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The alphavirus nsP gene region contains the ZAP sensitivity determinant. (A) Schematics of chimeric viruses generated. The GFP construct is the same across all viruses. (B) ZAP KO 293T cells with doxycycline-inducible expression of ZAPS or ZAPL were infected with GFP-expressing SINV, ONNV, or ONNV nsP/SINV sP chimeric virus at MOI = 0.1 PFU/cell for 18 h before their percentage of infection was determined by flow cytometry. Fold inhibition by ZAP relative to doxycycline-untreated (-dox) cells is shown here. Error bars represent the SD. Data representative of two independent experiments performed with biological replicates in triplicate wells. Asterisks indicate statistically significant differences (two-way ANOVA and Tukey’s multiple comparisons test: **, p < 0.01; ****, p < 0.0001). ns—not significant.
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Certain re-emerging alphaviruses, such as chikungunya virus (CHIKV), cause serious disease and widespread epidemics. To develop virus-specific therapies, it is critical to understand the determinants of alphavirus pathogenesis and virulence. One major determinant is viral evasion of the host interferon response, which upregulates antiviral effector...
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... It also interacts with various cellular proteins that may act as cofactors, enhancing its antiviral effects [29]. Furthermore, ZAP can augment other antiviral systems [30,31]. Recent studies, including our own, have demonstrated that flaviviruses such as Zika virus, JEV, and WNV are susceptible to ZAP antiviral effects in VERO cells [30,32,33]. ...
... However, the increased number of virus-positive cells ( Figure 8E,G,I) and infectious titers ( Figure 8D,H,J) in ZAP-KO cells suggests that these cells may be more effective for rescuing unmodified and modified flaviviruses and potentially viruses from other families sensitive to ZAP antiviral activity. ZAP was first characterized to inhibit murine leukemia virus [40] and later alphaviruses, filoviruses, influenza virus, porcine reproductive and respiratory syndrome virus, hepatitis B virus, human cytomegalovirus, human T-cell leukaemia virus type 1, and human immunodeficiency virus-1 (HIV-1) [31,34,[41][42][43][44][45][46][47][48][49]. ZAP targets CpG-rich regions of RNA, and ZAP binding to viral RNA mediates its degradation and translational inhibition [28,34]. ...
Classical methods for constructing infectious cDNA clones of flaviviruses are often hindered by instability and toxicity. The Infectious‐Subgenomic‐Amplicons (ISA) method is an advancement which utilizes overlapping DNA fragments representing viral genomic sequence and in‐cell recombination to bypass bacterial plasmid assembly. However, the ISA method has limitations due to the toxicity of some ISA DNA fragments in bacteria during synthetic production. We validated modified ISA strategies for producing toxic ISA Japanese encephalitis virus (JEV) and West Nile virus (WNV) DNA fragments. Three approaches were explored, including subdividing toxic DNA fragments into two sub‐fragments for synthetic clonal production, using a low‐copy bacterial plasmid, and subdividing the toxic DNA fragments into four short overlapping sub‐fragments, each up to 1.8 kb. The latter novel approach in ISA applications enabled the synthesis of entirely bacteria‐free ISA DNA fragments. Our results demonstrate that subdividing toxic fragments into sub‐fragments smaller than 1.8 kb for synthesis is the efficient strategy, circumventing the need for bacterial plasmids and ensuring rapid production of synthetic flaviviruses. This method also shortens the production timeline. We also compared the efficacy of JEV and WNV ISA in zinc finger antiviral protein 1 (ZAP) wild‐type and knockout cells and found that knockout cells may be more effective for ISA rescue of flaviviruses, including CpG‐enriched strains for live attenuated vaccines. The validated modified ISA strategies provide an efficient approach for producing synthetic JEV and WNV. This will enable rapid research during outbreaks of emerging flaviviruses by facilitating the quick generation of new virus variants.
... The zinc finger CCCH-type antiviral protein 1 (ZAP) is a cellular protein with broad antiviral activity [1][2][3][4][5]. ZAP targets CpG-rich regions of RNA and ZAP binding to viral RNA mediates its degradation and translational inhibition [3,6]. ...
... Recently, Riplet, a protein known for activating the retinoic acid-inducible gene I, was identified as a ZAP co-factor [14]. Initially characterized to inhibit murine leukemia virus [15], ZAP has shown efficacy against many viruses, including alphaviruses, filoviruses, influenza virus, porcine reproductive and respiratory syndrome virus, hepatitis B virus, human cytomegalovirus, human T-cell lymphotropic virus type 1, and HIV-1 [1,6,[16][17][18][19][20][21][22][23][24]. However, ZAP's antiviral efficiency is limited against certain RNA viruses, including vesicular stomatitis virus, poliovirus, enterovirus A71, herpes simplex virus type 1, and some flaviviruses [8,16,25]. ...
The zinc finger antiviral protein 1 (ZAP) has broad antiviral activity. ZAP is an interferon (IFN)-stimulated gene, which itself may enhance type I IFN antiviral response. In a previous study, Zika virus was identified as ZAP-resistant and not sensitive to ZAP antiviral activity. Here, we found that ZAP was associated with the inhibition of Zika virus in Vero cells, in the absence of a robust type I IFN system because Vero cells are deficient for IFN-alpha and -beta. Also, quantitative RNA-seq data indicated that endogenous ZAP is associated with altered global gene expression both in the steady state and during Zika virus infection. Further studies are warranted to elucidate this IFN-alpha and -beta independent anti-Zika virus activity and involvement of ZAP.
... ZAP inhibits a diverse range of virus genera, yet its antiviral activity can be specific to particular members in a genus, suggesting viral evasion or antagonism of ZAP inhibition [5,6]. For example, ZAP blocks many species of mosquito-borne alphaviruses to varying degrees, where Sindbis virus (SINV) and Ross River virus (RRV) are more sensitive than o'nyong'nyong virus (ONNV) and chikungunya virus (CHIKV) vaccine strain 181/clone 25 [7,8]. Alphaviruses have a positive-sense RNA genome, which can be immediately translated into viral proteins by host ribosomes upon entry into the host cell [9,10]. ...
... SINV (Toto1101) [67], SINV expressing luciferase (Toto1101/Luc and Toto1101/Luc:ts6) [39], SINV expressing enhanced green fluorescent protein (EGFP) (TE/5'2J/GFP) [68], RRV expressing EGFP (gift from Dr. Mark Heise, University of North Carolina) [69], ONNV expressing EGFP (gift from Dr. Steve Higgs, Kansas State University) [70], CHIKV vaccine strain 181/clone 25 expressing EGFP (gift from Scott Weaver, The University of Texas Medical Branch at Galveston) [70], VEEV vaccine strain TC-83 expressing EGFP (gift from Dr. Ilya Frolov, University of Alabama at Birmingham), and HIV-1 Bru ΔEnv pseudotyped with the glycoprotein from vesicular stomatitis virus have been previously described [8,66,72]. All alphaviral stocks were generated and titered in BHK-21 cells [39]. ...
... The genomic SINV DNA template was digested by XhoI and in vitro transcribed using SP6 RNA polymerase (New England Biolabs) and 0.5mM biotin-16-UTP (Roche Life Science, Penzberg, Germany) as previously described [33]. RNA biotinylation was confirmed by streptavidin-HRP dot blot as previously described [8]. ...
The host interferon pathway upregulates intrinsic restriction factors in response to viral infection. Many of them block a diverse range of viruses, suggesting that their antiviral functions might have been shaped by multiple viral families during evolution. Host-virus conflicts have led to the rapid adaptation of host and viral proteins at their interaction hotspots. Hence, we can use evolutionary genetic analyses to elucidate antiviral mechanisms and domain functions of restriction factors. Zinc finger antiviral protein (ZAP) is a restriction factor against RNA viruses such as alphaviruses, in addition to other RNA, retro-, and DNA viruses, yet its precise antiviral mechanism is not fully characterized. Previously, an analysis of 13 primate ZAP orthologs identified three positively selected residues in the poly(ADP-ribose) polymerase-like domain. However, selective pressure from ancient alphaviruses and others likely drove ZAP adaptation in a wider representation of mammals. We performed positive selection analyses in 261 mammalian ZAP using more robust methods with complementary strengths and identified seven positively selected sites in all domains of the protein. We generated ZAP inducible cell lines in which the positively selected residues of ZAP are mutated and tested their effects on alphavirus replication and known ZAP activities. Interestingly, the mutant in the second WWE domain of ZAP (N658A) is dramatically better than wild-type ZAP at blocking replication of Sindbis virus and other ZAP-sensitive alphaviruses due to enhanced viral translation inhibition. The N658A mutant is adjacent to the previously reported poly(ADP-ribose) (PAR) binding pocket, but surprisingly has reduced binding to PAR. In summary, the second WWE domain is critical for engineering a more potent ZAP and fluctuations in PAR binding modulate ZAP antiviral activity. Our study has the potential to unravel the role of ADP-ribosylation in the host innate immune defense and viral evolutionary strategies that antagonize this post-translational modification.
... Furthermore, multiple cellular cofactors have been identified that are required for ZAP's antiviral function [10][11][12][13][14]. However, there are still significant knowledge gaps regarding the specific factors that influence ZAP binding to viral RNA, as even between closely related viral species, differences in ZAP-mediated antiviral activity and RNA binding have been observed [15]. For example, ZAP exhibits varying antiviral activities against viruses within the Orthoflavivirus genus [16]. ...
The zinc-finger antiviral protein (ZAP) is a restriction factor that proficiently impedes the replication of a variety of RNA and DNA viruses. In recent years, the affinity of ZAP's zinc-fingers for single-stranded RNA (ssRNA) rich in CpG dinucleotides was uncovered. High frequencies of CpGs in RNA may suggest a non-self origin, which underscores the importance of ZAP as a potential cellular sensor of (viral) RNA. Upon binding viral RNA, ZAP recruits cellular cofactors to orchestrate a finely tuned antiviral response that limits virus replication via distinct mechanisms. These include promoting degradation of viral RNA, inhibiting RNA translation, and synergizing with other immune pathways. Depending on the viral species and experimental set-up, different isoforms and cellular cofactors have been reported to be dominant in shaping the ZAP-mediated antiviral response. Here we review how ZAP differentially affects viral replication depending on distinct interactions with RNA, cellular cofactors, and viral proteins to discuss how these interactions shape the antiviral mechanisms that have thus far been reported for ZAP. Importantly, we zoom in on the unknown aspects of ZAP's antiviral system and its therapeutic potential to be employed in vaccine design.
... The zinc finger CCCH-type antiviral protein 1, also known as ZAP, ZC3HAV1, or PARP13, is a cellular protein with broad antiviral activity. The protein was first characterized to inhibit murine leukaemia virus [12] and later alphaviruses, filoviruses, influenza virus, porcine reproductive and respiratory syndrome virus, hepatitis B virus, human cytomegalovirus, human T cell leukaemia virus type 1, and human immunodeficiency virus-1 [13][14][15][16][17][18][19][20][21][22][23]. The previous study in A549 cells showed JEV sensitivity to ectopic and endogenous ZAP [24]. ...
... It also interacts with various cellular proteins that may act as co-factors, enhancing its antiviral effects [32]. Furthermore, ZAP can augment other antiviral systems [14,26]. To better understand ZAP-viral RNA-cellular protein interactions, here we used Zika virus infection in wild-type and ZAP knockout VERO cells as a model. ...
One of the most recent advances in the analysis of viral RNA-cellular protein interactions is the Comprehensive Identification of RNA-binding Proteins by Mass Spectrometry (ChIRP-MS). Here, we used ChIRP-MS in mock-infected and Zika-infected wild-type cells and cells knockout for the zinc finger CCCH-type antiviral protein 1 (ZAP). We characterized ‘ZAP-independent’ and ‘ZAP-dependent’ cellular protein interactomes associated with flavivirus RNA and found that ZAP affects cellular proteins associated with Zika virus RNA. The ZAP-dependent interactome identified with ChIRP-MS provides potential ZAP co-factors for antiviral activity against Zika virus and possibly other viruses. Identifying the full spectrum of ZAP co-factors and mechanisms of how they act will be critical to understanding the ZAP antiviral system and may contribute to the development of antivirals.
... For instance, while LY6E effectively inhibits coronavirus fusion and VSV replication, its varied effects on other viruses, such as DENV and IAV, highlight the influence of viral tropism and cellular context. Similarly, flaviviruses provide another illustrative example where ZAP can inhibit certain viruses within the family but not others (116,142). While it has been described that some ZAP-resistant viruses encode proteins that actively antagonize the antiviral properties of ZAP, in other cases, the mechanisms by which these viruses evade ZAP inhibition remain unclear (116,(142)(143)(144)(145). ...
... Similarly, flaviviruses provide another illustrative example where ZAP can inhibit certain viruses within the family but not others (116,142). While it has been described that some ZAP-resistant viruses encode proteins that actively antagonize the antiviral properties of ZAP, in other cases, the mechanisms by which these viruses evade ZAP inhibition remain unclear (116,(142)(143)(144)(145). ...
The coronavirus disease 2019 (COVID-19) pandemic remains an international health problem caused by the recent emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of May 2024, SARS-CoV-2 has caused more than 775 million cases and over 7 million deaths globally. Despite current vaccination programs, infections are still rapidly increasing, mainly due to the appearance and spread of new variants, variations in immunization rates, and limitations of current vaccines in preventing transmission. This underscores the need for pan-variant antivirals and treatments. The interferon (IFN) system is a critical element of the innate immune response and serves as a frontline defense against viruses. It induces a generalized antiviral state by transiently upregulating hundreds of IFN-stimulated genes (ISGs). To gain a deeper comprehension of the innate immune response to SARS-CoV-2, its connection to COVID-19 pathogenesis, and the potential therapeutic implications, this review provides a detailed overview of fundamental aspects of the diverse ISGs identified for their antiviral properties against SARS-CoV-2. It emphasizes the importance of these proteins in controlling viral replication and spread. Furthermore, we explore methodological approaches for the identification of ISGs and conduct a comparative analysis with other viruses. Deciphering the roles of ISGs and their interactions with viral pathogens can help identify novel targets for antiviral therapies and enhance our preparedness to confront current and future viral threats.
... A cell-specific defect in RRV replication has been described before [28]. The authors showed that alphaviruses have differential sensitivity to the human antiviral effector molecule, zinc finger CCCH-type, antiviral protein 1 (huZAP) [29]. Therefore, we used HEK A cell-specific defect in RRV replication has been described before [28]. ...
... Therefore, we used HEK A cell-specific defect in RRV replication has been described before [28]. The authors showed that alphaviruses have differential sensitivity to the human antiviral effector molecule, zinc finger CCCH-type, antiviral protein 1 (huZAP) [29]. Therefore, we used HEK 293T ZAP KO cells, which are huZAP-deficient [22]. ...
... The delayed onset of RRV-mCherry replication in HEK 293T cells was also observed when virus genomic RNA was transfected into cells and shows that it is not caused by differences in cell entry of the two viruses. However, recently it was reported that alphaviruses have differential sensitivity to the antiviral effector molecule, zinc finger antiviral protein (ZAP) [28,29]. RRV and Sindbis virus (SINV) were more sensitive to endogenous ZAP than ONNV and CHIKV in HEK 293T cells. ...
A recombinant Ross River virus (RRV) that contains the fluorescent protein mCherry fused to the non-structural protein 3 (nsP3) was constructed, which allowed real-time imaging of viral replication. RRV-mCherry contained either the natural opal stop codon after the nsP3 gene or was constructed without a stop codon. The mCherry fusion protein did not interfere with the viral life cycle and deletion of the stop codon did not change the replication capacity of RRV-mCherry. Comparison of RRV-mCherry and chikungunya virus-mCherry infections, however, showed a cell type-dependent delay in RRV-mCherry replication in HEK 293T cells. This delay was not caused by differences in cell entry, but rather by an impeded nsP expression caused by the RRV inhibitor ZAP (zinc finger CCCH-Type, antiviral 1). The data indicate that viral replication of alphaviruses is cell-type dependent, and might be unique for each alphavirus.
... This preference for CpG-rich regions in RNAs has been interpreted as basis for ZAP specificity that could discriminate between self (host) and nonself (vir mRNAs, since CpG is generally underrepresented in mammalian genomes [85,88]. A hough ZAP binding to specific regions of the HIV-1 and SINV that are rich in CpG din cleotides has been reported, the basis for this specificity has not yet been fully defin [80,87,89]. ZAP binding requires not only the proper spacing of CpGs, but also, the n cleotide composition surrounding CpGs influences the ZAP's affinity for target RN ...
... This preference for CpG-rich regions in RNAs has been interpreted as the basis for ZAP specificity that could discriminate between self (host) and nonself (viral) mRNAs, since CpG is generally underrepresented in mammalian genomes [85,88]. Although ZAP binding to specific regions of the HIV-1 and SINV that are rich in CpG dinucleotides has been reported, the basis for this specificity has not yet been fully defined [80,87,89]. ZAP binding requires not only the proper spacing of CpGs, but also, the nucleotide composition surrounding CpGs influences the ZAP's affinity for target RNAs [86,90]. ...
... At the genomic scale, no obvious correlation was found between the sensitivity Viruses 2024, 16, 205 8 of 14 to the ZAP and the CpG composition among the main pathogenic alphaviruses analyzed. Therefore, some reports suggested that alphavirus evasion of the ZAP correlated with CpG suppression in specific regions of gmRNA, although this possibility remains to be confirmed [89]. Expanding the focus to include the insect-specific alphaviruses in the analysis could shed light on the phenomenon of CpG suppression in pathogenic alphaviruses. ...
Alphaviruses can replicate in arthropods and in many vertebrate species including humankind, but only in vertebrate cells do infections with these viruses result in a strong inhibition of host translation and transcription. Translation shutoff by alphaviruses is a multifactorial process that involves both host- and virus-induced mechanisms, and some of them are not completely understood. Alphavirus genomes contain cis-acting elements (RNA structures and dinucleotide composition) and encode protein activities that promote the translational and transcriptional resistance to type I IFN-induced antiviral effectors. Among them, IFIT1, ZAP and PKR have played a relevant role in alphavirus evolution, since they have promoted the emergence of multiple viral evasion mechanisms at the translational level. In this review, we will discuss how the adaptations of alphaviruses to vertebrate hosts likely involved the acquisition of new features in viral mRNAs and proteins to overcome the effect of type I IFN.
... ZAP has been shown to inhibit HIV-I by targeting multiple viral mRNA for degradation (Zhu et al., 2011). ZAP inhibits alphaviruses by targeting the CpG dinucleotides in the NSP2 region containing RNA (Nguyen et al., 2023). ZAP inhibits human cytomegalovirus by targeting its UL4/UL5 transcripts (Gonzalez-Perez et al., 2021). ...
Viral hepatitis is a major public health concern globally. World health organization aims at eliminating viral hepatitis as a public health threat by 2030. Among the hepatitis causing viruses, hepatitis B and C are primarily transmitted via contaminated blood. Hepatitis A and E, which gets transmitted primarily via the feco-oral route, are the leading cause of acute viral hepatitis. Although vaccines are available against some of these viruses, new cases continue to be reported. There is an urgent need to devise a potent yet economical antiviral strategy against the hepatitis-causing viruses (denoted as hepatitis viruses) for achieving global elimination of viral hepatitis. Although zinc was known to mankind for a long time (since before Christ era), it was identified as an element in 1746 and its importance for human health was discovered in 1963 by the pioneering work of Dr. Ananda S. Prasad. A series of follow up studies involving zinc supplementation as a therapy demonstrated zinc as an essential element for humans, leading to establishment of a recommended dietary allowance (RDA) of 15 milligram zinc [United States RDA for zinc]. Being an essential component of many cellular enzymes and transcription factors, zinc is vital for growth and homeostasis of most living organisms, including human. Importantly, several studies indicate potent antiviral activity of zinc. Multiple studies have demonstrated antiviral activity of zinc against viruses that cause hepatitis. This article provides a comprehensive overview of the findings on antiviral activity of zinc against hepatitis viruses, discusses the mechanisms underlying the antiviral properties of zinc and summarizes the prospects of harnessing the therapeutic benefit of zinc supplementation therapy in reducing the disease burden due to viral hepatitis.
Despite their role as innate sentinels, macrophages can serve as cellular reservoirs of chikungunya virus (CHIKV), a highly-pathogenic arthropod-borne alphavirus that has caused large outbreaks among human populations. Here, with the use of viral chimeras and evolutionary selection analysis, we define CHIKV glycoproteins E1 and E2 as critical for virion production in THP-1 derived human macrophages. Through proteomic analysis and functional validation, we further identify signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 subunit K (eIF3k) as E1-binding host proteins with anti-CHIKV activities. We find that E1 residue V220, which has undergone positive selection, is indispensable for CHIKV production in macrophages, as its mutation attenuates E1 interaction with the host restriction factors SPCS3 and eIF3k. Finally, we show that the antiviral activity of eIF3k is translation-independent, and that CHIKV infection promotes eIF3k translocation from the nucleus to the cytoplasm, where it associates with SPCS3. These functions of CHIKV glycoproteins late in the viral life cycle provide a new example of an intracellular evolutionary arms race with host restriction factors, as well as potential targets for therapeutic intervention.
