Analysis of the Zika and Japanese Encephalitis Virus NS5

Article pubs.acs.org/jpr

Cite This: J. Proteome Res. XXXX, XXX, XXX−XXX

Analysis of the Zika and Japanese Encephalitis Virus NS5 Interactomes Duangnapa Kovanich,† Chonticha Saisawang,† Potchaman Sittipaisankul,† Suwipa Ramphan,† Nuttiya Kalpongnukul,‡ Poorichaya Somparn,‡ Trairak Pisitkun,*,‡ and Duncan R. Smith† †

Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand Center of Excellence in Systems Biology, Research affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

‡

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S Supporting Information *

ABSTRACT: Mosquito-borne flaviviruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), are major human pathogens. Among the flaviviral proteins, the nonstructural protein 5 (NS5) is the largest, most conserved, and major enzymatic component of the viral replication complex. Disruption of the common key NS5-host protein−protein interactions critical for viral replication could aid in the development of broad-spectrum antiflaviviral therapeutics. Hundreds of NS5 interactors have been identified, but these are mostly DENV-NS5 interactors. To this end, we sought to investigate the JEV- and ZIKV-NS5 interactomes using EGFP immunoprecipitation with label-free quantitative mass spectrometry analysis. We report here a total of 137 NS5 interactors with a significant enrichment of spliceosomal and spliceosomal-associated proteins. The transcription complex Paf1C and phosphatase 6 were identified as common NS5-associated complexes. PAF1 was shown to play opposite roles in JEV and ZIKV infections. Additionally, we validated several NS5 targets and proposed their possible roles in infection. These include lipid-shuttling proteins OSBPL9 and OSBPL11, component of RNAP3 transcription factor TFIIIC, minichromosome maintenance, and cochaperone PAQosome. Mining this data set, our study expands the current interaction landscape of NS5 and uncovers several NS5 targets that are new to flavivirus biology. KEYWORDS: flavivirus, nonstructural protein 5, NS5, Japanese encephalitis virus, Zika virus

1. INTRODUCTION For a successful infection, viruses usually modify the host cellular environment in a number of ways to provide a more favorable platform and to co-opt factors necessary for their genome replication, protein production, and virion assembly. These processes typically include subverting host immune responses, remodelling host membranes and manipulating host signaling and metabolic pathways. Due to their minimal genomes, particularly those of RNA viruses, the manipulation of cellular processes by viruses chiefly occur through physical interactions between the viral and host proteins. The genus Flavivirus is a genus of 53 virus species in the family Flaviviridae.1 The flaviviruses are enveloped, positivesense single-stranded RNA viruses transmitted primarily through the bites of infected arthropods. Approximately 40 of the viral species are considered human pathogens, of which the medically important species are tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), West Nile virus (WNV), dengue virus (DENV), and Zika virus (ZIKV). While DENVs usually cause febrile illness and occasionally severe hemorrhagic fever, JEV and TBEV are known to be neurotropic and cause encephalitis. © XXXX American Chemical Society

ZIKV was previously considered to cause a mild febrile illness, however, after the recent worldwide outbreak, ZIKV has been recognized to cause a wide range of neurological complications including fetal microcephaly and Guillain-Barré syndrome.2 The flavivirus genome is nonsegmented with a size of around 10−11 kb in length. The genome is modified at the 5′ end with a m7GpppAm cap structure and mimics cellular mRNAs except for the lack of a poly-A tail. The genome is translated as a single polyprotein, which is subsequently cleaved by a combination of viral and host proteases into 3 structural (capsid, membrane, and envelope) and 7 nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The three structural proteins comprise the virion, while the NS proteins are primarily responsible for genome replication. Among the seven NS proteins, NS5 and NS3 are the two main components of flaviviral replication complexes (RCs), as they harbor all the enzymatic activities required for viral RNA synthesis and capping. NS5 is the largest flaviviral protein, consisting of a C-terminal RNAReceived: May 17, 2019 Published: June 14, 2019 A

DOI: 10.1021/acs.jproteome.9b00318 J. Proteome Res. XXXX, XXX, XXX−XXX

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Journal of Proteome Research

data were combined before protein identification by MaxQuant to increase identifications. A threshold of 1% FDR at the peptide and protein level, which is the standard statistical criterion for the database search, was used. Label-free quantification was performed by MaxQuant. In total, each pulldown data set consisted of three data points. Based on the assumption of a normal distribution, Student’s t test was used to compare each NS5 pull-down with control pull-down data sets. The details of the data analysis workflow are described in the following sections.

dependent RNA polymerase (RdRp) and an N-terminal guanylyltransferase (GTase) and methyltransferase (MTase) domain. The RdRp replicates the genomic RNA into the uncapped minus-strand RNA, which is subsequently used as a template to produce a large excess of the positive-sense RNA genome. The MTase then performs both guanine N-7 and ribose 2′−OH methylations for the capping of the newly synthesized positive sense RNAs. The precise composition of the flaviviral RCs are poorly understood, but likely includes key cellular proteins required for viral genome replication.3 Apart from its enzymatic functions, additional functions of flaviviral NS5 have been shown. These include evasion of host innate immune response,4,5 induction of the host inflammatory response6−8 and regulation of cellular splicing.9 Since NS5 is the most conserved protein among the flaviviral proteins and a similar enzyme is not present in host cells, the protein has been a promising target for antiflaviviral drug development. Several MTase and RdRp inhibitors have been shown to have potent activities;10 however, the drawback of these conventional antiviral drugs is the rapid emergence of drug-resistant viral strains. An alternative approach for antiviral therapeutics therefore is host-oriented drug targets. As several NS5 interacting proteins described in the literature are shared by different species of flaviviruses, the selective disruption of these shared cellular proteins could represent a novel and attractive cellular drug target for pan-flaviviral therapeutics. To this purpose, NS5 interactomes have been investigated by a number of high-throughput yeast two-hybrid (Y2H) assays, large-scale proximity dependent biotin identification (BioID), and affinity-purification mass spectrometry (AP-MS) either in NS5 overexpression systems or in infected cells.9,11−19 These approaches have yielded hundreds of putative NS5 interacting proteins; however, there are very few common interacting proteins. This is due to the fact that most high-throughput screenings have been focused mostly on DENV NS5. In this study, we sought to identify NS5 interacting proteins of the neurotropic JEV and ZIKV in human cells. Currently, there are two proteomics screens of ZIKV-NS5 interacting proteins,18,19 while for JEV, the NS5 interactome has been explored only with small-scale experiments.20−27 Our identification of the JEV- and ZIKV-NS5 interactomes will not only contribute to our fundamental understanding of how flaviviruses, especially the re-emerged ZIKV, manipulate cellular mechanisms and drive pathogenicity but also greatly expand the landscape of flavivirus-host protein−protein interactions (PPIs), which is a prerequisite for antiflaviviral drug design. We report here the identification of 137 human proteins interacting with JEV and/or ZIKV NS5. On top of that, we generated a JEV- and ZIKV-NS5 PPI network of 421 interactions involving 115 distinct human proteins. Our data highlighted the surprising versatility of NS5 to target a wide range of cellular proteins and processes to facilitate viral replication and infection.

2.1. Construction and Expression of EGFP-Tagged JEV and ZIKV NS5 and HA-Tagged Cellular NS5-Interacting Proteins

Genomic RNA was extracted from JEV Beijing-1 strain (GenBank Accession Number: L48961) using TRIzol reagent. The first-strand cDNA was synthesized from 1 μg of extracted RNA using ImProm-II reverse transcriptase (Promega). The cDNA primer was designed from the 3′UTR region of the JEV NS5 sequence. Full-length JEV NS5 protein was amplified from cDNA template using primers shown in Table S1. Fulllength ZIKV NS5 protein was amplified from a commercially synthesized DNA template (GenScript, Piscataway, NJ) encompassing the 3′-half of the Cambodian strain ZIKV genome (GenBank Accession Number AFD30972.1) using the primers shown in Table S1. The forward and reverse primers of both NS5 genes were designed to contain XhoI and KpnI restriction sites. The purified PCR products were digested with XhoI and KpnI and ligated into pEGFP-C2 vector (Clontech) downstream of the EGFP coding sequence to allow mammalian cell expression of EGFP-NS5. For construction of hemagglutinin (HA) tagged proteins, forward and reverse primers were designed to contain restriction sites as indicated in Table S1. For the cloning of N-terminal tagged PAF1 (NCBI Accession Number: NM_019088), LEO1 (NCBI Accession Number: NM_138792), MCM6 (NCBI Accession Number: NM_005915), and WDR92 (NCBI Accession Number: NM_138458), Kozak and HA-tag coding sequences were placed at the 5′-end after the restriction site of the forward primer. For the cloning of C-terminal tagged PPP6R3 (NCBI Accession Number: XM_006718608), OSBPL9 (NCBI Accession Number: AF392445), OSBPL11 (NCBI Accession Number: AF392454), and GTF3C2 (NCBI Accession Number: NM_001318909) forward primers were designed to contain the Kozak sequence, while the reverse primers were designed to include a four-amino acid linker GGSG and the HA-tag coding sequences. PCR products were cloned into pcDNA3.1+ vector (Thermo Fisher Scientific). All recombinant plasmids were transformed into E. coli DH5α. The candidate clones were first screened by a rapid size screening method. DNA sequences of all generated expression vectors were confirmed by sequence analysis (Macrogen, Seoul, Korea). The sequences of all primers used in this study are shown in Table S1. Protein expression was confirmed by fluorescent microscopy and Western blot analysis of transfected HEK293T/17 cells using anti-GFP (sc8334, Santa Cruz Biotechnology) or anti-HA (26183, Thermo Fisher Scientific) antibodies.

2. EXPERIMENTAL PROCEDURES This study aimed to identify JEV- and ZIKV-NS5 interacting proteins in human cells. NS5 interacting proteins were enriched by performing EGFP IPs using cell lysates from HEK293T/17 cells overexpressing EGFP-JEV-NS5 or EGFPZIKV-NS5. Control IPs were performed using cell lysates from HEK293T/17 cells overexpressing EGFP. Three biological replicates of each EGFP-NS5 and control IPs were performed. In addition, each biological replicate was injected twice and

2.2. Cell Culture and Transfection

HEK293T/17 cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco) and incubated culture at B


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ions (charge state: z ≥ 2) were isolated and fragmented (“Top10-Method”). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http:// proteomecentral.proteomexchange.org) via the PRIDE partner repository28 with the data set identifier < PXD009523>.

37 °C in the presence of 5% CO2. For transfections, 10 cm dishes were seeded with 5 × 106 cells 24 h prior to calcium phosphate transfection with 10 μg plasmid DNA coding for EGFP, EGFP-JEV-NS5, and EGFP-ZIKV-NS5. Each transfection reaction was performed independently in triplicate. At 24 h post-transfection, cells were observed using live cell immunofluorescence. Subsequently, cells were harvested and subjected to EGFP pull-down.

2.5. Label-Free Quantification

All raw files were analyzed together using MaxQuant (version 1.5.3.30)29 with enabled label-free quantification (LFQ) option as described by Keilhauer and colleagues.30 The two technical replicates of each biological replicate were combined before database search by MaxQuant. The derived peak list was searched using the Andromeda search engine31 against a concatenated database consisting of human UniProtKB/SwissProt and TrEMBL (downloaded on 24/3/2016, containing 151 869 entries), EGFP sequence (GenBank Accession Number AAB08058.1), and NS5 sequences from the aforementioned JEV Beijing-1 and ZIKV Cambodian strains. Briefly, a maximum of two missed cleavages was allowed and the minimum peptide length was set to seven amino acids. Main search peptide tolerance was set at 4.5 ppm and fragment ion mass deviation of 0.5 Da was allowed. Fixed modification was carbamidomethylation of cysteines and variable modifications were oxidation of methionine and N-terminal acetylation of protein. The false discovery rate (FDR) threshold for peptide and protein identifications was set to 1%. The match between runs option was enabled with a match-time window of 0.7 min and an alignment-time window of 20 min. Label-free quantification of proteins was performed using the MaxLFQ algorithm32 integrated into MaxQuant. FastLFQ option was enabled and minimum ratio count was set at 2. For each pairwise peptide intensity comparison, it was required that at least one of the two peptides was identified by MS/MS. LFQ intensities for respective protein groups were uploaded to Perseus and analyzed as described in ref 30. Briefly, reverse identifications, contaminants, and proteins “only identified by site” were filtered out from the data. Subsequently, the LFQ intensities were log transformed. Three biological replicates were grouped and identifications were filtered for proteins having valid values in all replicates in at least one IP group. Missing intensity values were imputed using random values drawn from the normal distribution of LFQ intensities. A Student’s t test was performed comparing each NS5 IP group to the control IP group. The differences between the logarithmic means in each NS5 IP group and control IP group were calculated and subsequently exponentially transformed to obtain the mean enrichment ratios. Subsequently, volcano plots were generated by plotting the log2 mean enrichment ratios of all proteins quantified in JEVor ZIKV-NS5 data sets against the corresponding negative log10 p-values. A protein was considered to be specifically enriched by NS5 IP if its mean enrichment ratio was significantly more than or equal to 2 (p ≤ 0.05).

2.3. EGFP Immunoprecipitation (EGFP IP)

EGFP, EGFP-JEV-NS5, and EGFP-ZIKV-NS5 IPs were performed using 10 μL GFP trap (ChromoTek GmbH, Munich, Germany), which consists of a single-chain anti-GFP VHH conjugated to agarose beads. Cells were lysed at 4 °C for 1 h on a rotary mixer in 1 mL of lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton x-100 supplemented with protease inhibitor cocktail). The lysate was cleared by centrifugation at 14 000 rpm. The GFP-trap beads were equilibrated with ice-cold lysis buffer before adding to the cell lysate. The IPs were performed for 2 h at 4 °C on a rotary mixer. The beads were washed 5 times with 1 mL lysis buffer each time. After centrifugation and removal of the lysis buffer, the beads were resuspended in 2× Laemmli SDS protein loading buffer containing DTT and boiled at 95 °C for 10 min to elute bound proteins for LC-MS/MS analysis. 2.4. Liquid-Chromatography Mass Spectrometry (LC-MS/MS) Analysis

IP samples were cleaned by running ∼2 cm into 4−15% trisglycine precast polyacrylamide gels (Biorad). Subsequently, the gels were stained with Coomassie blue (Bio-Safe Coomassie G250 stain, Biorad). Gel lanes were cut and subjected to in-gel tryptic digestion. Briefly, each gel lane was diced into smaller pieces and washed twice with 50 mM ammonium bicarbonate (NH4HCO3) in 50% acetonitrile. Proteins were reduced with 10 mM DTT in 50 mM NH4HCO3 at 56 °C for 1 h, followed by alkylation using 55 mM iodoacetamide in 50 mM NH4HCO3 in the dark for 30 min at room temperature. After washing with 50 mM NH4HCO3, followed by 100% acetonitrile for 2 cycles, gel pieces were incubated with trypsin (protein to enzyme ratio of 50:1, Promega) for 1 hr on ice, and then the digestion was undertaken at 37 °C in 50 mM NH4HCO3 overnight. The resulting peptides were dried in vacuo and resuspended in 1% formic acid and subjected to LCMS/MS. Peptides were analyzed using a quadrupole orbitrap QExactive Plus mass spectrometer (Thermo Scientific). Each sample was injected twice into the mass spectrometer to obtain two technical replicates. Briefly, analysis was carried out using reversed phase liquid chromatography coupled to a nanoflow electrospray ion source (EASY-nLC 1000 (Proxeon/Thermo Scientific)) with a 250 mm C18 column (internal diameter: 75 μm). Peptide separation was performed at a flow rate of 300 nL/min over 80 min (0 to 12% acetonitrile in 10 min, 12 to 35% in 50 min, 35 to 60% in 10 min, and 60 to 95% in 10 min). Survey full scan MS spectra (m/z 400 to 1600) of peptides were acquired in the Orbitrap at a resolution of 70 000 and m/z of 445.12003 was used as a lock mass. Higher energy collision dissociation was used as fragmentation mode. The mass spectrometer acquired spectra in data-dependent acquisition mode by automatically switching between MS and MS/MS acquisition. Signals with unknown charge state were excluded from fragmentation. The dynamic exclusion option was enabled (exclusion duration 60s). The ten most intense

2.6. Gene Ontology (GO) Annotation and Protein−Protein Interaction (PPI) Network Analysis

The list of NS5 interacting protein candidates was analyzed using DAVID 6.8 to identify statistical enrichment of specific GO terms from the cellular component (CC), molecular function (MF), and biological process (BP) categories.33 Each GO term was assessed if it was significantly enriched in the set of NS5 interacting proteins in comparison with the set of proteins annotated with this term within the human proteome background. PPI networks were constructed using STRING C


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Figure 1. EGFP immunoprecipitation (EGFP IP) in combination with label-free quantitative mass spectrometry analysis to identify NS5 interacting proteins. (A) Schematic representation of the LFQ and LC-MS/MS analysis methodology used in this study. HEK293T/17 cells were transfected with EGFP, EGFP-JEV-NS5, and EGFP-ZIKV-NS5, respectively. At 24 h after transfection, cells were lysed and EGFP, EGFP-JEVNS5, and EGFP-ZIKV-NS5 and their interacting partners were enriched using EGFP IP. Enriched proteins were identified using LC-MS/MS in label-free fashion. (B) HEK293T/17 cells were transfected with pEGFP-C2 plasmids expressing EGFP, EGFP-JEV-NS5, or EGFP-ZIKV-NS5, and their expression and localization were examined by confocal microscopy. In each image, the nucleus was DAPI stained and is shown in blue, while EGFP and EGFP-NS5 are represented in green. Scale bar, 10 μm (C) Following the EGFP IPs, enriched proteins from EGFP, EGFP-JEV-NS5, and EGFP-ZIKV-NS5 IPs were resolved by SDS-PAGE and Coomassie stained to confirm the sufficient enrichment of NS5 proteins and the correct molecular weight of EGFP-NS5.

v10.5 ((http://string-db.org/).34 Only the PPIs obtained from lab experiments or curated databases with medium interaction confidence score ≥0.4 were used to construct the NS5-host PPI network.

amine 2000 (Thermo Fisher Scientific). At 24 h posttransfection, cells were infected with 0.5 MOI of JEV or 5 MOI of ZIKV. Infection medium was serum-free DMEM and infection time was 2 h at 37 °C. After this period, infection medium was replaced with regular culture medium. JEVinfected cells were collected at 24 h postinfection while ZIKVinfected cells were collected at 48 h postinfection and stored at −80 °C until use. 2.7.2. HA-tag Immunoprecipitation. HA-tag immunoprecipitation was performed as described previously except lysis buffer with 1% Triton x-100 and anti-HA agarose (Pierce) were used instead of GFP-trap beads. Before the addition of the beads, three percent of each supernatant was preserved and diluted with 2x Laemmli SDS protein loading buffer containing DTT for subsequent immunoblot analysis. The beads were

2.7. Validation of NS5 Interacting Protein Candidates

2.7.1. Virus Stocks and Infection. JEV strain Beijing-1 (GenBank Accession Number: L48961) and ZIKV strain SV0010/15 (GenBank Accession Number: KX051562) were propagated in C6/36 cells (ATCC CRL-1660) and stored at −80 °C until use. The viral titer was determined by standard plaque assay. Then 24 h prior to transfection, 10 cm dishes were seeded with 3.5 × 106 HEK293T/17 cells. Cells were transfected with 5 μg of the indicated HA-tagged-NS5interacting-protein or empty expression vectors using lipofectD


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Journal of Proteome Research washed 7 times with 1 mL lysis buffer at 4 °C on a rotary mixer for 5 min each time, resuspended in 2x Laemmli SDS protein loading buffer containing DTT and boiled at 95 °C for 10 min to elute bound proteins. Immune complexes were resolved by 8 or 10% SDS-PAGE, blotted on to nitrocellulose membranes, and visualized with chemiluminescence after incubation of the blots with anti-HA (26183, Thermo Fisher Scientific), antiJEV NS5 (GTX131359, GeneTex), anti-ZIKV NS5 (GTX133312, GeneTex), anti-GAPDH (sc-32233, Santa Cruz Biotechnology), or anti-HSP70 (sc-1060, Santa Cruz Biotechnology).

immunofluorescence confocal microscopy. Immunofluorescence microscopy showed that EGFP, EGFP-JEV NS5, and EGFP-ZIKV NS5 localized to both the cytoplasm and the nucleus (Figure 1B). In the case of JEV NS5, the protein was predominantly localized in the cytoplasm with a small amount diffused throughout the nucleoplasm as previously shown in JEV-infected porcine kidney PS cells35 and HeLA cells.27 In the case of ZIKV NS5, nuclear localization was primarily associated with nuclear dots, as has been previously observed in Vero and HEK293T cells expressing ZIKV NS5.5,36 At 24 h post-transfection, lysates were prepared from the transfected cells. The EGFP IPs were conducted under nondenaturing conditions to recover proteins that directly or indirectly bind to NS 5s. The IP experiments included three replicates of control EGFP IPs using cell extract from HEK293T/17 cells expressing empty vector pEGFP-C2, three replicates of EGFP IPs using cell extract from HEK293T/17 cells expressing EGFP-JEV NS5, and three replicates of EGFP IPs using cell extract from HEK293T/17 cells expressing EGFP-ZIKV NS5. SDS-PAGE analysis of the GFP IPs followed by Coomassie staining revealed the presence of EGFP, EGFP-JEV NS5, and EGFP-ZIKV NS5 with the expected molecular masses, confirming the expression of these proteins and the successful enrichment of NS5 (Figure 1C). A label-free quantification (LFQ) approach was used to compare the EGFP-JEV-NS5 and EGFP-ZIKV-NS5 immune complexes to the control EGFP immune complexes to allow the identification of cellular proteins that specifically bound to the NS5 moiety within the EGFP-NS5 fusion proteins. The raw data for the entire study are available via ProteomeXchange with identifier PXD009523. The list of all protein groups identified and peptides assigned to each protein are provided in Tables S2 and S3 respectively. For any potential NS5 interacting protein, the mean enrichment ratio was used as a guide to the specificity of its interaction. Examination of this mean enrichment ratio for the two EGFP-NS5 data sets indicated that the majority of protein mean enrichment ratio was centered around 2 (1 on a log2 scale) (Figure 2). Therefore, a stringent cutoff threshold at the ratio of 2 was set to eliminate nonspecific interactions to either EGFP or the GFP-trap beads. Also, when a cellular protein was bound to only one of the two EGFP-NS5 but not EGFP, then the other EGFP-NS5 IP served as additional control experiment. Filtering and statistical tests were applied as described in materials and methods. Proteins exhibiting mean enrichment ratio greater than or equal to 2-fold with p-values less than or equal to 0.05 were therefore considered as potential NS5 interacting proteins. A total of 87 and 80 proteins were identified as potential JEV- and ZIKV-NS5 interacting proteins respectively (Figure 3 and Table S4). Interestingly, 30 proteins (approximately 35% of total protein identified in each data set), were enriched in both JEV- and ZIKV-NS5 IPs. These includes BCLAF1, CBR1, CDC73, CTR9, HNRNPH3, LEO1, MATR3, NOC2L, PAF1, PPP6R3, SF3B1, THRAP3, ZC3H18, ZRANB2, CEBPZ, HNRNPC, ISOC1, PITRM1, PLS3, PPP6C, PRPSAP1, PRPSAP2, PRRC2B, PTGES3, RBM14, RBMX, RRP8, SF3B3, SLC4A1AP, and TOP2B. We also compared our data with previously published works on JEV- and ZIKVNS5 interacting proteins. Of the 11 JEV-NS5 interacting proteins reported in the literature,20−27 only HNRNPA2B1 was identified in this study.26 For ZIKV, two proteins from the innate immune system, STAT2 and NLRP3 have been

2.8. siRNA-Mediated PAF1 Knockdown and Infection

3.5 × 105 HEK293T/17 cells were reverse transfected with 10 nM PAF1 siRNA (s29267, Thermo Fisher Scientific) or nontargeting control siRNA (SIC008, Sigma-Aldrich) using 2 μL of lipofectamine 2000 before seeded in a well of a 12-well plate. At 48 h post-transfection, cells were infected with ZIKV at 5 MOI 5 or JEV at 0.2 or 0.5 MOI using the infection conditions described above. JEV-infected cells were collected at 24 h postinfection while ZIKV-infected cells were collected at 48 h postinfection. Cells were lysed in 100 μL of lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 5% glycerol, 1% Triton x-100 supplemented with protease inhibitor cocktail) and protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific). For the analysis of E protein, 40 μg of lysate was diluted with 2x Laemmli SDS protein loading buffer and for the analysis of NS5 and PAF1, lysates were diluted with 2x Laemmli SDS protein loading buffer containing DTT. All samples were boiled at 95 °C for 10 min and resolved using 10% SDS-PAGE. Primary antibodies used were an antiflavivirus E protein antibody (4G2) produced from hybridoma D1-4G2-4-15 (ATCC HB112), an anti-PAF1 antibody (ab137519, Abcam), an anti-JEV NS5 antibody (GTX131359, GeneTex), and an anti-GAPDH antibody (sc-32233, Santa Cruz Biotechnology). The same blot was used for probing of each indicated target protein and GAPDH. Incubation with 1 mM sodium azide at 4 °C overnight was performed to inhibit the HRP activity on the blot prior to addition of the new antibody. Quantification of band intensities was performed using ImageJ software.27

3. RESULTS 3.1. Identification of Potential Interacting Proteins of JEV and ZIKV NS5

The flaviviral NS5 protein plays multiples roles during viral infection, mediating not only the capping and synthesis of the viral RNA genome (as reviewed in ref 3), but additionally modulating host cell responses including interfering with interferon signaling (as reviewed in ref 4). The latter activities are often a consequence of direct physical interaction of NS5 with host cell proteins. In order to investigate physical interactions of JEV and ZIKV NS5 with cellular proteins, we undertook EGFP immunoprecipitation (EGFP IP) based on expression of the respective EGFP-NS5 fusions in mammalian cells, coupled with ion intensity-based, label-free quantitative proteomics (Figure 1A). HEK2937T/17 cells were transfected with empty vector pEGFP-C2 (expressing EGFP only) or with EGFP-JEV-NS5 (expressing EGFP-JEV NS5 fusion protein), or with EGFP-ZIKV-NS5 (expressing EGFP-ZIKV NS5 fusion protein). Expression of EGFP, EGFP-JEV NS5, and EGFPZIKV NS5 in HEK293T/17 cells was confirmed by E


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interactome and hundreds of other flaviviral NS5 interacting proteins reported in literature.9,11−15,17−19,37 A total of 29 proteins from our study (BCLAF1, CAPN2, CBR1, CDC73, CCDC6, CTR9, EIF3L, FASN, HNRNPA0, HNRNPA1, HNRNPA2B1, HNRNPA3, HNRNPH3, HNRNPM, HTATSF1, IWS1, LEO1, MATR3, NOC2L, NPM3, NUMA1, PAF1, PRPF38A, SNRNP70, STAT2, SUPT16H, THRAP3, UBR5, and ZRANB2) were previously found to interact or copurify with the NS5 protein of other flaviviruses. Taken all together, we have identified in total 137 protein candidates, of which 81 are new flaviviral NS5 host targets. Notably, 56 proteins are putative NS5 host-binding partners of more than one species of flaviviruses (Table S4). To determine whether our identified NS5 interacting protein candidates are likely to play a role in flaviviral infection, we compared our combined list with the available data sets of flaviviral host factors identified by functional genomics screenings for YFV, WNV, DENV, and ZIKV.38−42 A total of 20 of our NS5 interactors (EIF3C, MAT2A, PRPS1, NPM3, WDR26, EIF3B, RUVBL1, RUVBL2, CSE1L, UBR5, CTDP1, TXNDC5, AGTPBP1, CTR9, DCAF5, EIF3G, SRSF7, SUGP2, TNPO1, and TSNAX) have been previously demonstrated as flaviviral host factors. Interestingly, 5 proteins (CTR9, DCAF5, SUGP2, TNPO1, and TSNAX) identified as ZIKV-NS5 interacting proteins in our study were previously identified as host factors required for ZIKV infection42 (Figure 3 and Table S4). 3.2. Features of the Cellular Proteins Targeted by JEV and ZIKV NS5

Molecular function, biological process and cellular component terms from Gene Ontology (GO) database were used to annotate the human proteins targeted by JEV and ZIKV NS5 (Figure 4 and Table S5). A cellular protein−protein interaction (PPI) network was determined by STRING (http://string-db.org/)34 to visualize protein complexes associated with NS5 (Figure 5). This PPI-based analysis revealed a high degree of connectivity, with 115 proteins forming a large interconnected network of 421 PPIs. Several large-scale screenings have shown that NS5 proteins heavily target host proteins involved in RNA synthesis, processing and transport.9,11−13,18,19 As expected, the most dominant annotation terms in our study were RNA binding, mRNA processing, and mRNA splicing (Figure 4, molecular function and biological process terms). These identified proteins included spliceosomal components and spliceosome associated proteins (SAPs) that formed the densest network connectivity as shown in Figure 5. The role of flaviviral NS5 in cellular splicing has recently been described9 and will be further discussed later. In addition, the study identified components of RNA polymerase II (RNAP2) transcription factor Paf1 complex (Paf1C), heterotrimeric protein phosphatase 6 (PP6) holoenzyme and phosphoribosylpyrophosphate synthetase (PRS), as conserved cellular targets of JEV and ZIKV NS5. Consistent with recent study,18 we identified components of CUL4-DDB1 ubiquitin ligase as ZIKV-NS5 binding proteins. Our ZIKV-NS5 interactome also revealed targeting to host proteins with a broad array of biological functions. These include lipid-binding/transfer proteins OSBPL9 and OSBPL11, two subunits of the RNA polymerase III (RNAP3) transcription factor TFIIIC (GTF3C2 and GTF3C4) and several metabolic enzymes, such as fatty acid

Figure 2. Volcano plots representing the analysis of JEV- and ZIKVNS5 IP experiments. (A) The log2 mean enrichment ratios of all proteins quantified in the JEV-NS5 IP data set were plotted against the corresponding -log10 p-values. (B) The log2 mean enrichment ratios of all proteins quantified in the ZIKV-NS5 IP data set were plotted against the corresponding -log10 p-values. Proteins passing the thresholds of p-value below 0.05 (≥1.3 on a negative log10 scale) and mean enrichment ratio ≥2 (1 on a log2 scale) were treated as putative NS5 interacting proteins and are represented in red. Red circles with black borders highlight interactions verified in current or previous studies.18,19,26,36

demonstrated previously to interact with NS5.7,36 Very recently, two global ZIKV-host interaction networks of all ten ZIKV proteins overexpressed individually in host cells were reported.18,19 Comparison of our data set with their ZIKV-NS5 data sets revealed a significant overlap with a total of 25 overlapping proteins. These include STAT2 as expected as well as ANKRD28, BCLAF1, CDC73, CTDP1, CTR9, DCAF5, DDA1, DHX38, GPATCH8, GSR, LEO1, MTA2, OSBPL11, OSBPL9, PAF1, PPP6R3, PRRC2B, RBM17, SLC4A1AP, THRAP3, UBR5, VPRBP, ZC3H18, and ZRANB2 (Table S4). Additionally, comparison of our JEV-NS5 data set with their ZIKV-NS5 data sets revealed more common NS5 interacting proteins between the two species. These include AGTPBP1, EIF3A, EIF3C, EIF3H, EIF3K, EIF3L, EIF3M, HTATSF1, KRR1, PDCD4, PRPF38A, SIRT1, and SNRNP70 (Table S4). We also explored the level of overlap between our NS5 F


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Figure 3. Cellular proteins identified by EGFP IP-MS as potential JEV- and ZIKV-NS5 cellular targets. A total of 137 JEV- and ZIKV-NS5 interacting protein candidates identified in this study are listed in black and white boxes, respectively. Overlapping candidates between JEV and ZIKV are listed in the intersection area of the two boxes (gray color). Candidates previously reported as flaviviral NS5 interacting protein candidates in previous screens are boxed with dashed-line rectangles and candidates previously described as flaviviral host factor candidates in previous functional genomics screens are circled with dashed-line ellipses.

approach, interactions between ZIKV NS5 and the two lipidshuttling proteins OSBPL9 and OSBPL11 were confirmed (Figure 6C,D). Since there is no previous link of flaviviruses to transcription factor of RNAP3, we therefore selected it to validate the interaction between ZIKV NS5 and the subunit 2 of TFIIIC (GTF3C2; Figure 6E). Among the previously validated NS5 interactors identified in our study are PAF1 and LEO1. These two components of Paf1C have been shown to interact with DENV, ZIKV, and WNV NS5.19 We validated these interactions for the JEV NS5 (Figure 6F,G). While no previous link to flaviviruses has been reported, we confirmed the interactions between JEV NS5 and MCM6, a member of minichromosome maintenance complex (MCM) and WDR92, a member of PAQosome (Figure 6H,I). Taken together, these data provide evidence of the validity of our NS5 AP-MS screen and confirm specific interactions of selected candidate proteins.

synthetase (FASN), glutathione-disulfide reductase (GSR) and S-adenosylmethionine synthetase 2 (MAT2A and MAT2B; Figure 5, dashed-line boxes) Our screen also revealed JEV NS5 association to eukaryotic initiation factor 3 (eIF3) complex, of which several components have been previously reported as candidate host ZIKV-NS5 binding proteins.19 In addition, our JEV-NS5 APMS screen discovered several cellular proteins, whose functions are completely unknown in relation to flaviviruses. These includes 5 subunits of the cochaperone PAQosome, subunit 6 of DNA replicative helicase minichromosome maintenance (MCM6) and the MCM associated protein, MCMBP (Figure 5, dashed-line boxes). 3.3. Validation of NS5 Interactions with Host Proteins

As a proof of principle, we validated 9 interactions identified by AP-MS. First, we validated specific interactions of one of the overlapping cellular targets of ZIKV and JEV NS5. HA-tag IP conducted in ZIKV- and JEV-infected cells overexpressing the HA-tagged protein phosphatase 6 regulatory subunit 3 (PPP6R3) confirmed the association between both NS5s to PP6 (Figure 6A,B). Additionally, using the same HA-tag IP

3.4. PAF1 Knockdown Reduces JEV Infectivity

Knockdown of PAF1 and other Paf1C components has been recently shown by immunostaining of E protein to increase DENV and ZIKV infectivity.19 We therefore examined whether the effect of the knockdown observed in the previous study was G


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Figure 4. Gene Ontology (GO) functional enrichment analysis of the JEV- and ZIKV-NS5 interacting proteins. Enriched GO terms identified by the DAVID bioinformatics database in the complete set of human proteome. Graph shows the−log10 p-values for each term. ZIKV and JEV GO terms are represented in gray and black, respectively.

interact with viral proteins of a number of different flaviviruses offers an attractive alternate target. The flaviviral NS5 protein has been a prime target for antiviral drug discovery, as it plays a central role in viral RNA replication and cellular immune invasion. Therefore, a thorough analysis of the NS5 interactome is fundamental to generate new insights into flaviviral pathogenesis as well as to provide a rich target environment for future drug development. Currently, the landscape of the flaviviral NS5 interactome in literature is based largely on identified DENV-NS5 interactions.9,11−16,19 Here, we present a JEV- and ZIKV-NS5 protein−protein interaction network in human cells. To our knowledge, currently the ZIKV-NS5 interaction map is based on the list of host proteins identified in two AP-MS based screens18,19 while for JEV, our work is the first report of a NS5host interaction map. The data from our proteomics screen not only sheds first light on the JEV-NS5 interaction landscape but also amplifies the current ZIKV-NS5 interactome in human cells. These newly identified NS5 interacting proteins greatly expand the ever-growing list of host proteins potentially involved in the flavivirus replication cycle. It is worth mentioning that 70 candidates or 50% of the proteins identified in our study have not been previously described in the literature either as NS5 interacting proteins or as flavivirus host factors. This partial overlap, which is typically found when merging data from large-scale studies, indicates that the current landscape of flavivirus-host interactome is far from complete and we have only just scratched the surface of the NS5-host interaction network. Nevertheless, our study has revealed several shared cellular targets between JEV and ZIKV NS5, of which several (BCLAF1, CBR1, CDC73, CTR9, HNRNPH3, LEO1, MATR3, NOC2L, PAF1, SF3B1, THRAP3, and ZRANB2) were previously identified in DENV and other flaviviruses.9,11−13,19 Mining this rich data set, novel cellular processes and protein complexes in flavivirus biology were revealed. Selected candidates will be discussed below at length.

conserved among DENV, ZIKV, and JEV. We first investigated the effect of PAF1 siRNA on PAF1 gene expression level in HEK293T/17 cells and found that PAF1 siRNA led to a clear reduction of PAF1 at 48 h after transfection with no apparent sign of morphology changes (Figure 7A). Next, we confirmed the reported ZIKV phenotype after PAF1 knockdown. In each replicate, HEK293T/17 cells were reverse transfected with PAF1 or control siRNA before infection with ZIKV. PAF1 and ZIKV E protein levels were examined by Western blot analysis and densitometry band quantification. As expected, PAF1 knockdown led to an approximate 2-fold increase in E protein level relative to a nontargeting siRNA control, indicating increased in infectivity as shown by Shah and colleagues19 (Figures 7B and S2). Subsequently, we investigated the effect of PAF1 knockdown on JEV infectivity. siRNA-transfected cells were infected with JEV at 0.5 MOI. Intriguingly, PAF1 knockdown led to an opposite phenotype. Instead of promoting infectivity as reported in DENV and ZIKV,19 PAF1 knockdown reduced JEV E and also NS5 protein levels by approximately 50% (Figures 7B and S2). We also investigated the effect of PAF1 knockdown at lower MOI (0.2 MOI) and obtained a similar result (Figures 7B and S2). These data indicated that there were different mechanisms through which PAF1 knockdown affected flavivirus infectivity, and that PAF1 promoted JEV infectivity as reflected by the reduction in viral protein synthesis after PAF1 knockdown.

4. DISCUSSION Among the 53 viral species of the genus Flavivirus,1 40 are considered human pathogens. While there are effective vaccines for YFV, TBEV, and JEV,43 vaccines for the others are either not broadly effective or are at low stages of development.44 Similarly, there is no specific drug to treat any flaviviral infection. While the viral proteins, particularly those that possess enzymatic activities offer potential drug targets, the low fidelity of replication of the genome of flaviviruses suggests that escape mutants would rapidly arise. Thus, targeting host cell proteins, and particularly proteins that H


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Figure 5. Protein−protein interaction network of the JEV- and ZIKV-NS5 interactomes. Gray and black nodes denote ZIKV- and JEV-NS5 interacting protein candidates identified in this study, respectively. Two-colored nodes represent overlapping candidates between JEV and ZIKV. Edges represent protein−protein interactions. Selected NS5-associated protein complexes are boxed with red dashed lines. Protein candidates that were previously reported as flaviviral NS5 binding protein candidates in previous screens are circled with gray rings. Please note that only connected NS5 interacting proteins are shown.

4.1. NS5 and Spliceosomes

sequences, the viruses definitely do not target the splicing machinery for processing their own genomes. There is growing evidence that DENV and ZIKV NS5 targets several components of splicing machineries9,11,19 and recently DENV infection has been shown to alter cellular mRNA splicing of several known antiviral factors.9 As expected, our study has revealed the potential interactions of ZIKV and JEV NS5 proteins to a plethora of spliceosomal proteins and SAPs,

Our analysis revealed a significant enrichment of spliceosomal components and spliceosome associated proteins (SAPs) in both data sets (the densest connectivity in Figure 5). Multiple RNA viruses utilize cellular splicing machinery to generate alternative splicing for their mRNAs.45−48 However, as the flaviviral genome is a positive-sense single-stranded RNA consisting of one open reading frame without intervening I


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Figure 6. Verification of selected JEV and ZIKV NS5 interacting proteins. HEK293T/17 cells were transfected with the indicated HA-tagged candidate protein expression vectors or empty vectors (negative control) and infected with JEV or ZIKV as indicated. Cell extracts were subjected to HA immunoprecipitation and immunoblotting using HA, JEV- or ZIKV-NS5 and GAPDH or HSP70 (loading control) antibodies as indicated. Immunoprecipitations of HA-tagged proteins in ZIKV infected cells validate ZIKV NS5 interactions with (A) PPP6R3, (C) OSBPL9, and (D) OSBPL11, and (E) GTF3C2. Immunoprecipitations of HA-tagged proteins in JEV infected cells validate JEV NS5 interactions with (B) PPP6R3 (F) PAF1, (G) LEO1, (H) MCM6, and (I) WDR92. Full blots are shown in Figure S1.

transcription regulatory function, Paf1C also participates in several cellular processes including gene expression and silencing, RNA maturation, DNA repair, and cell cycle regulation (as reviewed in ref 49). When combined the results from our and earlier studies,19 it becomes obvious that Paf1CNS5 interaction is conserved among all flaviviruses examined (DENV, ZIKV, WNV, and JEV). The antiviral role of Paf1C has been widely shown in HIV,50 Influenza virus A (IVA)51 and very recently in DENV and ZIKV.19 DENV-NS5 overexpression led to a reduction in Paf1C-driven transcription of several known antiviral genes as recruitment of the transcription complex to the genes was inhibited by binding of NS5 to the complex. In addition, knockdown of PAF1 as well as other Paf1C components led to an expected increase in

although some are different from those interacting with DENV NS5. All the above-mentioned studies combined, it becomes more likely that NS5 targeting splicing machinery is a conserved strategy flaviviruses adopted to counteract cellular antiviral responses, even though, the splicing proteins they target might be virus-specific. A list of DENV, JEV, and ZIKV targeted spliceosomal proteins and SAPs collected from literature and our study is shown in Table 1. 4.2. NS5 and RNAP2 Transcription Factor Paf1C

Paf1C is a transcription complex that comprises six subunits including PAF1, LEO1, CTR9, CDC73, RTF1, and WDR61. Paf1C coordinates all stages of the RNAP2 transcription cycle as well as the events following transcript synthesis. Besides the J


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Figure 7. Knockdown of PAF1 leads to an increase in infectivity by ZIKV but a decrease in infectivity by JEV. (A) Knockdown of PAF1 in HEK293T/17 cells was confirmed by Western blot analysis. (B) HEK293T/17 cells were reverse transfected with PAF1 or control siRNA and infected with ZIKV at 5 MOI or JEV at 0.5 or 0.2 MOI. Viral protein expression levels were examined by Western blot analysis and densitometry band quantification. Each protein was normalized to GAPDH. For each experiment, at least three replicates were performed. Bar graph shows relative fold changes of viral proteins relative to the control siRNA as the mean values of all replicates. Error bars represent standard deviation of the mean. Dashed line marks the position of the viral protein levels in control siRNA experiments. ** represents p < 0.01.

DENV as well as in ZIKV infectivity.19 However, in the case of JEV, instead of increasing infectivity as observed in DENV and ZIKV, knockdown of PAF1 reduced infectivity by half. It is very intriguing to see that even though the conserved host− virus PPIs imply a similar role of the protein complex in infection, the effect can still be different even among the closely related flaviviruses. Perhaps, what we observed is an indirect effect of the knockdown on JEV infection. Since Paf1C regulates all stages of RNAP2 transcription, knockdown of Paf1C subunits would result in expression alterations in a number of genes and some of those genes might encode host proteins required for JEV infection. Another possibility would be that Paf1C has an extra role in JEV viral RNA transcription as reflected by a decrease in viral protein production when knocked-down. Thorough investigations are warranted to define the unexpected proviral role of Paf1C in JEV infection.

unknown.54 Since tRNA transcription is a tightly regulated process, very much depending on the cell proliferative status,55 we hypothesized that ZIKV might target the RNAP3 transcription factor to regulate the host tRNA synthesis as in infected cells, and the demand for tRNAs and translation machineries are higher to facilitate viral protein synthesis. 4.4. NS5 and Protein Phosphatase 6

PP6 heterotrimeric holoenzyme consists of one catalytic (PPP6C), one of the three regulatory (PPP6R1-R3), and one of the three scaffolding ankyrin repeat domain-containing protein (ANKRD28, ANKRD44, and ANKRD52) subunits. The phosphatase stood out in our study as all the three subunits (PPP6C, PPP6R3, and ANKRD28) were identified in our screen. PP6 is a key regulator of a variety of cellular processes, ranging from DNA repair,56 pre-mRNA splicing57 to nuclear factor-κB (NF-κB) signaling.58 The only relationship between PP6 and viral infection was shown in IVA. PP6 regulatory and catalytic subunits were shown to copurify with IVA RdRp. Knockdown of the PP6 catalytic subunit was shown to delay IVA RNA accumulation, the process that heavily relies on newly synthesized and fully functional RdRp, suggesting PP6 acts directly on RdRp activity through dephosphorylation of the protein.59 In the case of flaviviruses, one possibility would be that NS5 is a PP6 substrate. NS5 is known to be serine/threonine phosphorylated in a number of flaviviruses60−63 and phosphorylation has been shown to be vital for NS5 activity, localization, and function.60,61 For example, phosphorylation of TBEV NS5 on Ser56, which is highly conserved among flaviviruses, has been shown to be essential for NS5MTase activity.60 The phosphorylation state of DENV NS5 has been observed to correlate with protein localization and interaction with NS3.61 Hypophosphorylated NS5 was found in the cytosol while hyperphosphorylated NS5

4.3. ZIKV NS5 and RNAP3 Transcription Factor TFIIIC

Apart from RNAP2 transcription complex, our study also reveals ZIKV-NS5 targeting to the RNAP3 transcription factor. Generally, RNAP3 transcribes genes encoding tRNAs, 5S rRNA, and other small RNAs. Recruitment of RNAP3 to these genes requires essential transcription factors including the sixsubunit TFIIIC.52 TFIIIC subunit 1 and 3 (GTF3C1 and GTF3C3) were previously reported to copurify with ZIKV NS518 while another two subunits (GTF3C2 and GTF3C4) were identified and GTF3C2 was confirmed in our study, indicating a functional relationship between RNAP3 transcription and ZIKV. Very little is known regarding the role of RNAP3 transcription factors in the context of virus infection. A previous study showed that vaccinia virus stimulated the expression of RNAP3 transcription factors TFIIIB and TFIIIC to induce tRNA synthesis.53 Another study showed that TFIIIC was targeted by IVA protein, but the function is still K


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Table 1. List of Spliceosomal Components and Spliceosome-Associated Proteins (SAPs) Identified as NS5 Interacting Proteins of DENV, JEV, and ZIKV 9,11,19

DENV common spliceosomal components

Sm core complex

U1 snRNP specific factors and U1 related factor U2 snRNP specific factors and U2 related factors

U5 snRNP specific factors

BCLAF1 HNRNPA1 HNRNPA3 HNRNPK HNRNPM HNRNPU PCBP1 RBMXL1 SRSF2 SNRPD1 SNRPD2 SNRPE SNRPA SNRNP70 SNRNPC PUF60 SNRPA1 SNRPB2 SF3A2 SF3B1 SF3B2 SF34B SNRNP40 SNRNF200 PRPF6 PRPF8 CD2BP2 DDX23

JEV (this study)

ZIKV (this study and refs 18 and 19)

BCLAF1 HNRNPA1 HNRNPA3 HNRNPC RBMX SRSF1 SRSF7 SRSF9 TRA2B ZC3H18

BCLAF1 HNRNPC HNRNPM RBMX ZC3H18

SNRNP70

SNRNP70

SF3A1 SF3B1 SF3B3

DENV U4/U6.U5 tri-snRNP related factors complex A specific factors complex B specific factors

9,11,19

EFTUD2 PRPF38A HTATSF1 THRAP3 MFAP1

JEV (this study) PRPF38A

PRPF38A

HTATSF1 SF1 THRAP3

HTATSF1

MAGOHB PNN

THRAP3 MFAP1 DHX35 GPATCH1 AQR BUD31 CRNKL1 PQBP1 PRPF19 ACIN1 THOC1

HNRNPA0 HNRNPA2B1 HNRNPH2 HNRNPH3 MATR3

CDC40 DHX38 HNRNPH3 HNRNPL MATR3 RBM14 RBM27

complex C specific factors Prp19 complex and Prp19-related factors

CHERP PUF60 RBM17 SF3B1 SF3B3 SNRPB2

was predominantly found in the nucleus.61 In addition, only the hypophosphorylated form of NS5 was found to coprecipitate with NS3.61 While there is a growing body of evidence on which cellular kinases phosphorylate NS5,64−67 little is known concerning the phosphatases that regulate NS5 phosphorylation. Since our study has revealed the hidden relationship between PP6 and the two flaviviruses, the biological significance of the phosphatase remains to be explored, as well as whether this interaction is also conserved with other medically relevant flaviviruses.

Exon junction complex (EJC)/ TREX step II factors

ALYREF

SAPs

DHX9 HNRNPH1 HNRNPH2 HNRNPH3 HNRNPR YBX3 PRPF39

ZIKV (this study and refs 18 and 19)

PNN RBM14

SRRM1 SUGP2 THOC7 YTHDC1

viral replication. In addition, an antifungal agent that exhibits off-target effect on OSBP was shown to inhibit DENV RNA replication.71 Taken together, these studies present that the lipid-shuttling proteins play a key role in virus-induced remodeling of intracellular membranes, which is vital for successful replication. Our newly validated ORP-ZIKV NS5 interactions are provocative to warrant further research on the role of ORPs in ZIKV infection. Screening for small molecules to block either the lipid-shuttling function or the interaction with NS5 are deserving of further investigation as interfering with such cellular function could prevent formation of viral replication complex.

4.5. NS5 Targeting Lipid-Binding/Transfer Proteins

Among the overlapping candidates between our study and the two ZIKV N5-host PPI mapping studies18,19 are the two oxysterol-binding protein (OSBP)-related proteins (ORPs), OSBPL9, and OSBPL11. We validated both interactions in ZIKV-infected cells. OSBP and ORPs constitute a large family of lipid-binding/transfer proteins that transports lipid over membrane contact sites to maintain distinct organelle membrane lipid compositions. ORPs can also act as lipid sensors with scaffolding function for signal protein complexes (as reviewed in ref 68). The molecular function of OSBP in (+)ssRNA viral infection has been explored in hepatitis C virus69 and the distantly related encephalomyocarditis virus.70 The two viruses target OSBP to modify host lipid landscape to generate specialized membranous compartments for replication.69,70 The study in DENV infection showed that knockdown of OSBP and several ORPs including OSBPL11 reduced

4.6. ZIKV NS5 Targeting Metabolic Enzymes

Viruses are obligate parasites that utilize host metabolic processes to reproduce. Elucidating the host metabolic processes that viruses are dependent on could provide novel therapeutic targets. We identified several metabolic enzymes potentially targeted by ZIKV NS5. These include guanosine monophosphate synthetase (GMPS), and phosphoribosyl pyrophosphate synthetase 1 (PRPS1), which are involved in nucleotide metabolism. Catalytic and regulatory subunits of Sadenosylmethionine synthetase (MAT2A and MAT2B, respectively) were also enriched in our ZIKV-NS5 pulldown. Previously, PRPS1 and MAT2A were identified as host factors required for efficient YFV propagation.38 MAT produces Sadenosyl methionine (SAM) which is the principal source of methyl groups in all living organisms. Flaviviral NS5MTase L


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represents EBOV transcription/replication.75 In our study, five subunits of PAQosome (all four of R2TP and WDR92), copurified with JEV NS5 (Figure 5, dashed-line boxes), suggesting the strong association between NS5 and the cochaperone. Since PAQosome is responsible for assembly of macromolecular complexes, we hypothesize that JEV NS5 might sequester the cochaperone to the RCs to assist in virion assembly, a process tightly coordinated with genome replication.76,77 Although this remains to be supported by relevant data in the future.

uses SAM as a methyl donor to generate the methylated 5′-cap, which as mentioned above is essential for RNA stability and translation. We postulate that ZIKV NS5 recruits MAT to the RCs to generate SAM for its viral RNA capping. Disruption of such interaction could then prevent viral RNA capping, resulting in unstable viral RNAs susceptible to degradation by the host immune response.72 Another ZIKV targeting host enzyme that caught our attention is glutathione-disulfide reductase (GSR). The NS5GSR interaction was identified by two complementary approaches from two independent studies (our ZIKV-NS5 AP-MS and ZIKV NS5 BioID18), thereby affirming the interaction. GSR is an essential enzyme that converts an oxidized dimer of glutathione (GSSG) to reduced glutathione (GSH). GSH is the most abundant reactive oxygen species (ROS) scavenger in the majority of cells. Recently, overexpression of ZIKV NS5 has been shown not only to directly interact with NLRP3 but also to trigger an increase in ROS production,7 ultimately leading to NLRP3 inflammasomederived interleukin 1β production, a key feature of inflammation during ZIKV infection.7,8 It was demonstrated that the NS5-NLRP3 interaction facilitated the assembly of NLRP3 inflammasome,7 however, the mechanism of how ZIKV NS5 promotes ROS production is still unknown. We hypothesize that NS5-GSR interaction might reduce or inhibit the enzyme activity, consequently resulting in the elevated level of ROS in the cells. Further study of the function of NS5-GSR interaction will definitely shed light on ZIKV pathogenesis along this NLRP3 inflammasome pathway.

5. CONCLUDING REMARKS In conclusion, this study provides novel data on the JEV- and ZIKV-NS5 interaction map in human cells. The functions of the JEV- and ZIKV-NS5 interacting proteins are surprisingly diverse, ranging from proteins involved in RNA processing, transcription, translation, phosphatase, lipid shuttling to chaperone machinery, and metabolic enzymes. The fact that NS5 is capable of putatively targeting such a variety of host proteins is startling. Some of the interactions have been previously identified in other flaviviruses, suggesting that they play vital roles in the flavivirus replication cycle. While further studies are warranted to define the functional relevance of the identified interactions, we have proposed possible roles of PP6, ORPs, MAT, GSR, TFIIIC, MCM, and PAQosome in infection. Future investigations focusing on elucidating the function of these new NS5 targets will shed new light on how these viruses utilize host machineries to promote successful replication and drive pathogenicity.

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4.7. Minichromosome Maintenance (MCM) DNA Helicase Complex

ASSOCIATED CONTENT

S Supporting Information *

Interestingly, our study revealed a JEV-NS5 association with the minichromosome maintenance (MCM) DNA helicase complex. The MCM heterohexamer is an essential DNA helicase consisting of six related ATPase associated subunits (MCM 2−7) required for both initiation and elongation of DNA replication. Up to date, there is little evidence to link MCM to the viral RdRp. Binding of the MCM to IVA RdRp stabilized the viral replication complex during its transition from initiation to elongation, potentially by scaffolding the RdRp with viral RNA.73 Subunit 7 (MCM7) was previously reported to copurify with DENV NS59 while we validated the interaction between JEV NS5 and subunit 6 (MCM6). Perhaps, MCM is also targeted by NS5 with the same purpose, even though IVA, a negative-sense ssRNA virus and positivesense ssRNA flaviviruses have profound differences in replication strategies.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.9b00318. Figure S1: Full blot of all IP-WB results shown in Figure 6 to validate selected JEV and ZIKV NS5 interacting proteins; Figure S2: Densitometric analyses of the expression of PAF1, ZIKV E, JEV E and NS5 proteins normalized to GAPDH; Table S1: Primers used in construction of EGFP-JEV NS5, EGFP-ZIKV NS5, PPP6R3-HA, OSBPL9-HA, OSBPL11-HA, GTF3C2HA, HA-PAF1, HA-LEO1, HA-MCM6, and HAWDR92 (PDF) Table S2: A list of all protein groups identified at 1% FDR together with their LFQ intensities (XLSX) Table S3: A nonredundant list of identified peptides meeting the 1% FDR cutoff together with their intensities (XLSX) Table S4: A combined list of JEV-NS5 and ZIKV-NS5 interacting proteins identified in this study together with their enrichment ratios (XLSX) Table S5: A list of genes annotated to each GO term and the p-values associated with each annotation term (XLSX)

4.8. Cochaperone Particle for Arrangement of Quaternary Structure (PAQosome) Complex

PAQosome is a large 11-subunit chaperone complex, consisting of two modules, the four-subunit R2TP and the five-subunit URI1 prefoldin, and the two associated proteins WDR92 and RPB5. The PAQosome is an important cochaperone that assists HSP90 in the assembly of large protein complexes such as box C/D and H/ACA snoRNPs, U4 and U5 snRNPs, telomerase, and all three nuclear RNA polymerases (as reviewed elsewhere74) . Very little is known regarding the role of PAQosome in viral infection. Very recently, the R2TP module has been shown to associate with the Ebola virus (EBOV) nucleoprotein, potentially to assist in capsid assembly, as knockdown of R2TP proteins showed moderate or no effect on EBOV minigenome activity, which

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AUTHOR INFORMATION

Corresponding Author

*Phone: +6692-537-0549. E-mail: [email protected]. ORCID

Duangnapa Kovanich: 0000-0001-5974-829X M


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Trairak Pisitkun: 0000-0001-6677-2271 Author Contributions

D.K. and D.R.S. formulated the study and wrote the manuscript. D.K. performed the NS5 AP-MS, data analysis, validation IP-WB and PAF1 knockdown experiments. D.K. and P.Si. performed laser scanning microscopy imaging. C.S. and S.R. contributed to cloning, and N.K. and P.So. contributed to LC-MS analysis. D.K., D.R.S., and T.P. discussed/interpreted results. All authors read and approved the final manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from Mahidol University, the Research Fund for DPST graduates (021/2559), and the Thailand Research Fund (MRG6080085) to D.K. D.R.S. was supported by the Thailand Research Fund (BRG6080006) and the Newton Fund as administered by the National Science and Technology Development Agency (FDS-CO-2561-6820-TH). T.P. was supported by Chulalongkorn Academic Advancement into its Second Century Project (CUAASC) grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Atitaya Hitakarun for helping in all aspects of experiments involving viruses.

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