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Multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity.

ABSTRACT: The zinc finger antiviral protein (ZAP) is a host factor that mediates inhibition of viruses in the Filoviridae, Retroviridae and Togaviridae families. We previously demonstrated that ZAP blocks replication of Sindbis virus (SINV), the prototype Alphavirus in the Togaviridae family at an early step prior to translation of the incoming genome and that synergy between ZAP and one or more interferon stimulated genes (ISGs) resulted in maximal inhibitory activity. The present study aimed to identify those ISGs that synergize with ZAP to mediate Alphavirus inhibition. Using a library of lentiviruses individually expressing more than 350 ISGs, we screened for inhibitory activity in interferon defective cells with or without ZAP overexpression. Confirmatory tests of the 23 ISGs demonstrating the largest infection reduction in combination with ZAP revealed that 16 were synergistic. Confirmatory tests of all potentially synergistic ISGs revealed 15 additional ISGs with a statistically significant synergistic effect in combination with ZAP. These 31 ISGs are candidates for further mechanistic studies. The number and diversity of the identified ZAP-synergistic ISGs lead us to speculate that ZAP may play an important role in priming the cell for optimal ISG function.

SUBMITTER: Karki S

PROVIDER: S-EPMC3353916 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Transcription Factor Promyelocytic Leukemia Zinc Finger Protein regulates Interferon Stimulated Gene expression

Project description:This SuperSeries is composed of the following subset Series: GSE15193: IFN-alpha Stimulated Gene Expression in Sensitive and Resistant Renal Cell Carcinoma Cell Lines GSE16196: Differential IFNa-stimulated gene expression profiles in PLZF-inducible U937T monocyte cells Refer to individual Series

2010-05-18 | E-GEOD-16197 | biostudies-arrayexpress

Transcription Factor Promyelocytic Leukemia Zinc Finger Protein regulates Interferon Stimulated Gene expression

Project description:This SuperSeries is composed of the SubSeries listed below.

2009-05-22 | GSE16197 | GEO

Zinc-finger antiviral protein inhibits XMRV infection.

Project description:BACKGROUND: The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including Moloney murine leukemia virus (MoMLV), HIV-1, and certain alphaviruses and filoviruses. ZAP binds to specific viral mRNAs and recruits cellular mRNA degradation machinery to degrade the target RNA. The common features of ZAP-responsive RNA sequences remain elusive and thus whether a virus is susceptible to ZAP can only be determined experimentally. Xenotropic murine leukemia virus-related virus (XMRV) is a recently identified ?-retrovirus that was originally thought to be involved in prostate cancer and chronic fatigue syndrome but recently proved to be a laboratory artefact. Nonetheless, XMRV as a new retrovirus has been extensively studied. Since XMRV and MoMLV share only 67.9% sequence identity in the 3'UTRs, which is the target sequence of ZAP in MoMLV, whether XMRV is susceptible to ZAP remains to be determined. FINDINGS: We constructed an XMRV-luc vector, in which the coding sequences of Gag-Pol and part of Env were replaced with luciferase-coding sequence. Overexpression of ZAP potently inhibited the expression of XMRV-luc in a ZAP expression-level-dependent manner, while downregulation of endogenous ZAP rendered cells more sensitive to infection. Furthermore, ZAP inhibited the spreading of replication-competent XMRV. Consistent with the previously reported mechanisms by which ZAP inhibits viral infection, ZAP significantly inhibited the accumulation of XMRV-luc mRNA in the cytoplasm. The ZAP-responsive element in XMRV mRNA was mapped to the 3'UTR. CONCLUSIONS: ZAP inhibits XMRV replication by preventing the accumulation of viral mRNA in the cytoplasm. Documentation of ZAP inhibiting XMRV helps to broaden the spectrum of ZAP's antiviral activity. Comparison of the target sequences of ZAP in XMRV and MoMLV helps to better understand the features of ZAP-responsive elements.

| S-EPMC3376128 | biostudies-literature

The zinc finger antiviral protein acts synergistically with an interferon-induced factor for maximal activity against alphaviruses.

Project description:Type I interferons (IFNs) signal through specific receptors to mediate expression of genes, which together confer a cellular antiviral state. Overexpression of the zinc finger antiviral protein (ZAP) imparts a cellular antiviral state against Retroviridae, Togaviridae, and Filoviridae virus family members. Since ZAP expression is induced by IFN, we utilized Sindbis virus (SINV) to investigate the role of other IFN-induced factors in ZAP's inhibitory potential. Overexpressed ZAP did not inhibit virion production or SINV-induced cell death in BHK cells deficient in IFN production (and thus IFN signaling), suggesting a role for an IFN-induced factor in ZAP's activity. IFN pretreatment in the presence of ZAP resulted in greater inhibition than IFN alone. Using mouse embryo fibroblast (MEF) cells deficient in Stat1, we showed that signaling through the IFN receptor is necessary for IFN's enhancement of ZAP activity. Unlike in BHK cells, however, overexpressed ZAP exhibited antiviral activity in the absence of IFN. In wild-type MEFs with an intact Stat1 gene, IFN pretreatment synergized with ZAP to generate a potent antiviral response. Despite failing to inhibit SINV virion production and virus-induced cell death in BHK cells, ZAP inhibited translation of the incoming viral RNA. IFN pretreatment synergized with ZAP to further block protein expression from the incoming viral genome. We further show that silencing of IFN-induced ZAP reduces IFN efficacy. Our findings demonstrate that ZAP can synergize with another IFN-induced factor(s) for maximal antiviral activity and that ZAP's intrinsic antiviral activity on virion production and cell survival can have cell-type-specific outcomes.

| S-EPMC2168828 | biostudies-literature

TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein (ZAP).

Project description:The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP's antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP's ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.

| S-EPMC5245905 | biostudies-literature

Mutagenesis analysis of the zinc-finger antiviral protein.

Project description:BackgroundThe zinc-finger antiviral protein (ZAP) specifically inhibits the replication of certain viruses, including murine leukemia virus (MLV), by preventing the accumulation of viral mRNA in the cytoplasm. ZAP directly binds to the viral mRNA through the zinc-finger motifs and recruits the RNA exosome to degrade the target RNA. RNA helicase p72 is required for the optimal function of ZAP. In an attempt to understand the structure-function relationship of ZAP, we performed alanine scanning analysis.ResultsA series of ZAP mutants was generated, in which three consecutive amino acids were replaced with three alanines. The mutants were analyzed for their antiviral activities against pseudotyped MLV vector. Out of the nineteen mutants analyzed, seven displayed significantly lower antiviral activities. Two mutations were in the very N-terminal domain, and five mutations were within or around the first and second zinc-finger motifs. These mutants were further analyzed for their abilities to bind to the target RNA, the exosome, and the RNA helicase p72. Mutants Nm3 and Nm63 lost the ability to bind to RNA. Mutants Nm 63 and Nm93 displayed compromised interaction with p72, while the binding of Nm133 to p72 was very modest. The interactions of all the mutants with the exosome were comparable to wild type ZAP.ConclusionsThe integrity of the very N-terminal domain and the first and second zinc-finger motifs appear to be required for ZAP's antiviral activity. Analyses of the mutants for their abilities to interact with the target RNA and RNA helicase p72 confirmed our previous results. The mutants that bind normally to the target RNA, the exosome, and the RNA helicase p72 may be useful tools for further understanding the mechanism underlying ZAP's antiviral activity.

| S-EPMC2847535 | biostudies-literature

Zinc Finger CCCH-Type Antiviral Protein 1 Restricts the Viral Replication by Positively Regulating Type I Interferon Response.

Project description:Zinc finger CCCH-type antiviral protein 1 (ZC3HAV1) is a host antiviral factor that can repress translation and promote degradation of specific viral mRNAs. In this study, we found that expression of ZC3HAV1 was significantly induced by infection with influenza A virus (IAV) and Sendai virus (Sev). It was shown that deficiency of IFNAR resulted in a dramatic decrease in the virus-induced expression of ZC3HAV1. Furthermore, transfection with poly(I:C) and treatment with interferon β (IFN-β) induced the ZC3HAV1 expression. Interference with the endogenous expression of ZC3HAV1 enhanced the replication of influenza virus by impairing the production of IFN-β and MxA, following the infection of influenza virus. In contrast, ectopic expression of ZC3HAV1 significantly restricted the replication of influenza virus by increasing the IFN-β expression. In addition, ZC3HAV1 also promoted the induction of tumor necrosis factor and interleukin 6. These results suggest that ZC3HAV1 is induced by IFN-β/IFNAR signaling during IAV and Sev infection and involved in positive regulation of IFN-dependent innate antiviral response.

| S-EPMC7456897 | biostudies-literature

Origin and evolution of the zinc finger antiviral protein.

Project description: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.

| S-EPMC8102003 | biostudies-literature

Inhibition of filovirus replication by the zinc finger antiviral protein.

Project description:The zinc finger antiviral protein (ZAP) was recently shown to inhibit Moloney murine leukemia virus and Sindbis virus replication. We tested whether ZAP also acts against Ebola virus (EBOV) and Marburg virus (MARV). Antiviral effects were observed after infection of cells expressing the N-terminal part of ZAP fused to the product of the zeocin resistance gene (NZAP-Zeo) as well as after infection of cells inducibly expressing full-length ZAP. EBOV was inhibited by up to 4 log units, whereas MARV was inhibited between 1 to 2 log units. The activity of ZAP was dependent on the integrity of the second and fourth zinc finger motif, as tested with cell lines expressing NZAP-Zeo mutants. Heterologous expression of EBOV- and MARV-specific sequences fused to a reporter gene suggest that ZAP specifically targets L gene sequences. The activity of NZAP-Zeo in this assay was also dependent on the integrity of the second and fourth zinc finger motif. Time-course experiments with infectious EBOV showed that ZAP reduces the level of L mRNA before the level of genomic or antigenomic RNA is affected. Transient expression of ZAP decreased the activity of an EBOV replicon system by up to 95%. This inhibitory effect could be partially compensated for by overexpression of L protein. In conclusion, the data demonstrate that ZAP exhibits antiviral activity against filoviruses, presumably by decreasing the level of viral mRNA.

| S-EPMC1865956 | biostudies-literature

The zinc finger antiviral protein directly binds to specific viral mRNAs through the CCCH zinc finger motifs.

Project description:The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. For this report, we mapped the viral sequences that are sensitive to ZAP inhibition. The viral sequences were cloned into a luciferase reporter and analyzed for the ability to mediate ZAP-dependent destabilization of the reporter. The sensitive sequence in MLV was mapped to the 3' long terminal repeat; the sensitive sequences in SIN were mapped to multiple fragments. The fragment of SIN that displayed the highest destabilizing activity was further analyzed by deletion mutagenesis for the minimal sequence that retained the activity. This led to the identification of a fragment of 653 nucleotides. Any further deletion of this fragment resulted in significantly lower activity. We provide evidence that ZAP directly binds to the active but not the inactive fragments. The CCCH zinc finger motifs of ZAP play important roles in RNA binding and antiviral activity. Disruption of the second and fourth zinc fingers abolished ZAP's activity, whereas disruption of the first and third fingers just slightly lowered its activity.

| S-EPMC525010 | biostudies-other

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