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

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.     

ZAP is a CRM1-dependent nucleocytoplasmic shuttling protein

The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MMLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. In this report, we demonstrate that ZAP is predominantly localized in the cytoplasm at steady state but shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. Two nuclear localization sequences (NLS) and one nuclear export sequence (NES) were identified. One NLS was mapped to amino acids 68-RARVCRRK-75 and the other mapped to a region including amino acids K405 and K406. The NES was mapped to amino acids 284-LEDVSVDV-291. These findings help to understand why ZAP specifically prevents the accumulation of viral RNA in the cytoplasm. These findings also suggest possible functions of ZAP in the nucleus.     

Indoleamine 2,3-dioxygenase mediates the antiviral effect of gamma interferon against hepatitis B virus in human hepatocyte-derived cells

Alpha interferon (IFN-α) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-γ) is a key mediator of host innate and adaptive antiviral immunity against hepatitis B virus (HBV) infection in vivo. In an effort to elucidate the antiviral mechanism of these cytokines, 37 IFN-stimulated genes (ISGs), which are highly inducible in hepatocytes, were tested for their ability to inhibit HBV replication upon overexpression in human hepatoma cells. One ISG candidate, indoleamine 2,3-dioxygenase (IDO), an IFN-γ-induced enzyme catalyzing tryptophan degradation, efficiently reduced the level of intracellular HBV DNA without altering the steady-state level of viral RNA. Furthermore, expression of an enzymatically inactive IDO mutant did not inhibit HBV replication, and tryptophan supplementation in culture completely restored HBV replication in IDO-expressing cells, indicating that the antiviral effect elicited by IDO is mediated by tryptophan deprivation. Interestingly, IDO-mediated tryptophan deprivation preferentially inhibited viral protein translation and genome replication but did not significantly alter global cellular protein synthesis. Finally, tryptophan supplementation was able to completely restore HBV replication in IFN-γ- but not IFN-α-treated cells, which strongly argues that IDO is the primary mediator of IFN-γ-elicited antiviral response against HBV in human hepatocyte-derived cells.     

Glycogen synthase kinase 3β (GSK3β) modulates antiviral activity of zinc-finger antiviral protein (ZAP)

Zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including HIV-1, Ebola virus, and Sindbis virus. ZAP binds directly to specific viral mRNAs and recruits cellular mRNA degradation machinery to degrade the target RNA. ZAP has also been suggested to repress translation of the target mRNA. In this study, we report that ZAP is phosphorylated by glycogen synthase kinase 3β (GSK3β). GSK3β sequentially phosphorylated Ser-270, Ser-266, Ser-262, and Ser-257 of rat ZAP. Inhibition of GSK3β by inhibitor SB216763 or down-regulation of GSK3β by RNAi reduced the antiviral activity of ZAP. These results indicate that phosphorylation of ZAP by GSK3β modulates ZAP activity.     

Zinc Finger Antiviral Protein Inhibits Murine Gammaherpesvirus 68 M2 Expression and Regulates Viral Latency in Cultured Cells

Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to specific viral mRNAs and repressing mRNA expression. Here we report that ZAP inhibits expression of murine gammaherpesvirus 68 (MHV-68) M2, which plays important roles in establishment and maintenance of viral latency. Downregulation of endogenous ZAP in cells harboring latent MHV-68 promoted lytic replication of the virus. These results suggest that ZAP inhibits M2 expression and regulates the maintenance of MHV-68 latency.     

DEXH-Box protein DHX30 is required for optimal function of the zinc-finger antiviral protein

The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by eliminating viral mRNAs in the cytoplasm. In previous studies, we demonstrated that ZAP directly binds to the viral mRNAs and recruits the RNA exosome to degrade the target RNA. In this article, we provide evidence that a DEXH box RNA helicase, DHX30, is required for optimal antiviral activity of ZAP. Pull-down and co-immunoprecipitation assays demonstrated that DHX30 and ZAP interacted with each other via their N terminal domains. Downregulation of DHX30 with shRNAs reduced ZAP’s antiviral activity. These data implicate that DHX30 is a cellular factor involved in the antiviral function of ZAP.     

Activation of pattern recognition receptor-mediated innate immunity inhibits the replication of hepatitis B virus in human hepatocyte-derived cells

Recognition of virus infections by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I), and melanoma differentiation associated gene 5 (MDA5), activates signaling pathways, leading to the induction of inflammatory cytokines that limit viral replication. To determine the effects of PRR-mediated innate immune response on hepatitis B virus (HBV) replication, a 1.3mer HBV genome was cotransfected into HepG2 or Huh7 cells with plasmid expressing TLR adaptors, myeloid differentiation primary response gene 88 (MyD88), and TIR-domain-containing adaptor-inducing beta interferon (TRIF), or RIG-I/MDA5 adaptor, interferon promoter stimulator 1 (IPS-1). The results showed that expressing each of the three adaptors dramatically reduced the levels of HBV mRNA and DNA in both HepG2 and Huh7 cells. However, HBV replication was not significantly affected by treatment of HBV genome-transfected cells with culture media harvested from cells transfected with each of the three adaptors, indicating that the adaptor-induced antiviral response was predominantly mediated by intracellular factors rather than by secreted cytokines. Analyses of involved signaling pathways revealed that activation of NF-κB is required for all three adaptors to elicit antiviral response in both HepG2 and Huh7 cells. However, activation of interferon regulatory factor 3 is only essential for induction of antiviral response by IPS-1 in Huh7 cells, but not in HepG2 cells. Furthermore, our results suggest that besides NF-κB, additional signaling pathway(s) are required for TRIF to induce a maximum antiviral response against HBV. Knowing the molecular mechanisms by which PRR-mediated innate defense responses control HBV infections could potentially lead to the development of novel therapeutics that evoke the host cellular innate antiviral response to control HBV infections.     

Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.     

Expression and RNA-binding of human zinc-finger antiviral protein

Zinc-finger antiviral protein (ZAP) is a recently isolated host antiviral factor that inhibits the replication of many viruses such as Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral mRNA in the cytoplasm. ZAP comprises four CCCH zinc-finger motifs, the second and fourth of which are responsible for protein activity based on their integrity. Thus far, there have been no reports on whether or not ZAP expressed in Escherichia coli is soluble. Therefore, we expressed N-terminal ZAP (NZAP, 254 amino acids) in E. coli as a fusion protein with several different cleavage sites and protein tags. Cleaved ZAP in soluble form strongly bound to RNA through its four CCCH zinc-finger motifs. Here, we provide evidence indicating that ZAP directly interacted with viral RNA. Each conserved zinc-finger motif of ZAP coordinates a zinc ion using three cysteines and one histidine. These findings suggest that ZAP recruits the cellular RNA degradation machinery for the degradation of viral RNA.     

In Vitro Anti-hepatitis B Virus Activity of 2',3'-Dideoxyguanosine

2',3'-dideoxyguanosine(DoG) has been demonstrated to inhibit duck hepatitis B virus(DHBV) replication in vivo in a duck model of HBV infection. In the current study, the in vitro antiviral effects of DoG on human and animal hepadnaviruses were investigated. Our results showed that DoG effectively inhibited HBV, DHBV, and woodchuck hepatitis virus(WHV)replication in hepatocyte-derived cells in a dose-dependent manner, with 50% effective concentrations(EC50) of 0.3 ± 0.05, 6.82 ± 0.25, and 23.0 ± 1.5 lmol/L, respectively. Similar to other hepadnaviral DNA polymerase inhibitors,DoG did not alter the levels of intracellular viral RNA but induced the accumulation of a less-than-full-length viral RNA species, which was recently demonstrated to be generated by RNase H cleavage of pgRNA. Furthermore, using a transient transfection assay, DoG showed similar antiviral activity against HBV wild-type, 3TC-resistant rtA181 V, and adefovirresistant rtN236T mutants. Our results suggest that DoG has potential as a nucleoside analogue drug with anti-HBV activity.     

ZAP融合蛋白抑制HIV病毒模型的构建及功能分析

ZAP是一种抗病毒因子,能够特异性结合病毒RNA并招募细胞中的RNA酶降解所结合的靶RNA,从而抑制某些病毒的复制,如鼠白血病病毒(MLV)、辛德比斯病毒(SIN).ZAP对HIV病毒抑制作用并不明显.Tat和Rev是HIV编码的两种可以特异性结合HIVRNA的蛋白质,将它们与ZAP构建成融合蛋白,使得融合蛋白通过Tat或Rev结合HIVRNA并通过ZAP降解HIVRNA,从而抑制HIV假病毒载体携带基因的表达.这一结果为抑制HIV病毒提供了一个新思路,也支持了ZAP招募mRNA降解机器降解靶RNA的模型.     

PR65A 调控抗病毒蛋白ZAP的活性

锌指结构抗病毒蛋白(ZAP)能够特异性地识别病毒RNA并促进其特异性降解,从而抑制病毒的复制．ZAP不能独立地发挥抗病毒的功能,需要招募细胞内很多因子,包括外切酶复合体(exosome)、RNA解旋酶p72 等．通过蛋白质组学分析方法,我们找到了与ZAP可能存在相互作用的一些蛋白质并证实蛋白磷酸酶2调节亚基A的?琢亚型(PR65A)与ZAP之间存在着直接的相互作用．细胞内PR65A的表达水平,降低削弱ZAP的活性, 表明PR65A是ZAP发挥最佳抗病毒活性所必需的．这丰富了我们对ZAP抗病毒作用机理的认识,同时也为更好地利用其抗病毒活性提供了重要的指导．     

Effects of mutations within and adjacent to the terminal repeats of hepatitis B virus pregenomic RNA on viral DNA synthesis.

The viral polymerase and several cis-acting sequences are essential for hepadnaviral DNA replication, but additional host factors are likely to be involved in this process. We previously identified two sequences, UBS and DBS (upstream and downstream binding sites), present in multiple copies in and adjacent to the pregenomic RNA (pgRNA) terminal redundancy, that were specifically recognized by a 65-kDa host factor, p65. The possible roles of these two sequences in hepatitis B virus (HBV) replication were investigated in the context of the intact viral genome. UBS is contained within the terminal redundancy of pgRNA, and the 5' copy of this sequence is essential for viral replication. Mutations within the central core of UBS ablate p65 binding and selectively block synthesis of plus-strand DNA, without affecting RNA packaging or minus-strand synthesis. The DBS sequence, which is located downstream of the pgRNA polyadenylation site, overlaps the core (C) protein coding region. All mutations introduced into this site severely affected viral replication. However, these effects were shown to result from dominant negative effects of mutant core polypeptides rather than from cis-acting effects on RNA recognition. Thus, the 5' UBS but not DBS sites play important cis-acting roles in HBV DNA replication; however, the involvement of p65 in these roles remains a matter for investigation.     

ZAP Inhibits Murine Gammaherpesvirus 68 ORF64 Expression and Is Antagonized by RTA

Zinc finger antiviral protein (ZAP) is an interferon-inducible host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1 and Ebola virus. ZAP functions as a dimer formed through intermolecular interactions of its N-terminal tails. ZAP binds directly to specific viral mRNAs and inhibits their expression by repressing translation and/or promoting degradation of the target mRNA. ZAP is not a universal antiviral factor, since some viruses grow normally in ZAP-expressing cells. It is not fully understood what determines whether a virus is susceptible to ZAP. We explored the interaction between ZAP and murine gammaherpesvirus 68 (MHV-68), whose life cycle has latent and lytic phases. We previously reported that ZAP inhibits the expression of M2, which is expressed mainly in the latent phase, and regulates MHV-68 latency in cultured cells. Here, we report that ZAP inhibits the expression of ORF64, a tegument protein that is expressed in the lytic phase and is essential for lytic replication. MHV-68 infection induced ZAP expression. However, ZAP did not inhibit lytic replication of MHV-68. We provide evidence showing that the antiviral activity of ZAP is antagonized by MHV-68 RTA, a critical viral transactivator expressed in the lytic phase. We further show that RTA inhibits the antiviral activity of ZAP by disrupting the N-terminal intermolecular interaction of ZAP. Our results provide an example of how a virus can escape ZAP-mediated immunity.     

Inhibition of filovirus replication by the zinc finger antiviral protein

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.     

Poly(ADP-ribose) potentiates ZAP antiviral activity

Zinc-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 an adenosine diphosphate-containing unit of PAR. Mutation of the PAR binding site in ZAP abrogates the interaction in vitro and diminishes ZAP activity against a CpG-rich HIV-1 reporter virus and murine leukemia 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.     

Disruption of Type I Interferon Induction by HIV Infection of T Cells

Our main objective of this study was to determine how Human Immunodeficiency Virus (HIV) avoids induction of the antiviral Type I Interferon (IFN) system. To limit viral infection, the innate immune system produces important antiviral cytokines such as the IFN. IFN set up a critical roadblock to virus infection by limiting further replication of a virus. Usually, IFN production is induced by the recognition of viral nucleic acids by innate immune receptors and subsequent downstream signaling. However, the importance of IFN in the defense against viruses has lead most pathogenic viruses to evolve strategies to inhibit host IFN induction or responses allowing for increased pathogenicity and persistence of the virus. While the adaptive immune responses to HIV infection have been extensively studied, less is known about the balance between induction and inhibition of innate immune defenses, including the antiviral IFN response, by HIV infection. Here we show that HIV infection of T cells does not induce significant IFN production even IFN I Interferon production. To explain this paradox, we screened HIV proteins and found that two HIV encoded proteins, Vpu and Nef, strongly antagonize IFN induction, with expression of these proteins leading to loss of expression of the innate immune viral RNA sensing adaptor protein, IPS-1 (IFN-β promoter stimulator-1). We hypothesize that with lower levels of IPS-1 present, infected cells are defective in mounting antiviral responses allowing HIV to replicate without the normal antiviral actions of the host IFN response. Using cell lines as well as primary human derived cells, we show that HIV targeting of IPS-1 is key to limiting IFN induction. These findings describe how HIV infection modulates IFN induction providing insight into the mechanisms by which HIV establishes infection and persistence in a host.     

A host factor that binds near the termini of hepatitis B virus pregenomic RNA.

The terminal regions of hepatitis B virus (HBV) pregenomic RNA (pgRNA) harbors sites governing many essential functions in the viral life cycle, including polyadenylation, translation, RNA encapsidation, and DNA synthesis. We have examined the binding of host proteins to a 170-nucleotide region from the 5' end of HBV pgRNA; a large portion of this region is duplicated at the 3' end of this terminally redundant RNA. By UV cross-linking labeled RNA to HepG2 cell extracts, we have identified a 65-kDa factor (p65) of nuclear origin which can specifically bind to this region. Two discrete binding sites were identified within this region; in vitro cross-competition experiments suggest that the same factor binds to both elements. One binding site (termed UBS) overlaps a portion of the highly conserved stem-loop structure (epsilon), while the other site (termed DBS) maps 35 nucleotides downstream of the hexanucleotide polyadenylation sequence. Both binding sites are highly pyrimidine rich and map to regions previously found to be important in the regulation of viral polyadenylation. However, functional analysis of mutant binding sites in vivo indicates that p65 is not involved in the polyadenylation of HBV pgRNA. Potential roles for the factor in viral replication in vivo are discussed.     

S-farnesylation is essential for antiviral activity of the long ZAP isoform against RNA viruses with diverse replication strategies

The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNAs and, through the recruitment of TRIM25, KHNYN and other cofactors, target them for degradation or prevent their translation. The long and short isoforms of ZAP (ZAP-L and ZAP-S) have different intracellular localization and it is unclear how this regulates their antiviral activity against viruses with different sites of replication. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), which transcribe the viral RNA in the nucleus and assemble virions at the plasma membrane, we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain in ZAP-L is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the residues in place of the catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks antiviral activity for CpG-enriched HIV-1 despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together jointly modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-L antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, which replicates in double-membrane vesicles derived from the endoplasmic reticulum. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in ZAP-L-mediated antiviral activity against divergent viruses with different subcellular replication sites.