A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

Domain: biology
Published: 2026-03-13
Editorial outcome: minor_revision

Abstract
Orthoflavivirus infections represent an increasing public health burden, with several members of the genus emerging or re-emerging globally. Several pre-clinical studies identified the non-structural protein 4B (NS4B), as the most promising target for the development of potent direct-acting antivirals. However, its functional roles in viral replication are still elusive. Here, we employ an integrated proteomic approach to systematically identify cellular targets of NS4B across eight prototypic orthoflaviviruses and characterize their influence on the human proteome. Using this approach, we mapped high-confidence NS4B-interacting human proteins across the genus, underlying potentially divergent and convergent mechanisms of host adaptation across orthoflaviviruses spanning diverse pathologies and vector preferences. Among these, we unveil a novel function for UBA5, the E1-activating enzyme of the UFMylation pathway, in orthoflavivirus replication. Mechanistically, we map associations of distinct viral proteins with multiple members of the UFMylation pathway, which are selectively recruited to sites of viral replication to promote infectious particle production. Finally, we demonstrate that pharmacological inhibition of UFMylation exerts potent antiviral activity in vitro and in vivo. This integrative study provides a rational framework for a system-level understanding of orthoflavivirus NS4B effector functions and sheds light on a conserved and unconventional role for UFMylation in orthoflavivirus replication.

Introduction
The genus Orthoflavivirus includes several clinically relevant emerging enveloped viruses with a positive-sense single-stranded RNA genome and re-emerging arboviruses, such as dengue virus (DENV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), Powassan virus (POWV), Usutuvirus (USUV), tick-borne encephalitis virus (TBEV) and West Nile virus (WNV)1. Orthoflaviviruses are encoding for a polyprotein that is co- and post-translationally processed into three structural (capsid, pre-membrane, envelope) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5)2. During infection, orthoflaviviruses hijack and rewire multiple host machineries to create a microenvironment conducive to viral RNA replication, including the biogenesis of replication organelles (ROs) within the endoplasmic reticulum (ER), where genome replication takes place2. The mature non-structural protein 4B (NS4B) is a small integral transmembrane protein that plays an essential, yet poorly characterized, role in viral replication via interactions with viral and host proteins3. NS4B is involved in the remodeling of the ER4, biogenesis of the viral ROs4–6, and modulation of mitochondria morphodynamics7.

Recently, NS4B has attracted renewed interest as small molecule inhibitors targeting its interactions with the viral protease/helicase NS3 have demonstrated highly potent antiviral activity in mice and primate infection models, with some currently being evaluated in human clinical trials8–11. The NS4B–host interactome has been previously characterized for individual viral species (DENV7,12, WNV13 and ZIKV14), identifying associations with cellular proteins involved in immune responses15–17, mitochondria–ER contact sites (MERCs)7,14, ER stress15,18 and autophagy18. However, the high heterogeneity of these data with respect to cellular backgrounds, expression systems and proteomic pipelines, has precluded the mapping of conserved or distinct specificities among orthoflaviviruses, which could help elucidating the molecular mechanisms underlying the broad range of diseases caused by this important group of arboviruses. Furthermore, only a limited number of NS4B-interacting proteins have been functionally or phenotypically characterized to date3. Altogether, we still know surprisingly little on the specific cellular machineries usurped by orthoflavivirus NS4B, and the molecular mechanisms underlying species-specific dysfunctional programs.

Here, we leverage an integrative systems biology approach, combining affinity-purification coupled with mass spectrometry and global proteomic profiling, to identify human proteins targeted by the NS4B of eight (re-)emerging orthoflaviviruses. This pan-viral NS4B interaction atlas, hereafter referred to as NS4Bome, elucidates hitherto unreported host-binding specificities. In addition to highlighting cellular factors that are selectively targeted by each viral species, this genus-wide atlas further categorizes cellular processes and proteins consistently hijacked across orthoflaviviruses. Among these, we identify a conserved pro-viral role for UFMylation – an emerging post-translational modification – in orthoflavivirus replication via multipartite interactions between viral proteins and UFMylation pathway components. Importantly, we demonstrate that pharmacological inhibition of UFMylation exerts potent antiviral activity in vitro and in vivo, improving disease outcomes in developing animals.

Collectively, these results provide a rational framework for the functional characterization of the most versatile orthoflavivirus protein and demonstrate the effectiveness of this approach in streamlining target identification efforts for the rational design of a new class of broad-spectrum antiviral inhibitors.

Results
A genus-wide map of orthoflavivirus NS4B human targets identifies strain-, species-specific and pan-orthoflavivirus associations with the human proteome

We employed a multi-layered proteomic approach, combining affinity purification coupled with quantitative mass spectrometry (AP-MS) and global proteomics (Fig. 1a), to map the extended orthoflavivirus NS4Bome and identify cellular target whose abundance is modulated by persistent viral protein expression. To this end, the C-terminally HA-tagged 2k-NS4B of eight prototypic orthoflaviviruses (DENV, ZIKV, JEV, WNV, YFV, TBEV, POWV and USUV) were stably expressed in human choriocarcinoma trophoblast JEG-3 cells. To account for possible differences between historical and contemporary isolates, the NS4B of two DENV serotype 2 (DENV2) strains were included (Supplementary Fig. 1). JEG-3 cells expressing the related Flaviviridae hepatitis C virus (HCV) NS4B, ZIKV capsid, Gaussia luciferase or empty vector were used as control for non-specific binding (Fig. 1a). Subcellular distribution and expression levels of HA-tagged NS4B were validated by immunofluorescence analysis, immunoblotting and analysis of intensity-based absolute quantification (iBAQ) values from the proteomic dataset, confirming comparable expression levels and a good separation of NS4B interactomes of individual species (Supplementary Fig. 1a–e). For each cell line, four replicates of affinity-purified complexes (NS4Bomes) and whole cell lysates (effectomes) were collected and analyzed by quantitative mass-spectrometry, identifying a total of 538 NS4B-interacting proteins and 514 host proteins differentially expressed upon stable NS4B expression (Figs. 1b, 2a and Supplementary Data 1, 2).

To prioritize reproducible, bait-specific protein-protein interactions (PPIs), we analyzed four replicates for each bait protein using very stringent cut-off criteria (log2 (fold-change) ≥ 5; two-sided Welch’s t-test p-value≤0.05; S0=4, FDR≤0.01, n=250). Collectively, this extended NS4Bome revealed interactions between 538 human proteins and 8 NS4B orthologues (Figs. 1b, 2a) with normalized intensities spanning 15 orders of magnitudes and normally distributed across the whole dynamic range of the human proteome (Supplementary Fig. 2a). The total number of PPIs displaying very high fold-enrichment levels (Log2 (fold-difference vs. G.luc HA control) > 5) varied significantly across baits, with up to ten-fold difference between the lowest (DENV-NS4B, 43 interactors) and the highest (WNV-NS4B, 386 interactors) number of PPI observed for individual species (Fig. 2a). Notably, these differences did not correlate linearly with either the expression levels of the baits, or the total number of preys identified (Supplementary Fig. 1c, Fig. 2a), suggesting a certain degree of divergence of NS4B-host-interactions within the Orthoflavivirus genus.

The systematic nature of the orthoflavivirus NS4Bome allows for the first time the identification and classification of human cellular targets into a core group of NS4B interacting proteins (broadly conserved, highly enriched interacting proteins), an extended group orthoflavivirus targets (proteins within pathways broadly targeted by the majority of the viral species) and a distinctive group of proteins selectively enriched in individual or a small number of species (sub- or species-specific binding profiles).

The core human interactome of orthoflavivirus NS4B

To define the core NS4Bome, we extracted from the extended NS4Bome proteins displaying very high enrichment levels (log2 (fold-enrichment) > 5) and significantly bound by at least 6 out of 8 viral species (Fig. 2a, Supplementary Fig. 2b). In total we mapped 74 host proteins within this class of interactors, including factors previously reported to interact with individual orthoflavivirus NS4Bs, such as BSG14, CHP114, STT3A19, SEC61G12, VDAC1/27 or TMEM41B14,20, as well as novel host interactors, such as GPX8, UNC50 and STX17 (Supplementary Fig. 2c, d). Beside interactions with discrete cellular proteins, the core NS4Bome revealed enrichment of distinctive cellular processes, including the translocon complex (SEC61B, SSR1, SSR3), the very-long chain fatty acid biosynthetic pathway (HSD17-B1-11-12, DHCR2 and DHCR7, FADS2 and FAR1) as well as mitochondrial respiration (COX6A1, NDUFA11, ATP5L) and dynamics (FIS1, MFN2). Furthermore, the oligosaccharyltransferase complex and signal recognition particle–dependent co-translational targeting to membranes and lipid biosynthesis were significantly overrepresented pathways, highlighting an evolutionary conserved targeting of processes involved in protein synthesis and lipid metabolism (Supplementary Fig. 2b, c, d).

Collectively, these proteins likely support evolutionary conserved mechanisms of host-adaptation across the Orthoflavivirus genus, providing cues into the key mediators of NS4B functions and a list of valuable targets for the rational design of pan-viral host-targeting antivirals.

The extended human interactome of orthoflavivirus NS4B

To identify pathways underlying functionally conserved mechanisms of host-adaptation across orthoflaviviruses, we additionally performed upset and gene ontology (GO) enrichment analysis on the extended NS4B PPI network. This approach revealed a higher number of shared interactions and targeted cellular pathways between WNV, YFV and ZIKV NS4B when compared to other members of the genus, suggesting a higher degree of functional conservation across these species (Fig. 2a). Furthermore, this analysis revealed significant enrichment of interactors associated with mitochondrial membrane organization and autophagy, consistent with the intricate relationship between orthoflaviviruses, the ER and mitochondria7,21–23 (Fig. 2b, c; and Supplementary Data 1). Notably, proteins involved in mitochondrial membrane organization, mitochondrial respiration and respiratory electron transport were amongst the most enriched cellular targets of the global PPI network, suggesting a distinctive recruitment of processes involved in oxidative phosphorylation via multiple interactions with mitochondrial membrane proteins.

Additionally, the extended NS4Bome illuminated unexpected interactions of NS4B with pathways involved in intracellular protein transport (COPII-mediated vesicular transport) and intercellular interactions (basigin interactions), residing in ER-distal organelles including the Golgi apparatus and the trans-Golgi network (Fig. 2c, Supplementary Fig. 2d). Importantly, it consolidates and expands the interconnections between NS4B and mitochondria-resident processes7,24, highlighting specific targeting of proteins involved in the mitochondrial respiratory chain.

Host processes and complexes targeted by individual orthoflaviviral NS4B proteins

In addition to proteins and pathways broadly targeted by the majority of orthoflavivirus NS4B, the extended NS4Bome enabled the identification of species-specific PPIs displaying highly selective binding profiles, providing a rational framework for mechanistic and functional follow-up studies on individual species. Examples of virus-specific interactions include proteins involved in protein sorting at the Golgi apparatus (VPS53 and VSP54, POWV), pyruvate dehydrogenase complex activity (PDP2, JEV) and ER membrane curvature (FAM134C, YFV) (Fig. 2d–g; and Supplementary Data 1). Interestingly, comparative analysis of PPIs between historical and contemporary strains of DENV2 (16681 and New Guinea C) also identified a number of isolate-specific PPIs, providing potential cues into differential host-responses and the replicative fitness observed in vitro25, and validating a large proportion of host interactors shared by both isolates such as TMEM165, ATP5L and NDUFA11 (Supplementary Fig. 1e).

To chart virus–host interactions into effector functions of viral proteins, we next analyzed the global proteomes of JEG-3 cells persistently expressing individual NS4Bs (“effectome”; Fig. 1a; and Supplementary Data 2). This approach identified 514 host proteins whose relative abundance was significantly modulated by expression of at least one NS4B orthologue. Intersecting individual NS4B interactomes and their corresponding effectomes, highlighted a subgroup of NS4B interacting proteins displaying altered steady state expression levels, providing direct links between NS4B-interactions and down-regulation of selected host proteins (Supplementary Fig. 2e–m). These include proteins previously associated with interferon (IFN) -dependent restriction of viral replication26 (IFITM3; HCV and YFV), providing a mechanistic basis for active counteraction of innate immune responses. This approach also revealed several host proteins involved in the ER-associated degradation machinery (i.e., DERL2; USUV and C6orf120; ZIKV, YFV, WNV) and selective components of the electron transport chain (i.e., MT-ND3; ZIKV, YFV, WNV), highlighting novel host proteins bound by NS4B and specifically targeted for degradation (Supplementary Fig. 2e–m and Supplementary Data 2).

Altogether, the orthoflavivirus NS4Bome provides an accurate cartography of NS4B-associated effector functions, providing an evidence-based framework to categorize and characterize broadly conserved as well as species-specific virus–host PPIs involved in orthoflavivirus replication.

Targeted RNAi-screening uncovers NS4B-interacting host factors impacting ZIKV replication

To assess the functional relevance of the newly identified NS4B-interacting host factors in viral replication, we selected 58 interactors for further phenotypic characterization in loss-of-function analysis. Candidate host-proteins were selected from each of the categories described above, spanning species-, sub- and pan-viral specificities (Fig. 3a, b). ZIKV was selected for functional follow-up experiments because of the specific cellular background closely resembling one of the known tissues targeted in vivo (placenta). JEG-3 cells were transduced with individual short hairpin RNA (shRNA)-encoding lentiviruses and infected with a ZIKV H/PF/2013 luciferase reporter virus27 (Fig. 3a). This approach identified 14 anti-viral and 5 pro-viral genes whose silencing significantly enhanced or impaired ZIKV replication, respectively, in the absence of overt cytotoxicity (Fig. 3c, red and blue shades; Supplementary Fig. 3a, b and Supplementary Data 3). Interestingly, these newly identified host dependency and host restriction factors include ZIKV-NS4B–interacting proteins displaying both higher (i.e., UBA5 and HSD17B1) and lower (HOOK2 and HSD17B7) enrichments than previously described and validated interactors (Fig. 3d, red and black circles, respectively), corroborating the value of the curated NS4Bome in detecting a wide-range of functionally relevant associations. Co-immunoprecipitation followed by western blotting (co-IP) confirmed that multiple proteins specifically associate with several orthoflavivirus NS4Bs (HOOK2, TECR, HSD17B1, HACD3; Supplementary Fig. 3c), largely mirroring AP-MS data (Fig. 3b). To further validate NS4B interactions in the context of productive viral infection, we engineered a ZIKV infectious clone carrying an internal HA-tag within NS4B (ZIKV-NS4BHA) (Supplementary Fig. 4a). We confirmed that the insertion of the HA-tag only slightly reduced replication levels (Supplementary Fig. 4b), and validated epitope tag stability and its co-localization with NS4B in infected cells (Supplementary Fig. 4c, d). Co-IP of endogenous host proteins in ZIKV-NS4BHA-infected JEG-3 cells confirmed specific NS4B interactions with HSD17B7, FPGS and UBA5 (Fig. 3e). Altogether, these experiments identify a new set of bona fide ZIKV-NS4B interactors functionally promoting or restricting viral replication.

UBA5 exerts a UFMylation-dependent pro-viral role in ZIKV replication

Among the strongest ZIKV-NS4B interactors, we identified ubiquitin-like modifier-activating enzyme 5 (UBA5), an E1 activating enzyme that specifically catalyzes the first step of UFMylation28. UFMylation is an emerging post-translational modification involved in regulation of the DNA damage response29–31, ER stress32, antiviral RIG-I signalling33, autophagy34, and protein synthesis through regulation of the ER translocon35,36.

To validate the pro-viral role of UBA5 in ZIKV replication, we generated stable UBA5 knock-down JEG-3 cells (Fig. 4a) and confirmed a significant reduction in viral RNA replication (Fig. 4b) and infectious particle production (Fig. 4c) using wild-type ZIKV H/PF/2013 as well as a luciferase-reporter ZIKV (Fig. 4d). Importantly, ectopic expression of FLAG-tagged UBA5 in UBA5-deficient cells rescued ZIKV replication, corroborating the specificity of the observed pro-viral effects. UFM1-reactive conjugates (UFC1-UFM1 and UBA5-UFM1) (Fig. 4f). Strikingly, ZIKV replication was even more severely impaired compared to UBA5 knock-down cells (Fig. 4g), and was fully rescued by ectopic expression of wild-type UBA5 (Fig. 4h). Interestingly, two distinct UBA5 mutants severely impaired in their ability to sustain functional UFMylation due to selective defects in UFM1 activation (UBA5_Mut1) or UFC1 binding38 (UBA5_Mut2), failed to rescue viral replication, demonstrating a functional role for UFMylation in viral replication. Altogether, these results unequivocally confirm a pro-viral role of UBA5 in viral replication via a UFMylation-dependent mechanism.

Multiple UFMylation pathway members are recruited to viral replication compartments via interactions with several viral proteins

To further characterize the impact of ZIKV infection on UFMylation, we next analyzed the migration profile of UFM1-reactive species under non-denaturing conditions (Fig. 5a). In agreement with previous reports38, we identified all the major UFM1-conjugates, including free UFM1, UFC1-UFM1 and UBA5-UFM1, alongside a few additional UFM1-reactive species. However, their relative abundance was not significantly affected by ZIKV infection (Fig. 5a). To further investigate potential perturbations within the UFMylation pathway, we next characterized the interactions of UFM1 with the core components of this pathway. As expected, FLAG-tagged mature UFM1 (FLAGUFM1ΔSC) was found to form complexes with UBA5, UFC1 and UFL1. However, none of these interactions was significantly modulated by ZIKV infection (Fig. 5b). Furthermore, analysis of crude fractions from mock- or ZIKV-infected cells, revealed no major differences in the degree of UFMylated RPL26 at the ER, suggesting comparable levels of UFMylation at the ER (Supplementary Fig. 5a). We next assessed potential co-opting of UFMylation components by viral replication complexes, characterizing the subcellular distribution of several pathway members in ZIKV-infected cells. Upon infection, both UFL1 and UFSP2, dramatically redistributed to viral replication sites and co-localized with NS4B and the viral protease/helicase NS3 (Fig. 5c). Notably, analysis of UFM1 eluates from infected cells, revealed a significant enrichment of both NS3 and its co-factor NS2B (Fig. 5d). Collectively, these results suggest that multiple UFMylation components are actively recruited to sites of viral replication via multipartite interactions with distinct elements of the viral replicase.

The UFMylation pathway exerts a conserved pro-viral function across the Orthoflavivirus genus

Having identified multiple interactions between viral proteins and members of the UFMylation pathway, we retrieved the interaction profiles of all detected UFMylation pathway members across our entire NS4Bome. This analysis revealed that, albeit with lower affinities, the NS4B proteins of other orthoflaviviruses such as DENV and YFV, also associate with UBA5 (Fig. 5e). Surprisingly, we identified additional associations of nearly all orthoflavivirus NS4Bs with at least one UFMylation pathway component, such as UFL1 (USUV, WNV, YFV, ZIKV), ODR4 (YFV, WNV) and UFSP2 (USUV, WNV, YFV, ZIKV, JEV) (Fig. 5e), suggesting a conserved targeting of the pathway through selectively divergent mechanisms. Unexpectedly, also the capsid protein of ZIKV (one of the control baits in our NS4Bome) associated very strongly with UFM1 itself and with CDK5RAP3, a substrate adaptor that directs UFMylation to the ribosomal protein RPL2639 (Fig. 5e).

To determine whether UFMylation is functionally required across the entire viral genus, we tested the replication fitness of multiple orthoflaviviruses in UBA5 KO JEG-3 cells (Fig. 5f). These experiments revealed a significant inhibition of viral replication across a broad range of viral species including DENV, YFV, JEV, ZIKV and WNV (40-99% inhibition compared to gNT control, Fig. 5f). In agreement with similar reports on other DNA viruses, such as Epstein-Barr virus (EBV)39, replication of herpes simplex virus 1 (HSV-1) was significantly up-regulated in UBA5 KO cells (Fig. 5f), likely as a result of reduced type I IFN responses33,40.

Altogether, these results demonstrate that UFMylation exerts a broadly-conserved pro-viral role in orthoflavivirus replication, through multipartite interactions between multiple UFMylation pathway members and several viral proteins.

Pharmacological inhibition of UFMylation results in antiviral activity

Several UFMylation inhibitors are being investigated for therapeutic applications, particularly in cancer and other diseases41–44. Among these, we evaluated the antiviral potential of DKM 2-93, a selective small-molecule inhibitor that covalently binds UBA5 catalytic cysteine, thereby inhibiting UFMylation45 (Fig. 6a, Supplementary Fig. 6a). DKM 2-93 significantly inhibited ZIKV replication in the low micromolar range without overt cytotoxicity (EC50=12,92 μM, CC50=81,23 μM; Fig. 6b). These effects were characterized by a reduction in both intracellular viral protein abundance (Fig. 6c) and infectious particle production by 10-fold (Fig. 6d), and could be phenocopied in multiple human cell lines (Fig. 6f, g and Supplementary Fig. 6b)

To identify the step(s) of the replication cycle targeted by UBA5 inhibition, we performed time-of-addition experiments, adding the compound before (pre-treatment), during (co-treatment) or after (post-treatment) virus absorption, and measured ZIKV replication after 48h. DKM 2-93 retained significant and comparable anti-ZIKV inhibitory activity when added up to 3h after infection, suggesting a role of UFMylation in a post-entry step of viral replication (Fig. 6e). To determine whether DKM 2-93 affects viral RNA (vRNA) synthesis and/or translation, we used a modified ZIKV subgenomic replicon (sgZIKV-R2A)27—a self-replicating vRNA lacking the coding sequences of structural proteins and therefore unable to produce viral particles—and evaluated the effect of the drug on luciferase expression following in vitro transcription and electroporation of these RNAs (Fig. 6f, g). Under these conditions, intracellular luciferase activity measured over time exclusively reflects efficient vRNA translation and replication, independent of viral entry and virus assembly or release. Luciferase activity at 4h post electroporation was not impacted by drug treatment, thus excluding DKM 2-93 effects on viral translation (Fig. 6f). Similarly, DKM 2-93 treatment did not significantly affect ZIKV replication up to 96h post-electroporation (Fig. 6g). In contrast, treatment of cells infected with a full-length ZIKV-R2A resulted in significant reduction of viral replication at late stages of infection (Fig. 6b), suggesting a selective inhibition of virion assembly or virus release. Collectively, these results indicate that functional UFMylation is critically required for late stages of the viral replication cycle, independent from viral entry, viral RNA translation and viral RNA replication.

UFMylation modulates viral replication via an IFN-independent mechanism

To shed light on potential mechanisms underlying UFMylation-dependent pro-viral functions, we next assessed the impact of UBA5 inhibition on cellular transcriptional responses. We analyzed transcriptomic changes in either control (shNT) or UBA5 knock-down cells (shUBA5), following mock or ZIKV infection (Supplementary Fig. 5). Geneset enrichment analysis of transcripts significantly regulated by UBA5 silencing, revealed an overall moderate impact on mRNA abundance, with only 15 and 11 transcripts robustly up- and down-regulated, respectively (|Log2(Fold-change)| > 2; Supplementary Fig. 5b). Among these, we observed an upregulation of genes involved in mitochondrial protein import (TIMM17A) and pre-mRNA processing (U2AF1L5, and RBMY1F) and down-regulation of genes involved in heme metabolism (HMOX1), cellular stress responses (TXNIP) and RNA binding (RBMY1E) (Supplementary Data 4, Supplementary Fig. 5b, c). These changes were largely observed also in ZIKV-infected cells, suggesting a lack of correlation with UFMylation-dependent pro-viral functions (Supplementary Fig. 5d, e). As expected, ZIKV infection robustly stimulated innate immune responses, as inferred by the analysis of an extended panel of 48 known IFN-stimulated genes (ISGs), including RIG-I, IFIT1, IFIT3 and IFNL2 (Supplementary Fig. 5f). Interestingly, silencing of UBA5 did not exacerbate innate immune responses in ZIKV infected cells, in agreement with recent reports describing UFMylation as a positive post-translational regulator of type I IFN responses34,46.

Altogether, these results suggest that the inhibition in viral replication observed UBA5-deficient cells is associated with a mild transcriptomics signature and is not due to exacerbated activation of innate anti-viral immune signalling.

UFMylation modulates mitochondrial respiration and morphodynamics

Orthoflaviviruses, including DENV and ZIKV, profoundly alter mitochondrial respiration in a time-dependent manner, correlating with changes in the stoichiometry of the electron transport chain and the abundance of Krebs cycle metabolites24. In addition, NS4B expression induces mitochondrial elongation by counteraction of the fission machinery, ultimately promoting virus replication7,47. Interestingly, our NS4Bome identified oxidative phosphorylation (OXPHOS) and mitochondrial processes-associated factors amongst the most enriched NS4B interactors broadly targeted by the majority of orthoflaviviruses (Fig. 2c), suggesting a possible functional link between UFMylation and mitochondria. To determine whether UFMylation inhibition perturbs the mitochondrial network, we analyzed several morphological features of the mitochondrial network in UBA5 KO (JEG-3 gUBA5) and control (JEG-3 gNT) cells (Fig. 7a–g). UBA5 depletion was associated with a significant increase in length (Fig. 7d, e), branching (Fig. 7f) and connectivity (Fig. 7g) of the mitochondrial network, without affecting their area or overall perimeter (Fig. 7b, c). Comparable results were obtained when JEG-3 cells were treated with DKM 2-93 (Supplementary Fig. 7a–g), further corroborating the specificity of this UBA5 inhibitor and the notion that UBA5 negatively regulates mitochondria elongation. Considering that enhanced elongation of mitochondria was reported to promote DENV and ZIKV replication in liver cells7,47, these results suggest that the proflaviviral role of UBA5 is not linked to its activity on mitochondria morphodynamics.

We next investigated the impact of UBA5 depletion and pharmacological inhibition on multiple parameters of mitochondrial respiration in Seahorse assays, by measuring the oxygen consumption rates (OCR) while sequentially adding selective inhibitors of the electron transport chain of the inner mitochondrial membrane (Fig. 7h–k). JEG-3 gUBA5 cells showed a significant reduction in basal respiration (Fig. 7i), ATP-linked respiration (Fig. 7j), and maximal respiration (Fig. 7k). These effects were fully phenocopied by treatment of JEG-3 cells with DKM 2-93, suggesting a direct modulation of mitochondrial respiration by UFMylation (Supplementary Fig. 7). In contrast, ZIKV infection elicited a reciprocal increase in all OCR parameters at 24 hpi in line with previous observations in liver cells47, confirming an upregulation of mitochondrial respiration at early stages of orthoflavivirus infections (Fig. 7l–o).

Together these results establish a novel connection between UFMylation and mitochondrial respiration, suggesting that UBA5 may contribute to ZIKV-induced stimulation of mitochondrial respiration to promote viral replication.

UFMylation inhibition exerts antiviral activity in vivo

We have recently established an in vivo zebrafish model of ZIKV infection, which phenocopies several patho-physiological features associated with ZIKV infections in humans, such as microcephaly as well as neural progenitor cells infection and depletion48. This model capitalizes two major advantages: (i) the rapid development of the embryonic/larval brain, which allows phenotypic characterization of host and viral determinants involved in ZIKV-induced neuropathogenesis, and (ii) time-effective quantitative assessment of antiviral drug efficacy in a large number of animals (2–3 days)48. To assess the antiviral potential of UFMylation pathway inhibition in vivo, zebrafish embryos were infected with ZIKV at the 2-4 cell stage and subsequently treated with non-cytotoxic concentrations of DKM 2-93 for two days, i.e. during the first stages of brain development (Fig. 8a). Treatment with NITD008, a pan-flaviviral NS5 polymerase inhibitor49, was used as control. The inhibition of UFMylation by DKM 2-93 significantly reduced ZIKV vRNA levels in whole larvae (pool of 6-10 larvae per condition per independent experiment) at 48h post-infection/fertilization compared to DMSO-treated animals (Fig. 8b, c). Importantly, the parallel analysis of ZIKV-induced morphological defects (such as abnormal morphology, curved tail and oedema) on 225 larvae revealed that this reduction in viral replication correlated with a significant reversion of virus-induced comorbidities in the infected larvae (from 58,65% to 31,17%, and from 22,96% to 18,18% of animals displaying moderate and severe phenotypes, respectively) when compared to DMSO-treated animals (Fig. 8d).

Collectively, these results demonstrate significant antiviral activity of DKM 2-93 in vivo and corroborate the functional relevance of the UFMylation pathway in ZIKV pathogenesis during organism development. This further highlights the therapeutic potential of the UFMylation pathway as a novel host-targeting antiviral strategy to control viral infections in vivo and attenuating neurovirulence in the developing brain.

Methods
Plasmids

The pWPI_puro constructs of the orthoflavivirus 2k-NS4B, Gaussia luciferase (GLuc), HCV-NS4B, and ZIKV-capsid, all C-terminally tagged with HA were synthesized as gBlocks fragments (Integrated DNA Technologies), and cloned into pWPI_puro vector using BamHI and SpeI sites. The pLKO constructs encoding non-targeting shRNA (plasmid SHC001; shNT-1), shRNA targeting the known virus restriction factor ATP6V0C (shATP6V0C-1), shRNA targeting UBA5 (clone ID: TRCN0000008018; shUBA5) and two shRNAs targeting UFM1 (shUFM1_1: TRCN0000232599 and shUFM1_2: TRCN 0000037004) were obtained from the MISSION TRC lentiviral library (Sigma-Aldrich).

A shRNA- and gRNA-resistant, N-terminally FLAG-tagged UBA5 (FLAGUBA5) and a guide RNA-resistant, N-terminally FLAG-tagged mature UFM1 (FLAGUFM1ΔSC) were synthesized as gBlocks fragments (Integrated DNA Technologies). FLAGUBA5 shRNA- and gRNA-resistant variants were generated by shuffling the codons in the shRNA and gRNA targeting region of the wild type UBA5 (shRNA: 901CCTCAGTGTGATGACAGAAAT921 and gRNA: 223GCCGTAGCAATAGTAGGTGT242) using the online tool ‘Synonymous Mutation Generator’ (http://jong2.pythonanywhere.com) and manually correcting the Codon Adaptation Index. shRNA resistant UBA5: 901CCCCAATGCGACGATCGCAAC921 and gRNA resistant UBA5: 223GCTGTCGCCATCGTCGGCGT242. UFM1 is translated in the cells in its precursor immature form that contains a serine followed by a cysteine amino acid residues at its C-term end. The cellular proteases UFSP2 cleaves these two residues during the UFM1 maturation process leaving the glycine residue as the neo C-term. Upon conjugation to target substrates, this glycine residue of the cleaved mature UFM1 forms an iso-peptide bond with a lysine residue of the target protein. UFM1ΔSC denotes the mature UFM1. The FLAGUFM1ΔSC was also made gRNA resistant in a similar way as FLAGUBA5. gRNA resistant UFM1: 15CTTCAAAATAACCCTCACCA34. FLAGUBA5 was PCR-amplified using the primers UBA5_Fwd: 5′-CGCGCCGGATCCGCCACCATG-3′ and UBA5_Rev: 5′-TAGTTTACTAGTCTACATATTCTTCATTTTGGCCATGAGGTCTTCCAAGC-3′, and cloned into the pWPI_puro vector using BamHI and SpeI sites. This construct served as a template to generate UBA5 mutants (UBA5_Mut1 and UBA5_Mut2) via overlap extension PCR, using the primers UBA5_Fwd, UBA5_Mut1_Fwd: 5′-GAAACGAGAAGGTGTTCGTGCAGCCAGTCTTCCT-3′, UBA5_Mut1_Rev: 5′-AGGAAGACTGGCTGCACGAACACCTTCTCGTTTC-3′ and UBA5_Mut2_Rev: 5′-GTAGTTTACTAGTCTACATATTCTTCCTTTTGGCCATCCTGTCTTCCAAGCTTTCACCAG-3′. Mature UFM1 was cloned similarly by inserting BamHI- and SpeI-digested gBlock fragments into the pWPI vector. The pWPI construct of FLAGUFM1ΔSC was used a template for the generation of the UFMylation deficient UFM1 mutant FLAGUFM1ΔGSC and de-UFMylation resistant UFM1 mutant FLAGUFM1G83A constructs using primers Fwd: 5′-AGCTTTGTTTAAACGGCGCGGATCCGCCACCATG-3′ and Rev: 5′-GCCCGTAGTTTACTAGTTTATTACACCCGATCTCTAGGAATAATCCG-3′ (for FLAGUFM1ΔGSC) or Rev: 5′-GCCCGTAGTTTACTAGTTTATGCCACCCGATCTCTAGGAATAATCCG-3′ (for FLAGUFM1G83A). The mutants were cloned using BamHI and SpeI sites.

To generate the CRISPR-Cas9 gRNA targeting UBA5 (gUBA5), UFM1 (gUFM1) and UFSP2 (UFSP2) the oligos 5′-CACCGAAGCAGCAGAACATACTCTG-3′ and 5′-AAACCAGAGTATGTTCTGCTGCTTC-3′ (for UBA5), 5′-CACCGCTTTAAGATCACGCTGACGT-3′ and 5′-AAACACGTCAGCGTGATCTTAAAGC-3′ (for UFM1), and 5′-CACCG AATAAGAGGAGGCCTTGATT-3′ and 5′-AAACAATCAAGGCCTCCT CTT ATT C-3′ (for UFSP2) were annealed and cloned into the pLentiCRISPRv2 plasmid, following previously described protocols67,68. Control non-targeting gRNA (gNT) pLentiCRISPRv2 and empty vector pLentiCRISPRv2 were previously described (Addgene).

The pFK_ZIKV_HPF201327 construct was used as a template to generate a pFK-based ZIKV H/PF/2013 construct carrying an HA epitope within NS4B (pFK_ZIKV-NS4BHA), between nucleotides 66 and 67 (corresponding to the site between the 22nd and 23rd amino acid residues of ZIKV NS4B protein). A two-step overlap extension PCR with the primers NS4A_SacII_Fwd: 5′-AAGCCGCGGCGGCCCAATTGCCGGAG-3′, NS4B_HA_Rev: 5′-AGCGTAATCTGGAACATCGTATGGGTATGATCCCTCCTCTCTCCTTCCCATTAG-3′, NS4B_HA_Fwd: 5′-TACCCATACGATGTTCCAGATTACGCTGGATCAGGGGCAACCATAGGATTCTCAATGGAC-3′ and NS5_EcoRI_Rev: 5′-AGTAGAATTCCAGGGCCGACATCTGGTTC-3′, was performed to introduce the HA epitope with the NS4B. The amplicon was cloned using SacII and EcoRI sites. The reporter construct pFK_ZIKV_R2A was previously described27.

Antibodies

The rabbit polyclonal anti-human UBA5 (12093-1-AP, WB: 1:1’000), HSD17B7 (14854-1-AP; WB, 1:1’000), HACD3 (28572-1-AP; WB, 1:1’000), mouse anti-Calnexin (66903-1-Ig; WB, 1:1’000; IF, 1:500), ChromoTek monoclonal rat anti-HA (7c9; 1:500) antibodies, and secondary anti-rat horseradish peroxidase (HRP)-conjugated antibodies (SA00001-15; WB, 1:1’000), were purchased from Proteintech. The rabbit monoclonal anti-human UFM1 (ab109305; WB, 1:1’000), UFC1 (ab189252; WB, 1:3’000; IF, 1:500), and UFSP2 (ab192597; WB, 1:1’000; IF, 1:500) antibodies were obtained from Abcam. The rabbit polyclonal antibody recognizing human UFL1 protein (NBP1-90691; WB, 1:1’000; IF, 1:500) was purchased from Novus Biologicals. The mouse monoclonal antibody recognizing ZIKV NS1 protein (GTX634158), and polyclonal rabbit anti-ZIKV NS2B (GTX133318), NS4A (GTX133704), and NS4B (GTX133311) antibodies (WB, 1:1’000), were purchased from GeneTex. The generation of the rat polyclonal anti-NS3 antibody by Medimabs (Montréal, Canada) was previously described (WB, 1:3’000; IF, 1:500)51. The mouse monoclonal antibodies recognizing FLAG (A8592; WB: 1:5000), FPGS (HPA050488; WB, 1:1000), HOOK2 (HPA043519; WB, 1:1000), TECR (HPA056488; WB, 1:1000), and HSD17B1 (HPA021032; WB, 1:1000), and secondary anti-mouse (A4416) and anti-rabbit (A6154) HRP-conjugated antibodies (WB, 1:10’000), along with agarose-conjugated anti-HA (A2095) and anti-FLAG (A2220) beads, were purchased from Sigma-Aldrich. The mouse monoclonal antibodies recognizing HA (2999; WB, 1:1000; IF, 1:100), the rabbit monoclonal anti-human COXIV (4850) antibody, and HRP-conjugated anti-human GAPDH (51332; WB, 1:5000) were purchased from Cell Signaling. The anti-human β-Actin monoclonal antibodies (sc-47778; WB, 1:5000) was purchased from Santa Cruz Biotechnology. Secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 568, and Alexa Fluor 647, for anti-rabbit (A21206), anti-mouse (A10037, A21235), and anti-rat antibodies (A21247, A21208), respectively, were purchased from Invitrogen (IF, 1:500).

Cell Culture

JEG-3 cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640; Sigma-Aldrich), supplemented with L-glutamine and sodium bicarbonate. HEK293T, Vero E6, HeLa, Huh7, and MRC-5 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich). Both media were supplemented with 10% fetal bovine serum (FBS; Capricorn) and 1% penicillin–streptomycin (Sigma-Aldrich). Cells were maintained at 37°C in a humidified incubator with 5% CO₂. All cell lines were authenticated by STR profiling (Eurofins Genomics).

Generation of stable cell lines

Stable JEG-3 cell lines expressing C-terminally HA tagged 2k-NS4B of eight different orthoflaviviruses (DENV2, JEV, USUV, WNV, YFV, ZIKV, POWV and TBEV), Gaussia luciferase (GLuc), HCV-NS4B, and ZIKV-capsid were generated by lentiviral transduction. Stable JEG-3 cell lines expressing short hairpin RNAs (shRNAs) targeting either ATP6V0C (shATP6V0C), UBA5 (shUBA5) or UFM1 (shUFM_1 and shUFM1_2), as well as a non-targeting control (shNT), were also generated by lentiviral transduction. JEG-3 cells (30,000 cells per 500µL per well in a 24-well plate) were transduced with lentiviruses encoding the respective shRNAs at a multiplicity of infection (MOI) of 3. Twenty-four hours post-transduction, the medium was replaced with fresh complete RPMI supplemented with 1µg/mL puromycin (Sigma-Aldrich). Three days later, once all cells in the non-transduced control well were eliminated, puromycin-resistant cells were expanded. To generate JEG-3_shUBA5 cells reconstituted with shRNA-resistant FLAG-tagged UBA5 (JEG-3_shUBA5+FLAGUBA5), lentiviral transduction was performed using the corresponding overexpression construct. JEG-3 cells overexpressing FLAG-tagged mature UFM1 (JEG-3_FLAGUFM1ΔSC), UFMylation deficient UFM1 mutant (Mut1, JEG-3_FLAGUFM1ΔGSC) and de-UFMylation resistant UFM1 mutant (Mut2, JEG-3_FLAGUFM1G83A) were generated similarly by transducing cells with lentiviruses carrying the relevant overexpression plasmid. Knockout JEG-3 cell lines were generated using CRISPR-Cas9. Cells were transduced with lentiviruses encoding guide RNAs targeting UBA5 (JEG-3 gUBA5), a non-targeting control (JEG-3 gNT). Following knockout validation by western blotting, single-cell clones were obtained by serial dilution in 96-well plates. Clonal populations were monitored for two weeks and subsequently expanded.

Lentivirus production

Lentiviral particles were produced by co-transfecting HEK293T cells with the appropriate pLKO-, pWPI-, or pLentiCRISPRv2-based transfer plasmids and packaging vectors (pMD2-VSV-G and either psPAX2 or pCMV-GagPol). Subconfluent monolayers of HEK293T cells were seeded in 6-well plates or 10-cm2 dishes, and co-transfected with the packaging plasmids pMD2-VSV-G and psPAX2 (for shRNA transfer plasmids) or pCMV-GagPol (for overexpression plasmids), and the transfer plasmid encoding the gene of interest. Plasmids were mixed at a 1:0.5:1.5 ratio (transfer:packaging:envelope), and polyethylenimine (PEI) was added at a PEI:DNA ratio of 3:1. The DNA–PEI complexes were incubated at room temperature for 20 min before being added dropwise to cells. Culture media were replaced 16h post-transfection. Lentivirus-containing supernatants were collected at 48, 56, and 72h post-transfection, pooled, 0.45µm-filtered and stored at −80°C until use. Viral titers were estimated by colony-forming unit (CFU) assay on HeLa cells as previously described69.

Lentiviral shRNA Library Production and shRNA Screen

Three different shRNAs targeting each of the 58 MS hits and 4 controls (non-targeting shRNA-encoding plasmids SHC001 and SHC002—shNT-1 and shNT-2, and plasmids encoding shRNA targeting the known host dependency factor ATP6V0C—ATP6V0C-1 and ATP6V0C-2) were selected from the MISSION TRC lentiviral library (Sigma-Aldrich; a total of 178 shRNAs; Supplementary Data 1). Lentiviruses encoding individual shRNAs were produced in HEK293T cells by co-transfection with the packaging plasmids psPAX2 and pMD2-VSV-G, and the shRNA-encoding pLKO plasmids. Lentiviruses were harvested at 48, 56, and 72h post-transfection. For quality control, ~25% of the shRNA library was randomly selected to measure lentiviral titers. JEG-3 cells were seeded at 20,000 cells in 500µL per well in 24-well plates for the screen. Cells were transduced with lentiviruses (MOI≈3; one shRNA/well) in triplicate wells and three independent biological replicates. Three days post-transduction, cells were infected with the ZIKV H/PF/2013 reporter strain expressing Renilla luciferase (ZIKV-R2A, MOI=0.1). Forty-eight hours post-infection, wells were washed once with PBS and luciferase activity measured as previously described70. Luminescence values for the target shRNAs were normalized to those of the non-targeting shRNAs, and hits were selected based on the criteria of a 50% difference in viral replication with two out of three shRNAs or >75% difference with one out of three shRNAs. Cell viability was assessed by a resazurin-based cell viability assay at 72h post-transduction on plates transduced with lentiviruses in parallel. Individual shRNAs were considered toxic if the cell viability (% of shNT-1 or shNT-2) was ≤75%.

Virus stock production

Stocks of dengue virus (strain UVE/DENV-2/2018/RE/47099), Japanese encephalitis virus (strain UVE/JEV/2009/LA/CNS769), West Nile virus (UVE/WNV/UNK/CF/Ar B 3573/82), Yellow fever virus (strain Asibi), Zika virus (strain H/PF/2013), and herpes simplex virus 1 (strain F) were produced in Vero E6 cells. Stocks of ZIKV H/PF/2013 reporter strains, either expressing Renilla luciferase (ZIKV-R2A), carrying an HA epitope within NS4B (ZIKV-NS4BHA) were produced by electroporating Vero E6 cells with in vitro-transcribed viral RNA. In both cases, culture medium was replaced the day after infection or electroporation with DMEM supplemented with 2% fetal bovine serum (FBS) and 15 mM HEPES (no HEPES for HSV-1). Supernatants were collected 4–6 days post-infection, clarified through 0.45µm filters, aliquoted, and stored at −80°C. Viral titers of infectious clones were determined by plaque assay; while titers of reporter viruses were determined by 50% tissue culture infectious dose (TCID₅₀) assay as previously described70.

In vitro transcription (IVT) and electroporation of RNA

Plasmid DNA (10µg) encoding the transcript was linearized with XhoI (New England Biolabs) and purified using the phenol–chloroform extraction method. The ethanol-precipitated DNA was air-dried and resuspended in nuclease-free water. In vitro transcription was carried out using the RiboMAX™ Large Scale RNA Production System-T7 (Promega) with linearized plasmid DNA as template. Reactions (100µL) were assembled with 3.125 mM each of rATP, rCTP, and rUTP; 0.625 mM rGTP; 1X T7 transcription buffer; 1:10 diluted T7 enzyme mix; and 1 mM Anti-Reverse Cap Analog (ARCA; New England Biolabs), and incubated for 30 min at 37°C. Subsequently, 2.5 mM rGTP was added, and the reaction was incubated for an additional 6h at 37°C. Transcription was terminated by treatment with 10 U RQ1 RNase-free DNase (Promega) for 30 min at 37°C. RNA was extracted with acidic phenol–chloroform, precipitated with isopropanol, and resuspended in nuclease-free water. RNA concentration was determined using a Nanodrop, and integrity was assessed by agarose gel electrophoresis.

For electroporation of ZIKV full-length RNA (ZIKV H/PF/2013 reporter strain expressing Renilla luciferase) and subgenomic replicons into Huh7 (or Vero E6 cells for virus stock production), cells were resuspended at a density of 1×10⁷ cells/mL (1.5×10⁷ cells/mL for Vero E6 cells) in Cytomix buffer (120 mM KCl, 0.15 mM CaCl₂, 10 mM potassium phosphate buffer pH 7.6, 25 mM HEPES pH 7.6, 2 mM EGTA, 5 mM MgCl₂) supplemented with 2 mM ATP and 5 mM glutathione. For each reaction, 200µL of resuspended cells were mixed with 5µg RNA (400µL cells and 10µg RNA for virus stock production), and electroporated using a Gene Pulser system (Bio-Rad) in a 0.2-cm gap cuvette (0.4-cm cuvette for 400µL volume). Cells were immediately transferred to 6 mL complete DMEM, and 500µL of the suspension was seeded per well in a 24-well plate. For virus stock production, cells were pooled from two electroporations into 25 mL DMEM and seeded into a 15-cm dish.

Virus titrations

Plaque assays were performed by seeding Vero E6 cells (2×10⁵ cells in 500µL per well) in 24-well plates 24h prior to infection. Confluent monolayers were infected with serial tenfold dilutions of virus-containing supernatants and incubated for 1h at 37°C. Following infection, the inoculum was removed and replaced with Minimum Essential Medium (MEM; Gibco) supplemented with 2% FBS and 1.5% carboxymethylcellulose (Sigma-Aldrich). At 4 days post-infection, cells were fixed with 5% formaldehyde for 30 min at room temperature, washed, and stained with 1% crystal violet solution (in PBS with 20% ethanol) for 30 min. After rinsing with water, plaques were counted, and viral titres were calculated as plaque-forming units (PFU) per millilitre.

Antibody-based TCID₅₀ (50% tissue culture infectious dose) assays were performed by seeding Vero E6 cells (2.5×10⁴ cells in 180µL per well) in 96-well plates 24h prior to infection. Tenfold serial dilutions of virus supernatants were prepared in-plate (six replicates per dilution). At 4 days post-infection, cells were fixed with 5% formaldehyde for 30 min at room temperature, washed, and permeabilised with 0.5% Triton X-100 in PBS for 10 min. Cells were then blocked with 2.5% milk in PBS containing 0.25% Tween-20 (PBS-T) for 30 min at room temperature. After a brief wash, cells were incubated with a protein A–purified anti-orthoflavivirus envelope protein monoclonal antibody (clone D1-4G2-4-15; 1:250 dilution in PBS; in-house, stock concentration 500ng/µL) for 2h at room temperature, followed by incubation with a secondary anti-mouse HRP-conjugated antibody (1:200 dilution in PBS) for 1h. Foci were visualized by adding carbazole solution and titers calculated using the Spearman–Kaerber method as previously described71.

Proteomics sample preparation, data analysis and processing

Confluent monolayers of JEG-3 cells stably expressing HA-tagged 2k-NS4B of each individual orthoflavivirus or controls (n=4 biological replicates/sample) were collected and processed for HA-immunoprecipitation using anti-HA–conjugated agarose beads (Sigma-Aldrich) as described before14. Eluates from affinity-purified complexes (interactomes) and whole-cell lysates (effectomes; 50µg total) were denatured by incubation in 40µL U/T buffer (6 M Urea, 2 M Thiourea, 100 mM Tris-HCl, pH 8.5), and reduction and alkylation carried out with 10 mM DTT and 55 mM iodoacetamide in 50 mM ABC buffer (50 mM NH₄HCO₃ in water pH 8.0), respectively. After digestion with LysC (WAKO Chemicals USA) (IPs: 0.5µg; whole-cell lysates: 1µg) at room temperature for 3h, the suspension was diluted in ABC buffer, and the protein solution was digested with 1µg trypsin (Promega) (IPs: 0.5µg; whole-cell lysates: 1µg) overnight at room temperature. Digestion was stopped with 1% TFA, and peptides were purified on StageTips containing three C18 Empore filter discs (3M) and analyzed by liquid chromatography coupled to tandem mass spectrometry on a timsTOF Pro (Bruker).

Ultra-high-performance liquid chromatography and trapped ion mobility spectrometry quadrupole time of flight settings

Samples were analysed on a nanoElute (plug-in v.1.1.0.27; Bruker) coupled to a trapped ion mobility spectrometry quadrupole time of flight (timsTOF Pro) (Bruker) equipped with a CaptiveSpray source. Peptides (500ng) were injected into a Trap cartridge (5 mm × 300 μm, 5 μm C18; Thermo Fisher Scientific) and next separated on a 25 cm × 75 μm analytical column, 1.6 μm C18 beads with a packed emitter tip (Ion Opticks). The column temperature was maintained at 50°C using an integrated column oven (Sonation GmbH). The column was equilibrated using 4 column volumes before loading samples in 100% buffer A (99.9% Milli-Q water, 0.1% formic acid (FA)). Samples were separated at 400 nl min−1 using a linear gradient from 2 to 17% buffer B (99.9% ACN, 0.1% FA) over 60 min before ramping up to 25% (30 min), 37% (10 min) and 95% of buffer B (10 min) and sustained for 10 min (total separation method time, 120 min). The timsTOF Pro was operated in parallel accumulation-serial fragmentation (PASEF) mode using Compass Hystar v.5.0.36.0. Settings were as follows: mass range 100–1700 m/z, 1/K0 start 0.6 V⋅s/cm2 End 1.6 V⋅s/cm2; ramp time 110.1 ms; lock duty cycle to 100%; capillary voltage 1600 V; dry gas 3 l min−1; dry temperature 180°C. The PASEF settings were: 10 tandem mass spectrometry (MS) scans (total cycle time, 1.27 s); charge range 0-5; active exclusion for 0.4 min; scheduling target intensity 10,000; intensity threshold 2500; collision-induced dissociation energy 42 eV.

Raw data processing and analysis

Raw MS data were processed with the MaxQuant software v.1.6.17 using the built-in Andromeda search engine to search against the human proteome (UniprotKB, release 2019_10) containing forward and reverse sequences concatenated with the individual viral open reading frames of orthoflavivirus 2k-NS4B or control baits manually annotated, and searched with the label-free quantitation (LFQ) algorithm. Additionally, the intensity-based absolute quantification (iBAQ) algorithm and match between runs option were used. In MaxQuant, carbamidomethylation was set as fixed and methionine oxidation and N-acetylation as variable modifications. Search peptide tolerance was set at 70 p.p.m. and the main search was set at 30 p.p.m. (other settings left as default). Experiment type was set as TIMS-DDA with no modification to the default settings. Search results were filtered with a false discovery rate of 0.01 for peptide and protein identification. Perseus software versions v.1.6.10.4 and v.1.6.15.0 were used to process the data further. Protein tables were filtered to eliminate the identifications from the reverse database and common contaminants. When analyzing the MS data, only proteins identified on the basis of at least one peptide and a minimum of three quantitation events in at least one experimental group were considered. The iBAQ protein intensity values were normalized against the median intensity of each sample (using only peptides with recorded intensity values across all samples and biological replicates) and log-transformed; missing values were filled by imputation with random numbers drawn from a normal distribution calculated for each sample (width 0.3, downshift 1.8). Principal component analysis was used to identify and remove outliers. In total, three individual samples were removed from the NS4Bome (JEV_3, POWV_2 and WNV_1), and the Effectome (Gluc_3, JEV_4 and YFV_3).

Significantly up- or down-regulated proteins within NS4B-expressing cell lines (Effectome) were identified using a two-sided FDR-adjusted (n=250) Student’s t-test (|log2(Fold-change)|>2; p value≤0.05; S0=1, FDR≤0.05) using Gaussia luciferase as control (Supplementary Data 1).

Significant protein-protein interactions for each bait were identified using a two-sided FDR-adjusted (n=250) Welch’s t-test (P value≤0.05; S0=4, FDR≤0.01) using Gaussia luciferase as control (Supplementary Data 2). Only proteins measured in at least 3 biological replicates of each bait were considered. Furthermore, inspection of unimputed intensity profiles of all significant interactors across control baits (ZIKV-capsid, HCV-NS4B and Mock) was used to monitor for subcellular specificity and organellar selectivity.

For identification of high-confidence core interactors of orthoflavivirus NS4B, only proteins displaying a log2 (fold-change) ≥ 5 for each bait were considered. To identify shared host proteins among different NS4Bs in the extended NS4Bome network (Fig. 1b) and all intersection and functional enrichment analysis (Figs. 2, 3, Supplementary Fig. 1, 2), the high-confidence network was relaxed to include significantly enriched proteins across other NS4B interactors (log2 (fold-change) ≥ 2).

To identify selective, virus-specific host interacting proteins (Fig. 2d–g), the multi-volcano H analysis available within Perseus, setting all the other baits as background control. The s0 and FDR parameters of the multi-volcano analysis for Class A (higher confidence, s0=1, FDR=0.01) and Class B (lower confidence, s0=1, FDR=0.05) were chosen by visual inspection, aiming for a low number of significantly depleted proteins in any of the experiments (Supplementary Data 1).

For Fisher’s exact tests, annotations were extracted from UniProtKB, Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG) and CORUM (Supplementary Fig. 2d). For functional enrichment of cellular processes and pathways within the “core” NS4Bome (Supplementary Fig. 2a) STRING network analysis and gene ontology analysis were performed using STRING v11.072. For functional enrichment of cellular processes and pathways within the extended NS4Bome (Fig. 2b, c) GO analysis was performed using Metascape73. Complete enrichment analyses are available in Source Data and representative categories are visualized.

Oxygen consumption rate measurements

The day before the assay, Seahorse XF 96-well sensor cartridges (Agilent) were hydrated overnight at 37°C in a non-CO₂ incubator using 200µL of Seahorse XF calibrant (Agilent) per well. Simultaneously, UBA5 knockout JEG-3 (JEG-3_gUBA5) and control JEG-3_gNT cells were seeded at a density of 50,000 cells per well into a Seahorse XF 96-well cell culture microplate (Agilent) and incubated overnight at 37°C in a 5% CO₂ atmosphere. To evaluate the effect of ZIKV infection on mitochondrial respiration, infected and uninfected JEG-3 cells (50,000 cells/well) were similarly seeded in the Seahorse XF microplate and incubated overnight at 37°C with 5% CO₂. For experiments involving the UBA inhibitor DKM 2-93, JEG-3 cells were seeded at a lower density of 20,000 cells per well, three days before the assay. One day after seeding, the cells were treated with either 32µM DKM 2-93 or DMSO (control) and incubated for 48h under the same conditions. At least 5 replicates per condition were included in each experiment. On the day of the assay, the cells were washed with 200µL of Seahorse XF RPMI medium supplemented with 1mM pyruvate, 2mM glutamine, and 10mM glucose. Washed cells were then incubated in 180µL of the supplemented Seahorse XF RPMI medium for 1h at 37°C in a non-CO₂ incubator. Oxygen Consumption Rates (OCR) was measured using the Seahorse XF Cell Mito Stress Test kit (Agilent) following the manufacturer’s protocol. Briefly, 20µL of 10mM oligomycin, 22µL of 10mM FCCP, and 25µL of 5mM rotenone/antimycin A were loaded into the designated injection ports of the hydrated sensor cartridges. Sensor cartridges were then positioned onto the Seahorse XF 96-well cell culture microplate. Sequential drug addition (10-fold dilution) and time-lapse OCR measurements were carried out using a Seahorse XFe96 analyzer (Agilent). OCR data were analyzed using Wave 2.6.1 software (Agilent) to calculate basal respiration, ATP-linked respiration, and maximal respiration. For each experiment, the values were first normalized to total mean protein concentration, measured with Bradford assay on all replicates (μg per condition), and then to the mean basal OCR of the control condition from each independent experiment.

Mitochondria morphology analysis

JEG-3 mitochondria were labelled in standard immunofluorescence using anti-COX-IV primary antibodies (Cell Signaling, #4850S). Mitochondria images were acquired using an LSM780 confocal microscope (Carl Zeiss Microimaging) at the Confocal Microscopy Core Facility of INRS, and subsequently analyzed using the Mitochondria Analyzer plugin in ImageJ74. For images containing multiple cells, individual cell areas were first selected using the ROI Manager. The single channel containing the mitochondrial staining was then selected and converted into a binary image. Thresholding was performed using the 2D Threshold option in the Mitochondria Analyzer Manager. For each experiment, the thresholding method and C-value were previously optimized. After thresholding, images were analyzed using the 2D Analysis option. For statistical analysis, all values were processed using GraphPad Prism software. Normality was first assessed for each dataset, in which case an unpaired t-test or one-way ANOVA was used. Alternatively, the Mann-Whitney test or Kruskal-Wallis test was applied.

Zebrafish infection experiments

All the zebrafish experiments were performed in compliance with the guidelines of the Canadian Council for Animal Care (CCAC) and under the approval of institutional ethic committee (“Comité institutionnel de protection des animaux” (CIPA); certificate number: #2005-01) and biosafety committee (“Comité de biosécurité”; certificate number: #2016-12) of Institut National de la Recherche Scientifique (INRS). Adult zebrafish (Danio rerio, TL fish line) were maintained at 28°C with a 12h/12h light/dark cycle in the aquatic facility of the National Laboratory of Experimental Biology at INRS. Fertilized eggs were collected and maintained in petri dishes containing E3 medium at 28.5°C. At the 2–4 cell stage, embryos were injected with 4 nL containing ZIKV H/PF/2013 particles (~100 PFU in total) or DMEM (vehicle) using the FemtoJet 4i microinjection system (Eppendorf). At 4h post-infection, embryos were treated with 20µM DKM 2-93, 100µM NITD008 (Tocris Small Molecules) or DMSO diluted in E3 medium. Two days post-infection, embryos were dechorionated with forceps if unhatched, then fixed overnight at 4°C with 4% formaldehyde in PBS. Morphological assessment was performed using a Stemi 305 microscope (Zeiss) as described before48. Phenotypes were classified according to the severity of several morphological features (e.g., curved tail, edema, head size and shape), exactly as we previously described48. For viral RNA absolute quantification, on the second day post-infection, embryos were pooled by condition, ensuring equal numbers of animals per experimental group (6-10 embryos per group). RNA was extracted from whole non-fixed larvae using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. For cDNA synthesis, 500ng of RNA was used with the QuantiTect Reverse Transcription Kit (Qiagen). Amplification and absolute quantification of ZIKV cDNA was perforemd using the QX200 ddPCR EvaGreen Supermix (Bio-Rad) on the QX200 ddPCR System (Bio-Rad). The primers specific to ZIKV H/PF/2013 sequence were 5′-AGATGAACTGATGGCCGGGC-3′ and 5′-AGGTCCCTTCTGTGGAAATA-3′.

RNA-seq analysis

Total RNA was isolated from JEG-3 cells using TRIzol (Invitrogen) as per manufacturer instructions. High RNA integrity was confirmed by applying the Bioanalyzer Total RNA 6000 Nano Assay (Agilent, 5067-1511). Per sample, 1µg total RNA (quantified by Qubit RNA HS Assay, Thermo Fisher, Q32852) was pretreated applying the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) and further processed via the CORALL Total RNA-Seq Library Prep Kit (Lexogen, user guide version 095UG190V0130) in combination with Unique Dual Indexing Add-on Set B1 (Lexogen) according to manufacturer’s instructions (applying 16 cycles of Library Amplification PCR, step 4.3, user guide version 117UG228V0201). Libraries were quality controlled on a TapeStation D1000 Assay (Agilent, D1000 ScreenTape 5067-5582 with D1000 Reagents 5067-5583) and were sequenced on an Illumina NextSeq 2000 instrument (NextSeq 2000 P3 Reagents (200 Cycles), product number: 20040560) in a paired-end mode (2×107 bp). After demultiplexing via bcl2fastq, 13.5–23.5 million read pairs assigned to each sample (compare table Supplementary Data 1). All samples passed quality control by fastqc (Andrews S. (2010). FastQC: A Quality Control Tool for High Throughput Sequence Data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and multiqc75 and were subjected to downstream analysis.

Gene abundance for each sample was quantified with human gene annotations from gencode (version 43) for GRCh38 genome assembly using salmon76 (v1.10.0), and imported using R package tximport77. Counts normalization and multi-factor differential expression (DE) analysis was performed using DESeq2 package78. Replicates were evaluated based on PCA plot and outliers were removed. Design matrix with interaction term “~ group + condition + group:condition” for multi-factor DE analysis, where condition refers to (CT/shNT1 and SH/shUBA5, 5 sample in control vs 4 in treated) and group refers to the sub-group in each of the conditions (mock and Infected) was used as experiment design for DESeq2. Null variance of Wald test statistic output by DESeq2 was re-estimated using R package fdrtool79 to calculate p values (and adjusted using Benjamini-Hochburg method) for the final list of differentially expressed gene. FDR (BH-adjusted p values)<0.1 was used a criterion for the final DE gene list. Volcano plots were created using EnhancedVolcano R package (Blighe K, Rana S, Lewis M (2024). EnhancedVolcano: Publication-ready volcano plots with enhanced colouring and labeling. R package version 1.24.0, https://github.com/kevinblighe/EnhancedVolcano) and Gene ontology enrichment analysis was performed using R package clusterProfiler80. To find overlapping interferon gene signatures in shNT1 and shUBA5 in infection condition, samples were compared with their respective controls using design matrix “~ condition”. R statistical computing language (version 4.3.3) was used to perform abovementioned steps, except gene quantification.

Anisomycin treatment and cell fractionation by sequential detergent extraction

Crude extracts of JEG-3 cells were prepared as recently described81. On the day of the treatment, the spent media in the cell culture dish was replaced with fresh media containing 200nM Anisomycin (ANS, Sigma-Aldrich). ANS is an elongation inhibitor, that at substoichiometric concentrations, stochastically stalls a subset of ribosomes causing uninhibited upstream ribosomes to collide with them, triggering Ribosome-associated Quality Control (RQC). Following the addition of ANS, the cells were incubated for 20 min at 37°C to induce RPL26 UFMylation. Post incubation, the cells were harvest by trypsinisation, washed once with ice cold PBS and resuspended in fractionation buffer (50 mM HEPES pH 7.5 and 150 mM NaCl; 600µL of the buffer was used for JEG-3 cells harvested from one confluent 10-cm cell culture dish) freshly supplemented with protease inhibitor cocktail. One-third of the resuspended cells were incubated on ice for 10 min with 1% NP40 followed by centrifugation at 20,000×g to obtain the whole cell lysate (WCL) fraction. The remaining two-thirds were resuspended in the fractionation buffer containing 0.005% digitonin for 5 min to permeabilize the plasma membrane, followed by centrifugation at 8000×g for 5 min. The supernatant containing the cytosolic fraction was collected (Cyto), and the pellet was washed once with 500µL fractionation buffer with no detergents before resuspending in 100µL fractionation buffer containing 1% NP40. After incubating for 10 min on ice and centrifugation at 20,000×g for 10 min, the supernatant containing the membrane fraction (ER) was collected. Equal volumes of collected fractions were analyzed by SDS-PAGE and immunoblotted with anti-UFM1 antibodies.

Luciferase Assay

Luciferase assay was used to measure viral replication in cells infected with the ZIKV H/PF/2013 reporter strain expressing Renilla luciferase (ZIKV-R2A) or electroporated with full-length viral RNA or subgenomic replicons. On the day of harvest, cells were washed once with PBS, lysed in luciferase lysis buffer (1% Triton-X-100, 25 mM Glycyl-Glycine pH 7.8, 15 mM MgSO₄, 4 mM EGTA, 1 mM DTT added directly prior to use), and plates were frozen at −20°C until analysis. Plates were eventually thawed on ice and luciferase activity was measured using a M200 Tecan plate reader (Tecan Life Sciences) which was programmed to inject 80µL of luciferase assay buffer (25 mM glycyl glycine, pH 7.8, 15 mM KPO₄ buffer pH 7.8, 15 mM MgSO₄, 4 mM EGTA, 1 mM coelenterazine freshly added before the assay) into each well. The luciferase activity was then measured using default settings.

Western blotting

Protein samples were harvested in RIPA buffer, and protein concentrations were determined using the Pierce 660 nm Protein Assay kit (Pierce). Normalized samples were denatured in 1X sample buffer (200 mM Tris, pH 8.8, 5 mM EDTA, 0.1% bromophenol blue, 10% sucrose, 3% SDS, and 1 mM DTT) for 5 min at 95°C. Samples were loaded onto 10%, 12%, or 15% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to a nitrocellulose membrane using a Mini Trans-Blot Cell wet-blotting apparatus (Bio-Rad). The membrane was blocked with 5% non-fat dry milk in PBST (0.5% Tween-20 in 1X PBS) and incubated with primary antibodies overnight at 4°C (or for 1h at room temperature in the case of HRP-conjugated primary antibodies). After three washes with PBST, the membrane was incubated with HRP-conjugated secondary antibodies for 1h at room temperature. The membrane was developed using Western Lightning Plus-ECL reagent (PerkinElmer) and imaged using an Intas Chemocam Imager 3.2 (Intas).

Co-immunoprecipitation

Frozen cell pellets were thawed on ice and incubated with lysis buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris-Cl pH 8.0, 1X cOmplete protease inhibitor cocktail (Roche)) for 30 min on ice. All subsequent steps were performed at 4°C unless otherwise stated. The lysates were centrifuged for 15 min at 20,000×g. The protein concentration was determined, and the normalized lysates were incubated with pre-cleared beads for 4h. After incubation, the beads were washed three times with lysis buffer and three times with wash buffer (150 mM NaCl and 50 mM Tris-Cl pH 8.0). Immunocomplexes were eluted in 2X sample buffer at room temperature and analyzed by Western blotting.

Immunofluorescence microscopy

Cells grown on coverslips for immunofluorescence analysis were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. The fixed cells were permeabilized with either 0.2% or 0.5% (v/v) Triton X-100 in 1X PBS for 10–15 min and blocked with 5% milk in 1X PBS-Tween 20 (0.25%) or 5% bovine serum albumin (BSA) and 10% goat serum (Thermo-Fisher) for 30–60 min at room temperature. Blocked coverslips were incubated with primary antibodies for 2h at room temperature, followed by incubation with Alexa Fluor–conjugated secondary antibodies. Nuclear DNA was stained with 4’,6-diamidino-2-phenylindole (DAPI, Molecular Probes). Coverslips were mounted using Fluoromount-G mounting medium (Southern Biotechnology Associates), and cells were visualized using a ECLIPSE Ti2-E Inverted Microscope with Yokogawa CSU-W1 Borealis Spinning Disk Confocal or an LSM780 confocal microscope (Carl Zeiss Microimaging). Images were analyzed using Fiji/ImageJ software74.

Quantitative real-time PCR

Total cellular RNA was extracted using the NucleoSpin RNA II kit (Macherey-Nagel), and cDNA was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Life Technologies), following the manufacturer’s protocols. Quantitative real-time PCR (qRT-PCR) was performed on 7500 Fast Real-Time PCR System (Applied Biosystems). For each primer set, reactions were conducted in triplicate using SYBR Green master mix (A25742, Applied Biosystems) with the following primers binding to a region within the ZIKV E coding sequence: Fwd: 5′-CCGCTGCCCAACACAAG-3′ and Rev: 5′-CCACTAACGTTCTTTTGCAGACAT-3′, and within the GAPDH coding sequence: Fwd: 5′-GAAGGTGAAGGTCGGAGTC-3′ and Rev: 5′-GAAGATGGTGATGGGATTTC-3′.

Resazurin cell viability assay

Cell viability was assessed by adding resazurin (final concentration 0.167 mg/mL, Sigma-Aldrich) to the cells. After incubation for 1h at 37°C, the reduction of the non-fluorescent dye resazurin to the highly fluorescent dye resorufin, resulting from the metabolic activity of living cells, was quantified by measuring absorbance at 598 nm on a Tecan M200 plate reader (Tecan Life Sciences). Absorbance values were normalized to those of the control cells, and cells were considered viable if their absorbance was at least 75% of the control.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 10 software, unless otherwise stated.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Rajasekharan et al. take a multiomics approach to better understand the role of the orthoflavivirus non structural protein NS4B in viral replication. By expressing NS4B of many orthoflaviviruses in JEG3 cells, authors generate a map of virus-specific and common interacting partners. To gain further insight into the role of NS4B binding, authors perform an RNAi screen in which a number of identified binding partners are depleted, and ZIKV infectivity is assed. Authors then focus more specifically on one interacting host factor, UBA5, an E1 activating enzyme specific for UFM1. Authors find no major modifications to the UFMylation pathway upon ZIKV challenge, however do notice some relocalization of UFMylation proteins to the NS3 and NS4B positive replication sites. In further characterizing binding of UFMylation host factors to orthoflavivirus NS4B, authors find NS4B of DENV and YFV also bind UBA5, while NS4B of many other viruses bind additional UFMylation machinery. Pharmacological inhibition of UBA5 and therefore the UFMylation pathway, inhibit ZIKV infection, specifically during the virion production/egress phase. While innate immune signaling was not altered by UFMylation perturbation in ZIKV challenged cells, it was determined that UFMylation is altering mitochondrial respiration rates, and shape, which may play a role in ZIKV infection. Lastly, in zebra fish treated with an UFMylation inhibitor during ZIKV challenge, a reduction in the pathology is observed. In all, authors have identified novel NS4B binding partners, across multiple orthoflaviviruses, and provided insight into a potential druggable host factor for the therapeutic treatment of patients with orthoflavivirus infections. This study is thoughtfully designed, and conclusions are strongly supported by experimental evidence. In addition, this study is a valuable resource providing multiomics data that may prove useful to many in the orthoflavivirus field.\nMajor points:\n1) A major finding of this study is the involvement of UBA5, and the UFMylation pathway, in ZIKV infection. Through a series of logical and well-controlled experiments, the authors conclude that UBA5’s function appears to be related to later steps in the viral life cycle; post entry, translation and RNA replication. The most well-characterized role of UFMylation is in ribosome quality control. Flavivirus infection activates the UPR and extensively remodels the ER to stimulate RNA replication and assembly. While the replicon experiments suggest that translation and replication are not affected by knockout, it is still conceivable that there is a link between flavivirus translation at the ER and viral assembly. It is possible that polyprotein biogenesis and ER-membrane insertion requires the UFMylation machinery to deal with stalled ribosomes, possibly stalled on the large, structurally complex viral RNA. Addressing this in the discussion or, ideally, experimentally, by testing whether ribosome stalling occurs during flavivirus infection e.g. by looking at UFM1 conjugation to RPL26 in the ER fraction, would strengthen the manuscript.\n2) The authors demonstrate a defect in mitochondrial respiration in UBA5 KO cells and show that ZIKV replication stimulates respiration. While intriguing, it remains rather speculative to claim that this correlation is relevant for the viral assembly phenotype. It is also unclear how specific interactions with NS4B would mediate this. The authors should be more nuanced in their discussion toning down some conclusions.\n3) Authors state that ZIKV infection causes components of the UFMylation machinery to redistribute to replication sites. This is based on colocalization in infected cells using immunofluorescence. It is unclear if this is an active recruitment and if a larger proportion of these proteins are now at the ER compared to mock infected cells, or whether this simply reflects the fact that the ER-derived replication organelle takes up a larger area in the cell. This data should not be overinterpreted.\n4) In figure 3 silencing of SEL1L resulted in an increase of viral infection and RPN2 has a variable effect. This contrasts with prior literature where they were identified as strong proviral factors (PMID: 31273220, PMID: 28720733, PMID: 33980593). Could the authors please discuss this.\n5) The authors should mention and discuss another recent study independently identifying Ufmylation factors involved in flavivirus infection (PMID: 40459261).\n6) Line 155: “suggesting an evolutionary divergence of NS4B-host-interactions within the Orthoflavivirus genus.” This is an overstatement. The proteomic data uses arbitrary cutoffs, so a false negative does not mean much. Also, the affinity purifications were performed only in one cancer cell line.\nMinor points:\n1) Figure 1B is a very nice figure- it should be made larger (perhaps its own figure) so that readers can really dive into all the interactions.\n2) The definition of “viral replication” varies among virologist- either it refers to a specific stage (copying of the RNA genome) in viral infection, or it can refer to the entire viral lifecycle. Throughout the manuscript authors use “replication” to describe the viral life cycle and to describe RNA replication. This is confusing.\n3) Line 226: Protein levels are altered as a result of two major changes to the cell- changes to mRNA expression, or changes in degradation kinetics. Authors use the phrase “targeted for degradation” without monitoring mRNA expression or preventing protein degradation with a lysosomal or proteasomal inhibitor, and therefore this is not supported by the data.\n4) “Virus replication” is used for multiple axis titles (figure 4b-d, and others), as well as “Virus titer” (fig. 5F). Please use consistent and more specific naming, and please refer back to minor point 2. “virus titer”, “infectious viral particles”, or “virions” seem like appropriate axis titles for as assay that measures PFUs. “Viral RNA” for qPCR, “luciferase activity “for luciferase assays.\n5) Figure 4b: UBA5 addback doesn’t appear to restore ZIKV replication in the RT-qPCR experiment. Could the authors please address this.\n6) There doesn’t appear to be much of a change in the number of severe pathology cases in ZIKV infected Zebra fish, with or without the UFMylation inhibitor, however this is never addressed.\n7) If available, it would be great to see longer exposures for the western blots, and for the western blot sizes to be increased.\n8) Figure 4h: With one of the biological replicates, it appears as though one of the UBA5 addbacks did not restore ZIKV replication, and is significantly different than the other two replicates. This should be repeated for an additional biological replicate.\n9) Please make the methods section clearer. For example, I was unclear what the delta SC construct was, or how the UBA5-FLAG construct was made shRNA resistant. How was the NS4B mutant with the internal HA tag made?\n10) Figure 5b: Western blots have been cut or boxed in a way that prevents the reader from viewing them, specifically the UBA5 and UFC1 blots.\n11) Figure 6C: Samples from the drug treated cells are not loaded equally to the DMSO treated cells. Please load equal amounts to make conclusions about NS1 expression and viral inhibition under drug treatment.\n12) Figure 6d: There could be a concern that the drugs are still present in the harvested supernatant, but this depends on the specific dilutions used for the plaque assay. Please comment on the dilutions.\n13) Figure 7a: please discuss in greater detail in manuscript.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This study by Rajasekharan et al investigates the role of the UFMylation pathway in flavivirus restriction using a combination of genetic knockout, chemical inhibition, and in vivo validation approaches. The authors propose that UFMylation functions as host dependency pathway for these viruses and characterize the small molecule UBA5 inhibitor DKM 2-93 as a potential therapeutic compound. While the proteomics in this study are rigorously performed, the mechanism of UFMylation's role in NS4B-dependent virus replication is less convincing. Some of the concerns are outlined below:\nGeneral comments:\n1. The primary weakness in this style of proteomics is the reliance on viral protein overexpression studies conducted outside the context of infection. This approach has fundamental limitations for flavivirus research, as demonstrated by the poor reproducibility between studies examining NS4B interactions through overexpression approaches (PMID: 30037810, PMID: 28317380, PMID: 30177828). Multiple studies have now shown that NS4B interaction partners differ substantially when the protein is expressed alone versus in the presence of other viral proteins such as NS4A (PMID: 27524440, PMID: 29902443). A better experimental design would involve proteomics analysis in the context of authentic viral infection, as demonstrated in previous studies (PMID: 25926641; bioRxiv doi: 10.1101/2024.04.03.587877). The authors should compare their NS4B interaction data with findings from these infection-based studies and their own previous work (PMID: 30177828) to assess the degree of overlap and discuss potential differences. If UFMylation machinery was not identified in previous infection-based studies, this could indicate that the interactions observed here are cell type specific effects or potential artifacts of the overexpression system.\n2. Another critical gap in the manuscript is the lack of clarity regarding the mechanism by which UFMylation supports viral replication. Is NS4B itself modified by UFMylation, or does the interaction with UBA5 facilitate modification of other viral or cellular proteins? How do UFMylation substrates change during infection? The authors propose that UFMylation enhances mitochondrial respiration to support viral replication, but this hypothesis requires more direct experimental validation through alternative approaches to modulate respiration. Additionally, if mitochondrial function is the key mechanism, the authors should explain why NS4B specifically needs to interact with UBA5 and whether this interaction occurs at mitochondrial membranes.\n3. The findings would be significantly strengthened by validation in multiple cell types beyond JEG3 cells, particularly given the potential for cell-type-specific UFMylation interactions. The restriction to a single cell type limits the generalizability of the conclusions and raises questions about the broader relevance of the UFMylation-flavivirus interaction.\nSpecific comments:\nFigure 1 and 2: Key validation data are missing regarding NS4B expression levels and subcellular localization. The authors should show that expressed NS4B achieves physiologically relevant expression levels comparable to those observed during infection and maintains proper subcellular distribution. The resolution of Supplementary Figure 1 is insufficient to assess localization patterns. Additionally, the temporal dynamics of the NS4B-UBA5 interaction during the viral lifecycle are not characterized.\nFigure 4: In Figure 4a-c, the extent of both the reduction and the restoration of viral titers are very modest. The level of NS2B in the UBA5-depleted samples show minimal reduction in NS2B levels and that does not change in the reconstituted samples. Similarly, the infectious virus production is between 2-4 fold and rescue seems to be partial. The authors should move this to the supplementary section, since the KO data (Figure 4h) seems more convincing. What is also puzzling is that while reconstitution of the WT UBA5 in the depleted (shRNA) and deleted (gRNA) background seem equivalent (Figure 4f), the viral titres are rescued in the gRNA background (Figure 4h) but not in the shRNA background (Figure 4b). What is the explanation for this?\nThe authors should determine which step in the viral lifecycle is affected by systematically measuring entry, replication and release in the KO and reconstituted cells.\nFigure 5: The imaging data (panel c) lacks sufficient resolution for meaningful colocalization analysis. The authors to provide higher resolution data and quantify colocalization between UFMylation components and viral proteins. It would also be useful to see whether this is happening at mitochondrial membranes, given the effect of UFMylation on increased mitochondrial respiration.\nFigure 6: While the effects of the chemical inhibitor on viral proteins and infectious particles are clear (panels 6c-d), the time-of-addition experiments show much more modest effects (<2-fold reduction compared to DMSO control). What is the explanation for this discrepancy?\nFigure 7: The analysis of Figure 7a should be performed by co-staining for NS4B and UFMylation components to directly visualize their spatial relationship in the context of the proposed mechanism.", "reviewer_4": "This manuscript provides a comprehensive analysis of cellular proteins with viral NS4B of multiple orthoflaviviruses and incorporates information from the host factors that are perturbed during orthoflaviviral infection. They focus on the role of UBA5 and the UFMylation pathway in promoting infection, with emphasis on ZIKV, in in vitro and in vivo model systems, using genetic ablation as well as a pharmacological inhibitor. In addition, they also describe a role for UFMylation in regulating mitochondrial morphodynamics that they postulate could potentially be hijacked or mediated by viral proteins during infection. Overall, the study identifies a new role for a post-translation modification pathway during orhtoflavivirus infection that could serve as a potential target for the design of pan-orthoflaviviral drugs.\nThe methods are well-described and the supplementary tables cover required information. While the manuscript provides sufficient evidence for what the current title describes, there are a few suggestions/ queries that, if addressed, would greatly benefit the work overall.\nMajor comments:\n1. As the authors might already be aware, a recent article published in JVI addresses a very similar question (https://doi.org/10.1128/jvi.00654-25). While both manuscripts focus on the UFMylation pathway, they differ in their approaches and breadth and also their targets. The JVI manuscript would need to be cited here and this manuscript would also benefit from additional discussion from the author’s perspectives on a parallel UFMylation mechanism in orthoflaviviruses.\n2. Readability – the abstract and introduction are concise and well written. However, the first and second results sections require constant back and forth, especially between Figures 1b and 2a, affecting the readability of the manuscript. The first part of the results section could also benefit from some language editing to make it easier for readers to comprehend.\n3. The transition from Figure 2 to Figure 3 results is a big jump, going from a pan-orthoviral proteomics study to performing functional follow-up only on ZIKV. What was the reason to choose ZIKV as the virus of choice for the mitochondrial studies? While a narrower scope for a RNAi screen is understandable on a technical level, the manuscript should give a rationale for the choice of virus for functional follow-up. Additionally, the figure and section title should more accurately describe the scope of the work in this section; as written, it reads like an over-statement of the presented work. This is also true for the discussion of the RNAi results in the Discussion.\n4. Authors haven’t shown that the UBA5 inhibitor used in the study, DKM 2-93, successfully inhibits UFMylation in their experiments. Specificity of the inhibitor in affecting UFMylation downstream by lack of formation of UFM-conjugates would need to be demonstrated in both in vitro and in vivo experiments.\n5. Are there other E1 enzymes known for UFMylation in humans? The UBA5 inhibitor used is effective in micromolar concentrations which is higher than what is conventionally considered ‘low’. The authors do show that viability isn’t affected but what could be the reason for such high amounts of inhibitor required to show an antiviral effect?\n6. While the authors have shown that pharmacological inhibition of UBA5 restricts ZIKV replication in zebrafish, demonstrating that genetic ablation or UBA5 knockdown has the same effect would further substantiate their in vivo results.\nMinor comments:\n1. Incorrect figure cited in Line 130 (should be Supplementary Figure 1b).\n2. Looks like incorrect virus may be mentioned in Line 214, (JEV instead of YFV?) based on Figure 2g.\n3. Authors use the term ‘replication’ to denote RNA copies as well as viral titers interchangeably in the y-axes of figures and also in the text (for example – Figure 4c, g, h v/s Figure 5f). Maintaining consistency throughout the manuscript will prevent confusion.\n4. The previous point is especially important in the title of Figure 6 – the title reads “Pharmacological inhibition of the UFMylation pathway impairs ZIKV replication” while 6f and 6g clearly indicate that the viral replication process is not hindered by DKM 2-93 treatment. The results section for this figure explains this clearly. Please modify this figure title.\n5. Text referring to Figure 3c and Supplementary Figure 3a mentions ‘5 pro-viral genes’ throughout the manuscript (Lines 247, 470) while the figure has 6 genes indicated. Kindly clarify.\n6. Figure 1b –only the high confidence interactors have been denoted. While this could be a stylistic choice by the authors to emphasize the specific interactions, having all the interactions shown here would make the figure a lot more appealing (the empty circles, especially prominent in the “Mitochondria” bubble, don’t convey much information and therefore don’t substantially help the figure). Additionally, this Figure has more than 200 interactors labelled and the figure legend indicates only high confidence interactors have been denoted. Kindly clarify the discrepancy in numbers.\n7. Figure 2a and Line 189 indicate different numbers of interactors.\n8. Figure 4b – shUBA5 v/s exogenous supplementation does not show significant difference in viral RNA levels, while Figure 4c shows successful rescue in viral titers. Can the authors comment on this?\n9. Can the authors provide a better rationale in the manuscript how they chose UBA5 from the proteomics dataset? The extended NS4B effectome and the extended NS4Bome indicate that UBA5 was only picked up in ZIKV samples (Supp Tables 1 and 2, and Figure 1b).", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point response to reviewers’ comments\nManuscript Number: NCOMMS-25-34251-T\nRe.: Rebuttal to reviewer’s comments for\n“A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for\nUFMylation in orthoflavivirus replication” by Rajasekharan S. et al.\nWe thank the 5 reviewers for their constructive feedback and suggestions that helped to further\nimprove the manuscript. To address their comments, we have now performed a series of\nadditional experiments and analyses in addition to revisions of results, discussion, and\nmethods.\nThese experiments include: 1) a fractionation-based approach to analyze the impact of viral\ninfection on RPL26 UFMylation at the ER, 2) the requested additional biological repetitions of\nselected experiments, 3) drug-testing in additional cell lines, 4) extensive additional\nexperiments to address the role of NS4B in cellular respiration and the differential UFMylation\nlandscape in viral-infected cells and 5) validation of UFMylation inhibition in drug-treated cells.\nJust to contextualize our efforts, these new analyses also included generation and\ncharacterization of novel knock-out cell lines and over 20 hours of additional mass-\nspectrometry experiments, including manual inspection of MS/MS spectra of modified\npeptides.\nWe feel that the new data are highly valuable and have strengthened our manuscript,\nconfirming our previous results. However, in our view, the new observations on differential\nUFMylation rather form the basis for an entirely new study, and are beyond the scope of this\nstudy which has its main focus on the characterization of the NS4B interaction landscape\nacross multiple orthoflaviviruses and already includes:\na) a genus-wide interaction and effectome atlas of orthoflavivirus NS4Bs,\nb) a targeted RNAi screen identifying novel host-restriction and -dependency factors,\nc) generation and characterization of a novel replication-competent ZIKV NS4B-tagged virus,\nd) validation of key protein-protein interactions in infection,\ne) detailed analysis of UFMylation pathway with respect to transcriptional landscape\n(RNAseq), mitochondrial morphology, cellular respiration and overall pathway activity using\nRNAi and CRISPR/Cas9-based systems, co-IP-WB and immunofluorescence analysis\nf) in vitro and in vivo drug-repurposing experiments demonstrating the antiviral potential of\nUFMylation inhibitors and characterizing the replication step(s) affected.\nIn light of this, we suggest not to include the new UFMylation data in this manuscript as albeit\nextremely interesting they require further additional validation and mechanistic experiments\nwhich will be the basis of future studies.\nPlease find uploaded in the online submission system the merged document highlighting the\nchanges to the first version of the manuscript. Point-by-point responses to reviewers’\ncomments are indicated below in dark blue."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have satisfactorily addressed my concerns.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The revised version has largely addressed the concerns raised.\nWhat is still missing from the study is that the defect in the late stage of the viral lifecycle is inferred.\nAssembly/release/spread is not directly measured by intracellular vs extracellular infectious units, particle:PFU ratio, intracellular E/prM accumulation with reduced release etc.\nFig 6a legend describes DKM 2-93 as “competitively” binding the catalytic cysteine. DKM 2-93 is described in the primary\nliterature to covalently modify the catalytic cysteine of UBA5 (Cys250). That's not the same thing.", "reviewer_4": "The authors have largely amended their manuscript to address the reviewer’s comments. However, there are a few areas that can still be improved.\n1. Figure 1b is now much more interpretable and interesting to go through. This change is great.\n2. While the authors have addressed the issue of readability by combining results sections 1 and 2 that makes it easier, they might want to consider rearranging their figures to make it easier for readers.\n3. The justification for doing follow up studies with ZIKV is still weak. The use of JEG-3 for their APMS study is then confusing as this background is not ideal for 4/8 viruses chosen by the authors. While the experiments that follow make sense, how they address choosing ZIKV is still not convincing.\n4. The authors have provided western blotting data to confirm inhibition of UFMylation pathway. This definitely helps but the use of 16uM for their experiments is tricky as it doesn’t seem to inhibit the pathway as effectively as 32uM, but it does inhibit viral protein synthesis.\n5. The authors have explained the difficulties with generating a UBA5 KO zebrafish model, but they say they have included these considerations in the discussion, which doesn’t seem to be the case.\n6. The revised manuscript largely addresses the queries of all reviewers. However, some justifications still seem like overinterpretations. I would like to ask the authors to revise the manuscript to make sure not exaggerate some of their findings.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point response to reviewers’ comments\nManuscript Number: NCOMMS-25-34251A\nRe.: Rebuttal to reviewer’s comments for\n“A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for\nUFMylation in orthoflavivirus replication” by Rajasekharan S. et al.\nWe thank the 5 reviewers for their constructive feedback and suggestions that helped to further\nimprove the manuscript. Please find below a point-by-point reply to their remaining comments.\nPlease find uploaded in the online submission system the merged document highlighting the\nchanges to the second version of the manuscript. Point-by-point responses to reviewers’\ncomments are indicated below in dark blue."}]

Decision letter