽

Influenza A ◽

Human Influenza ◽

Mammalian Cell Lines

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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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