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Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein}
Article:
Abstract:
CCCH-type zinc-finger antiviral protein( ZAP) is a host factor that restricts the infection of many viruses mainly through RNA degradation, translation inhibition and innate immune responses. So far, only one flavivirus, yellow fever virus, has been reported to be ZAP-resistant. Here, we investigated the antiviral potential of human ZAP( isoform ZAP-L and ZAP-S) against three flaviviruses, Japanese encephalitis virus( JEV), dengue virus( DENV) and Zika virus( ZIKV). Infection of JEV but not DENV or ZIKV was blocked by ZAP overexpression, and depletion of endogenous ZAP enhanced JEV replication. ZAP hampered JEV translation and targeted viral RNA for 3′-5′ RNA exosome-mediated degradation. The zinc-finger motifs of ZAP were essential for RNA targeting and anti-JEV activity. JEV 3′-UTR, especially in the region with dumbbell structures and high content of CG dinucleotide, was mapped to bind ZAP and confer sensitivity to ZAP. In summary, we identified JEV as the first ZAP-sensitive flavivirus. ZAP may act as an intrinsic antiviral factor through specific RNA binding to fight against JEV infection.
Introduction:
Zinc-finger CCCH-type containing, antiviral 1( ZC3HAV1) also known as zinc-finger antiviral protein( ZAP) was first discovered in rats as a host antiviral protein against Moloney murine leukemia virus[1]. Later, multiple RNA and DNA viruses, like retroviruses, filoviruses, alphaviruses, and hepatitis B virus were shown to display sensitivity to ZAP[2–6]. However, ZAP does not induce a universal antiviral state, since viruses such as herpes simplex virus 1( HSV-1), vesicular stomatitis virus and yellow fever virus( YFV) are resistant to ZAP[4]. Furthermore, viruses within the same family may exhibit different sensitivity to ZAP. For example, in the family Picornaviridae, ZAP inhibited coxsackievirus B3 but not poliovirus infection[4, 7]. In humans, ZAP contains two major isoforms( ZAP-L and ZAP-S), which differ in the C-terminal region through alternative splicing[8]. In the N-terminus of ZAP, there are four CCCH-type zinc-finger motifs, which are required for RNA binding and antiviral property[9]. ZAP exhibits antiviral activity generally via posttranscriptional RNA regulation, such as mRNA decay and translation inhibition. ZAP recruits the RNA processing exosome complex and poly( A)-specific ribonuclease( PARN) to degrade the target RNA from the 3′-end[6, 10]. ZAP also interacts with p72 RNA helicase to recruit decapping enzymes DCP1/DCP2 and exoribonuclease XRN1 to degrade the target RNA from the 5′-end[6, 11]. Besides targeting RNA accumulation, ZAP can block translation of incoming viral RNA of Sindbis virus( SINV)[4] probably by interrupting the interaction between translational initiation factors eIF4A and eIF4G[12]. Moreover, ZAP-S can associate with retinoic acid-inducible gene I( RIG-I), a key sensor to recognize viral RNA, to promote the innate immune response and contribute to its antiviral potential[13]. Flaviviruses include numerous important human pathogens such as YFV, West Nile virus( WNV), Zika virus( ZIKV), dengue virus( DENV) and Japanese encephalitis virus( JEV), causing endemic or pandemic outbreaks in tropical and subtropical areas[14]. Flaviviral virions are enveloped and contain a single-stranded, positive-sense RNA genome with 5′-cap but not 3′-poly( A) tail. Flavivirus RNA encodes a single open reading frame( ORF) flanked by 5′- and 3′-untranslated regions( UTRs), which contain RNA secondary structures required for viral translation and transcription[15, 16]. YFV was resistant to the antiviral activity of rat ZAP[4], but the susceptibility of other flaviviruses to ZAP is not clear. In this study, we determined the antiviral activity of ZAP against JEV, DENV, and ZIKV and found different viral responses to ZAP. We further demonstrated the antiviral mechanism of ZAP against JEV infection.
Discussion:
ZAP exhibits antiviral activity against a variety of viruses, but flaviviruses were not known to be sensitive to ZAP until this study. Here, we demonstrate that overexpression of human ZAP( isoforms ZAP-L and ZAP-S) inhibited JEV infection and downregulation of endogenous ZAP enhanced JEV replication, indicating the intrinsic antiviral potential of ZAP. We also found that, similar to YFV[4], DENV and ZIKV are resistant to ZAP, supporting the notion that ZAP is not a universal antiviral factor even for viruses within the same family. Viruses may use different strategies to evade the ZAP-mediated antiviral action, but it is still not fully understood what determines the sensitivity of a virus to ZAP. Influenza A virus NS1 protein antagonizes ZAP-S by interrupting its binding to target mRNA[19]. Murine gammaherpesvirus 68 RTA inhibits the antiviral activity of ZAP by disrupting the N-terminal intermolecular interaction of ZAP[20]. HSV-1 UL41 endoribonuclease was identified as an antagonist of human ZAP by degrading its mRNA[17]. Enterovirus 71 3C protease mediates the cleavage of ZAP protein[18]. In our hands, DENV neither downregulated the expression of ZAP nor blocked the antiviral activity of ZAP( S4 and S5 Figs). However, we were able to detect the interaction of ZAP with JEV RNA( Figs 2F and 3B) but not with DENV RNA( S7 Fig), consistent with the antiviral potential of ZAP against JEV but not DENV. Thus, the resistance of DENV to ZAP might be resulted from a failure of ZAP to bind with DENV viral RNA. Recently, viral RNA with high CG dinucleotide content was found to be targeted by ZAP, and ZAP bound directly and selectively to RNA sequences containing CG dinucleotides[32]. The known ZAP-sensitive virus genomes contained relatively higher CG frequencies, such as ZAP-sensitive SINV( 0. 9) vs. ZAP-resistant YFV( 0. 38)[32]. Interestingly, the ZAP-sensitive JEV genome also has a relatively higher CG frequency( 0. 61) as compared to that of ZAP-resistant ZIKV( 0. 46) and DENV( 0. 36). Moreover, we detected the interaction of ZAP with JEV RNA( Figs 2F and 3B) but not with DENV RNA( S7 Fig). Thus, JEV RNA enriched with CG dinucleotides may be targeted by ZAP binding to trigger the antiviral effect. Our crosslinking-immunoprecipitation-sequencing assay showed several peaks of ZAP binding in the JEV 3′-UTR, and domain II of JEV 3′-UTR with high frequencies of CG dinucleotide was further mapped as the major ZRE( Figs 7 and 8). Since domain II also contains stem-loop and pseudoknot structures[31, 33], the contribution of RNA secondary structures in addition to CG dinucleotides in ZAP recognition cannot be ruled out. ZAP-S serves as a key regulator of RIG-I-mediated innate immune responses for type I IFN production to limit the infection of influenza A virus and Newcastle disease virus. Besides, ZAP-S also enhances NF-κB and IRF3 signaling downstream of RIG-I for the induction of proinflammatory cytokines, like TNF-α and IL-6[13]. In this study, we showed that ZAP promoted the production of IFN-β, TNF-α and IL-6 during JEV infection( Fig 6A–6C). It has been speculated that elevated IFNs, TNF-α, and IL-6 during viral infection correlated with the severity and outcome of viral diseases. Similarly, IFNs and proinflammatory cytokines including TNF-α and IL-6 were elevated in patients with acute Japanese encephalitis[34–36]. Thus, the induction of IFN and proinflammatory cytokines/chemokines by ZAP may contribute to host defense responses as well as the JEV-induced inflammatory states and pathogenesis during JEV infection. ZAP restricted JEV infection mainly by posttranscriptional regulation such as blocking protein translation and enhancing RNA degradation. Through interaction with XRN1 and exosome components, like Rrp46( EXOSC5), Rrp40( EXOSC3) and Rrp42( EXOSC7)[6, 10], ZAP utilizes cellular RNA decay machineries to destabilize both viral and cellular RNAs. ZAP binds with 5′-UTR of HIV-1 nef mRNA and 3′-UTR of cellular TRAILR4 mRNA and then requires RNA exosome, and potentially XRN1, to degrade the target RNAs[6, 25]. The anti-JEV effect of ZAP was diminished by knockdown of the exosome component( Fig 5D–5F), indicating the involvement of 3′-5′ RNA decay in the ZAP antiviral pathway. Thus, ZAP can bind with JEV RNA and target the viral RNA to the 3′-5′ RNA exosome complex for RNA degradation. Furthermore, XRN1-dependent RNA decay is known to generate subgenomic flavivirus RNA( sfRNA)[37, 38], which can then block XRN1 activity and alter host mRNA stability[39]. Interestingly, XRN1 was not involved in the anti-JEV activity of ZAP( Fig 5A–5C), probably due to the interplay between XRN1 and sfRNA in JEV-infected cells prevailing the involvement of XRN1 in the antiviral action of ZAP. Since ZAP not only restricts viral infection but also regulates cellular mRNA abundance[25], we cannot exclude the possibility that the blocking effect of ZAP on JEV is due to the altered cellular mRNA and protein expression. Overall, we have identified JEV as the first flavivirus sensitive to human ZAP and provide insight about the antiviral mechanism of ZAP against viral RNA without 3′-poly( A) tail.
In addition to innate and adaptive immunities , many cellular proteins also exert antiviral activity against viral invasion . Human zinc-finger antiviral protein ( ZAP ) is a cellular restriction factor against many viruses but its role with regard to the flavivirus family is largely unknown . We tested the antiviral potential of ZAP against three flaviviruses and found that Japanese encephalitis virus ( JEV ) was ZAP-sensitive , while dengue virus and Zika virus were ZAP-resistant . ZAP specifically targets JEV viral RNA and induces translation repression and RNA degradation . Our findings highlight the ZAP-mediated anti-JEV mechanisms and extend the antiviral spectrum of ZAP to include a member of the Flavivirus genus .