Proviral NUP153 binding to viral proteins and RNA regulates structural–nonstructural protein ratios in orthoflavivirus infection

Domain: neuroscience
Published: 2026-04-08
Editorial outcome: major_revision

Abstract
Orthoflaviviruses are RNA viruses that cause serious diseases in humans, with currently no antivirals available. Targeting host factors is emerging as an attractive antiviral approach. However, as a first step, there is a need to understand which host proteins are hijacked and for what purpose. Here, using a combination of fluorescence microscopy, knock-down, crosslinking immunoprecipitation sequencing, mass spectrometry, and in vitro and biophysical assays, we identify nucleoporin-153 (NUP153) as a proviral factor during orthoflavivirus infection. We show that NUP153 is recruited to the virus amplification site on the endoplasmic reticulum to impact the structural to nonstructural viral protein ratios. We find that NUP153 interacts with both the viral proteins NS3 and NS5, and a highly conserved G-rich motif on the viral RNA. These interactions specifically promote the production of viral structural proteins, leading to an efficient virion assembly, virus release and spread to new cells. We propose that NUP153 acts as a key regulator in viral protein ratios, a mechanism that appears conserved among orthoflaviviruses.

Introduction
Orthoflaviviruses, such as West Nile virus (WNV), yellow fever virus (YFV), dengue virus (DENV), tick-borne encephalitis virus (TBEV), and Zika virus (ZIKV)1–3 are arthropod-borne RNA viruses that are responsible for high morbidity and mortality across the world. Although vaccines have been developed against some of these viruses, no clinically approved antivirals are currently available against them.

Orthoflaviviruses are 40–60 nm spherical enveloped viruses that contain a single-stranded positive-sense RNA genome of approximately 11 kb, which is translated into a single polyprotein that is proteolytically processed into three structural proteins [the capsid (C) protein, the precursor form of the membrane protein (prM), and the envelope (E) protein] and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5)2,4,5. Following entry into the cell, orthoflaviviruses reprogram the host cellular machinery and induce major structural rearrangements of the endoplasmic reticulum (ER) to support viral replication, protein translation, and virion assembly, and we define this as the viral amplification site4,6,7. To accomplish the reprogramming, viral proteins hijack host proteins. Gaining insights into the mechanisms by which orthoflaviviruses exploit their host environment to promote viral propagation could lead to the identification of targets for host-directed therapies to combat infections.

Nucleoporins (NUPs) are the building blocks of the nuclear pore complex (NPC), which is one of the largest complexes in the cell, reaching a size of 120 MDa in human8. The NPC is composed of multiple copies of approximately 30 different NUPs9. The main function of the NPC is to create a channel across the nuclear envelope by fusing the inner and outer nuclear membranes for transport of proteins and RNA molecules across the nuclear membranes10. One of the NUPs, NUP153, is located at the nuclear basket of the NPC. It is involved in multiple RNA and protein export pathways, and is required for correct assembly of the NPC11,12. Interestingly, during hepatitis C virus (HCV) infection NUP153 is recruited to the ER-derived cytoplasmic structures where it regulates the transport of viral proteins into the HCV replication complexes13. Orthoflaviviruses also utilize ER membranes for their replication, and might also utilize NUP153 as a host factor during infection.

Here we demonstrate that NUP153 is a host factor exploited by both tick- and mosquito-borne orthoflaviviruses. Using a combination of fluorescence microscopy, knock-down, crosslinking immunoprecipitation sequencing, mass spectrometry, in vitro biophysical assays and mass spectrometry, our data shows that during flavivirus infection, NUP153 expression is upregulated and the protein is recruited to the viral amplification site on the ER, where it interacts with viral proteins NS3 and NS5, and a conserved G-rich motif on the viral RNA. These interactions impact the ratio of structural to nonstructural proteins to promote effective virion production, which is different from NUP153’s proposed role during HCV infection13. Importantly, the results reveal a proviral role of NUP153 in orthoflavivirus infection.

Results
Viral infection upregulates NUP153 expression and causes NUP153 relocation to the viral amplification site
NUP153 plays a role in the lifecycle of some viruses13,14, but its function during orthoflavivirus infection is poorly understood. To shed light on NUP153’s role during orthoflavivirus infection, we first analyzed its localization during tick-borne orthoflavivirus infection, as the protein is typically found in the nuclear envelope (NE). We infected cells with TBEV and Langat virus (LGTV; commonly used as a BSL2 model for TBEV) as representative tick-borne orthoflaviviruses and analyzed the cellular localization of NUP153 using microscopy (Fig. 1A)15. We found that in the infected cells, the levels of NUP153 appeared increased and that a fraction of NUP153 colocalized with the viral NS3 protein in the cytoplasm. Quantification of the NUP153 signal revealed a 3-fold increase of NUP153 levels during TBEV and LGTV infection, both in the nucleus and the cytoplasm, as compared to the levels in the mock-treated samples (Fig. 1B, C). However, no difference in protein ratio between cytoplasm/nucleus was detected between mock and infected cells (Supplementary Fig. 1A). The upregulation of NUP153 at the level of RNA was verified in HEK293T cells infected with both LGTV and TBEV (Supplementary Fig. 1B). Upregulation could be caused by either the virus or the host innate responses. To distinguish between these scenarios, we analyzed the RNA transcript of virus, interferon β (IFNβ), and NUP153 in A549 cells or A549 cells knocked out for the mitochondrial antiviral-signaling protein (MAVS), which is an adapter protein of importance for the innate immune defense against viruses16. TBEV replicated to the same extent in both cell types (Fig. 1H), and A549 cells readily upregulated IFNβ to high levels but not in cells with knocked out MAVS expression, in line with its expression being related to the innate immune response (Fig. 1I). NUP153 expression was instead significantly upregulated independently of MAVS expression (Fig. 1J). Furthermore, we confirmed that the conventional role of NUPs in the export of cellular mRNA was not compromised in infected cells (Supplementary Fig. 1C–F).

During flavivirus infection, the viral protein NS3 is present at the cytosolic side of the host ER, along with viral dsRNA, in the replication vesicles6,17. To determine if NUP153 is recruited to the ER during infection, we conducted super-resolution microscopy using structured illumination microscopy (SIM) on TBEV-infected cells (Fig. 1D, E). Interestingly, the fluorescent signal from both calnexin (ER marker) and NUP153 was increased in infected cells (Fig. 1D, E). The SIM analysis revealed that NUP153 protein has a reticular pattern in the cell which overlapped both with calnexin and viral NS3 during infection (Fig. 1D). We also found that a fraction of the NUP153 staining overlapped with dsRNA, the viral RNA replication intermediate, as a specific marker of RNA replication vesicles (Fig. 1E). Next, we quantified the amount of colocalization of NUP153, calnexin, NS3, and dsRNA (Fig. 1F, G). NS3 and NUP153 almost completely overlap with each other, and we found that both proteins also colocalized with dsRNA (Fig. 1G). Furthermore, our colocalization analysis also revealed that while most of the dsRNA signal colocalized with both NS3 and NUP153, most of NUP153 and NS3 did not colocalize with dsRNA, indicating that these proteins can also be found outside of the replication vesicles in the remaining of the ER as shown by their colocalization with ER marker calnexin (Fig. 1F). Collectively, the above data indicated that viral infection promotes ER expansion and ER localization of NUP153.

NUP153 is a proviral factor during early infection
NUP153 is an essential cellular protein. To investigate its role in TBEV infection, we used the auxin-induced degron (AID) system to rapidly degrade endogenous neonGreen-tagged NUP153 in DLD-1 cells [NUP153(NG)AID] without affecting the integrity of the NPC18. Using this system, DLD-1 cells were genetically modified to express AID and neonGreen-tagged NUP153, which can be rapidly degraded conditionally upon addition of auxin18. These cells were compared with DLD-1 control cells, which is the parental cell line. First, we evaluated the degradation efficiency, and full degradation of the NUP153(NG)AID protein occurred within 2 h of auxin treatment (Fig. 2A, B, and Supplementary Fig. 1A, B). Secondly, we evaluated the cellular distribution using immunofluorescence analysis, and the neonGreen signal overlapped almost completely with the NUP153 signal in these cells, supporting that the neonGreen signal is a reliable marker for NUP153 localization (Fig. 2C, D). Further, we verified the recruitment and colocalization of NUP153 with NS3 at the viral amplification site in NUP153(NG)AID and DLD-1 cells (Fig. 2E, F, and Supplementary Fig. 1C).

Using the AID system, we depleted NUP153 and analyzed the effect on LGTV infection. In these experiments, the infection level was quantified by measuring the amount of viral E-positive cells with immunofluorescence analysis and viral titers in the supernatant. We found that the viral infection was reduced by 50% in the absence of NUP153 (Fig. 2G) and viral titers were reduced by 80% (Fig. 2H). Furthermore, we analyzed the effect on TBEV infection after depletion of NUP153 at different timepoints to investigate the importance of NUP153 activity during the viral lifecycle. We observed that the addition of auxin 2 h prior to infection, and at 2 hpi or 4 hpi resulted in approximately 40% reduction of infection in NUP153(NG)AID cells but not in uninduced cells or the parental DLD-1 control cells (Fig. 2I and Supplementary Fig. 2D) and viral titers were reduced by 80–95% (Fig. 2J). Auxin addition at 6 hpi led to no significant reduction in the number of infected NUP153(NG)AID cells, and had no detectable effect on the number of subsequently released viral particles (Fig. 2K and Supplementary Fig. 2E). Because we detected differences between the reduction in number of infected cells compared to the released viral particles following depletion of NUP153 we measured the amount of TBEV RNA at 2, 8, 12, and 16 h post infection in cells induced with auxin 1 hpi (Fig. 2L). We found that depletion of NUP153 led to approximately 50% reduction in TBEV viral RNA at 16 hpi, however, no reduction at 8 or 12 hpi (Fig. 2L), indicating that NUP153 depletion does not affect early replication but more likely affects virus assembly and release.

To verify that the reduction of viral infection was dependent on NUP153 expression, NUP153(NG)AID cells were either transfected with plasmids expressing either enhanced green fluorescent protein (eGFP) or eGFP-tagged NUP153. Complete restoration of infected cells was seen by eGFP-NUP153 plasmid transfection (Fig. 2M). Taken together, these results indicated that although NUP153 is an important proviral factor during the early stages of viral infection, it does not affect viral replication perse, and that particles released are highly dependent on NUP153 expression.

TBEV infection affects the NUP153 interactome
NUP153 thus plays an important role in viral propagation and is recruited to the viral amplification site on the ER during infection. To understand the underlying mechanism, we employed an unbiased proteomic approach to examine the effect of infection on the NUP153 interactome. Cells transfected with eGFP-NUP153 or eGFP-encoding plasmids were infected with TBEV or mock-treated. The interacting proteins were enriched by protein GFP pulldown and analyzed by mass spectrometry. The analysis revealed subsets of proteins that specifically interacted (directly or indirectly) with NUP153 during either mock treatment or TBEV infection (Fig. 3A), as well as 242 proteins that interacted with NUP153 under both conditions. Pathway analysis (KEGG) of the shared protein set revealed that the NUP153 interactors were involved at different stages of protein production, from mRNA processing (spliceosome, 37 proteins) to nucleocytoplasmic transport (21 proteins) and translation (ribosome, 47 proteins) (Fig. 3B). Comparison of the data from mock-treated samples with the TBEV infected cells revealed that a set of proteins, involved in gene expression (spliceosome, mRNA surveillance, and ribosome), RNA degradation, and nucleocytoplasmic transport, was either lost or decreased during infection (Supplementary Fig. 3A). Statistical analysis of the 242 proteins found under both conditions revealed differential enrichment in mock-treated and TBEV infected samples (Fig. 3C). Notably, during infection, we observed a shift in the cellular interactome of NUP153 towards actin cytoskeleton proteins, as well as a set of ribosomal and rRNA processing proteins (Fig. 3D). Several of these proteins participate in cell cycle regulation (examples EBNA1BP2, LYAR, and RPS12) and are also involved in the translation process (examples DDX21 and NACA), either directly at the ribosome or by regulating ribosomal biogenesis19–26.

TBEV and other viruses induce translational shut-off in the host cells27,28. We investigated whether NUP153 is involved in this process, as we noted that infection altered its interactions with several proteins that are involved at different stages of gene expression. To quantify the absolute translation levels, we measured the incorporation of O-propargyl puromycin (OPP) into elongating polypeptides in the presence or absence of NUP153 using the AID system. We found that while TBEV infection resulted in a 40% reduction of global translation levels, NUP153 depletion did not affect these levels (Fig. 3E). To verify that the effect we observed was not due to reduced cell viability, we analyzed cell viability using trypan blue following TBEV infection and absence of NUP153 and detected no significant differences (Fig. 3F). These data indicate that infection changes the interactome of NUP153 in the cell and that NUP153 is not involved in the translational shut-off caused by virus infection.

NUP153 associate with viral protein NS3 and NS5
Approximately 50% of human proteins identified in the NUP153 pulldown experiment were endogenous RNA-binding proteins (Supplementary Table 1). In addition, we identified three RNA-binding viral proteins (C, NS3, and NS5) (Fig. 3A)29,30. To determine whether NUP153 requires RNA binding as a bridge to interact with these viral proteins, we performed co-immunoprecipitation assay in the presence or absence of RNase A (Fig. 3G). All three viral proteins co-immunoprecipitated with NUP153 in the presence of RNA, but the interaction between NUP153 and C was reduced after RNase A treatment, indicating that NS3 and NS5 bind to NUP153 independent of RNA, whereas the C interaction is likely RNA dependent. Furthermore, we also find that RNase A treatment resulted in decreased C protein in the input (Fig. 3G), suggesting that RNA could be important for this protein stability. Immunofluorescence analysis revealed that, while the colocalization of NS3 and NUP153 was more pronounced in the cytoplasm than in the nucleus (Figs. 1A and 3I), NUP153 colocalized very strongly with NS5 in the nucleus and cytoplasm during infection (Fig. 3H, J). Next, we transfected cells with constructs encoding FLAG-tagged NS2B3, NS3, or NS5, and used immunofluorescence microscopy to analyze the distribution of these proteins and NUP153 (Supplementary Fig. 3B, C). We observed that the presence of NS3 and NS5 resulted in upregulation of NUP153 levels, while the presence of NS2B3 only resulted in a minimal increase of the NUP153 signal. Accordingly, this could indicate that nuclear localization of the viral proteins might be required for upregulation of expression. Overall, we find that NUP153 interacts with viral proteins NS3 and NS5 in an RNA-independent manner. These interactions could be involved in the upregulation and recruitment of NUP153 to the viral amplification site that we observed during infection.

The RNA-binding domain of NUP153 is important for NUP153 recruitment to the viral amplification site and binds viral RNA
NUP153 is a large multi-domain protein31. The N-terminal domain contains NE-binding site (amino acids 1–144), NPC-targeting domain (amino acids 39–439), and an RNA-binding domain (RBD) (amino acids 250–400). The middle domain contains four zinc fingers, and the C-terminal domain contains FG repeats (Fig. 4A)31,32. To investigate which region of NUP153 is important for the recruitment of the protein to the viral amplification site during infection, we transfected HEK293T cells with constructs encoding different eGFP-tagged NUP153 truncation variants, and then infected the cells with LGTV (Fig. 4B). Similar to what was previously reported, in uninfected cells, the variants containing the NE-binding site and NPC-targeting domain (eGFP-NUP153 1–650, eGFP-NUP153 1–250 and eGFP-NUP153 1–650-RBD) correctly localized at the NE, while the variant lacking these domains (eGFP-NUP153 880–1475 and eGFP-NUP153 RBD) did not and, instead, were detected diffused in the cytoplasm or in the nucleus respectively (Fig. 4B)33,34. Further, the cellular localization of eGFP-NUP153 1–650, but not eGFP-NUP153 1–250, eGFP-NUP153 1–650-RBD and eGFP-NUP153 RBD, changed during infection (Fig. 4B), indicating that the RNA-binding domain is required but not sufficient for the recruitment of NUP153 to the viral amplification site.

Next, we performed a crosslinking immunoprecipitation (CLIP) experiment to investigate whether NUP153 can interact with viral RNA (Fig. 4C). First, protein expression of the NUP153-GFP constructs were verified by WB (Supplementary Fig. 4A). As a control we verified that NUP153 yet not eGFP was able to bind cellular RNA encoding for actin (Supplementary Fig. 4B). The analysis of the interaction with viral RNA revealed that full-length NUP153, as well as the N-terminal (eGFP-NUP153 1–650), the construct only expressing the RBD (eGPF-NUP153 RBD) and C-terminal (eGFP-NUP153 880–1475) variants can bind viral RNA (Fig. 4C). As the FG repeat-containing C-terminal domain of NUP153 binds multiple proteins, including viral proteins (HIV), the interaction with viral RNA may have been mediated by indirect interaction involving additional binding partners35. The eGFP-NUP153 1–250 and the eGFP-NUP153 1–650-RBD variants, which both lack the RNA-binding domain, did not bind viral RNA, suggesting that the RNA-binding region36 is required for viral RNA binding (Fig. 4C). We also investigated whether the interaction between NUP153 and viral RNA is RNA polarity-specific. We observed that NUP153 bound both positive- and negative-strand viral RNA, although the interaction with the positive-strand was more pronounced than that with the negative-strand (Fig. 4D).

Furthermore, we performed RNA electrophoretic mobility shift assay (RNA EMSA) using purified RBD domain of NUP153 (His-eGFP-NUP153 RBD) and in vitro transcribed viral TBEV genome to confirm that the RBD domain alone could interact with viral RNA without other cellular components. The interaction with TBEV viral RNA was confirmed, finding a shift at ~14 µM (Fig. 4E).

The RBD is thus responsible for the viral RNA interaction, and the N-terminal domain 1–650 is important for the redistribution of NUP153 to the ER during infection. However, it is unclear which domains are important for viral infection. Therefore, NUP153(NG)AID cells were transfected with the different NUP153 mutants in the presence or absence of auxin and infected with TBEV. We found that only the full length and the NUP153 1–650 could rescue the infection (Fig. 4F). Because the RBD domain alone was not able to rescue infection, we hypothesize that the remaining N-terminal domain is required for recruitment to the replication site and interaction with the viral proteins. To investigate this, we performed a co-immunoprecipitation assay with the N-terminal (eGFP-NUP153 1–650) and C-terminal (eGFP-NUP153 880–1475) domains of NUP153 and found that the N-terminal domain is required for the interaction with viral protein NS3 (Supplementary Fig. 4C).

Together, these observations indicated that the RBD of NUP153 is required for binding to viral RNA. However, the entirety of the N-terminal domain of NUP153 (1–650aa) is required for interaction with viral protein NS3, recruitment to the viral amplification site and the proviral function.

NUP153 bind two conserved regions in the viral genome
To determine if there was a specific site on the viral RNA that interacted with NUP153, we analyzed cells transfected with eGFP-NUP153 1–650 encoding construct and infected with TBEV, using CLIP-sequencing (CLIP-seq) (Fig. 5A). We opted to use the eGFP-NUP153 1–650 instead of the full-length construct to minimize indirect RNA binding arising from proteins bound to the FG-domain of NUP153 (Supplementary Fig. 4D). NUP153 primarily bound host RNA, however, approximately 1.4% of reads mapped to the TBEV genome (Fig. 5B), particularly the positive strand (Fig. 5C). Statistical analysis of the positive-strand reads revealed association with three viral RNA regions, depicted as peaks in Fig. 5C. The first peak was located at the end of the E protein coding region (peak 1, 2118–2264 nt), a region that is highly conserved among orthoflaviviruses, while the two other peaks (peak 2, 5760–5948 nt; and peak 3, 6950–6970 nt) were located in the NS3 and NS4B coding sequences, respectively (Fig. 5C and Supplementary Fig. 4E–G).

The RNA-binding domain of NUP153 has previously been shown to associate with G- and GC-rich regions of RNA molecule11,36. G-rich sequences can fold into G-quadruplex (G4) structures, a type of four-stranded nucleic acids secondary structure that can fold in certain G-rich sequences37. Peak 1 contains two predicted G4 structures (Fig. 5D boxes and Supplementary Fig. 6A). The second of which is highly conserved among orthoflaviviruses sequences (Supplementary Fig. 4E)38,39. We used in vitro transcription to generate RNA fragments containing the wildtype (WT) peak 1, a mutated construct (11G1C) lacking the second highly conserved G4 and a construct lacking all orthoflavivirus conserved Gs (28G) (Fig. 5D). We also in vitro transcribed three versions of peak 2, the WT peak, a mutated construct lacking the GA pattern that we identified to be conserved among orthoflaviviruses (8G8A) and a construct lacking all conserved Gs (19G) (Supplementary Fig. 5A). RNA-EMSA was used to determine the interaction of the NUP153 RBD with the different RNA fragments corresponding to peak 1 and 2 and their mutants. We found that WT fragments of peaks 1 and 2 bound better to the NUP153 RBD compared to the 28G and 19G fragments, respectively, that lacked all conserved Gs (Fig. 5E–H and Supplementary Fig. 5B, C), indicating that Gs in these regions are important for the interaction with NUP153. Additionally, we found that removing the conserved GA pattern (8G8A) in peak 2 does not affect binding to the RBD, suggesting that it is not important for the interaction. These results showed that NUP153 bind specific regions in the TBEV genome, which contains conserved Gs among orthoflaviviruses, and these are important for effective binding.

NUP153 interact with a conserved G-rich region on viral RNA if the G-quadruplex (G4) structure is destabilized
In peak 1, we predicted one highly conserved G4 motif among all orthoflaviviruses, however we do not know whether this region can indeed fold into G4s and if this motif is required for interaction with NUP153. To test if peak 1 can fold into G4 we generate longer in vitro transcribed fragments of the wild type (WT) peak and a mutated construct (11G1C) lacking the second, highly conserved G4 (Supplementary Fig. 6B) and transfected these into cells and measured their ability to form G4s, by quantifying the mean intensity signal after detection with anti-G4 antibody (1H6). We detected G4 signal from both transfections, however, the signal intensity of WT RNA was significantly higher compared to 11G1C RNA, suggesting that the WT sequence has higher propensity to fold into stable G4 structures (Fig. 6A, B). To verify the existence of the two different G4s, we then in vitro transcribed the WT and mutant versions of peak 1 and analyzed the properties using circular dichroism (CD) spectroscopy (Fig. 6C, D). G4s formed in RNA oligonucleotides are reported to predominately form parallel G4s, with positive maximum around 264 nm, and their stabilization is dependent on potassium ions40. The CD analyses showed a positive maximum at 264 nm for the WT and the 11G1C mutant peak 1 in the presence of KCl, while in its absence, the maximum shifted to 268 nm for the WT and 272 nm for the 11G1C (Fig. 6C). These data suggested that both the WT sequence and the mutant 11G1C may form parallel G4 structures in the presence of KCl. The small shift in the positive maximum of the WT fragment in the absence of KCl could be explained by the formation of complex secondary structures along the RNA molecule, including RNA stem-loop structures, which produces overlapping CD spectra with RNA G4s41,42. In contrast, the 28G derivative, which lacked all (28) conserved guanines (including the two G4s) but contained 17 none-conserved guanines showed a maximum at 272 nm in all conditions (Fig. 6C) indicating that 28G mutant cannot form RNA G4 secondary structures.

To further confirm the effect of KCl in G4 formation and stabilization in these sequences, we conducted thermal stability analysis using temperature-induced CD melting experiments. These experiments showed that the melting temperatures (Tm) of both WT and 11G1C sequences were significantly increased upon KCl addition by around 3 and 6 °C, respectively, while no change in Tm was observed for 28G mutant in presence of KCl (Fig. 6D).

Even though the analysis indicated that peak 1 can form a G4, it was not clear whether the G4 was bound by NUP153. In vitro, several sequences are able to form G4s, yet most of these are found unfolded in vivo, potentially through a complex mechanism involving proteins that bind G-rich RNA regions40. We therefore quantified eGFP-NUP153 1–650 binding to the different RNA fragments in vitro in the presence or absence of KCl. We found that eGFP-NUP153 1–650 bound significantly more WT RNA in the absence of KCl than in the presence of KCl (Fig. 6E). Similarly, the absence of KCl improved the eGFP-NUP153 1–650 binding to the 11G1C derivative, which lacked the second G4 motif. In contrast, only low levels of binding were observed with the 28G derivative. This interaction was not significantly affected by KCl (Fig. 6E). This indicated that peak 1 form G4 motifs in the presence of KCl, but NUP153 interacted with peak 1 in the absence of G4 secondary structure.

Similar to peak 1, peak 2 (in the NS3 coding sequence) is conserved among orthoflaviviruses and contains a predicted G4 structure (Supplementary Fig. 6A)37; however, the G4 is not conserved (Supplementary Fig. 4F). The total percentage of guanines in peak 2 is similar to that in peak 1 (31% vs. 32%) (Supplementary Fig. 6C) but, while 56% of these residues are conserved in peak 1, only 31% are conserved in peak 2. No conserved guanines were located in peak 3 (Supplementary Fig. 4G). To investigate whether the conserved guanines or GAs identified in peak 2 are important for the binding to NUP153, we performed an in vitro binding assay with WT and mutated RNA sequences. We found that in the presence of KCl, peak 2 WT, 8G8A, and 19G RNA derivatives (Supplementary Fig. 5A) bound eGFP-NUP153 1–650 to a similar degree as peak 1 WT in the absence of KCl (Fig. 6E, F). This indicated that the binding of NUP153 to peak 2 was not mediated by the conserved guanines or GAs, which is in line with the RNA EMSA results in Fig. 5G.

Taken together, the above findings indicated that NUP153 binds positive-stranded RNA, and specifically, conserved guanines in peak 1, in the absence of a G4.

NUP153 is a proviral factor during orthoflavivirus infection
Since the guanines needed for NUP153 binding in peak 1 are conserved among all orthoflaviviruses, we next sought to investigate the importance of NUP153 during infection with other prominent orthoflaviviruses. We first evaluated the intracellular localization of NUP153 during infection with ZIKV, JEV, WNV, YFV, and DENV2. We found that, similar to TBEV and LGTV infection, infection with other orthoflaviviruses induced the expression of NUP153 and recruited NUP153 to the cytosolic fraction (Fig. 7A, B). Furthermore, depletion of NUP153 using the AID-system resulted in a reduced number of infected cells in all the tested orthoflaviviruses and a strong reduction in progeny particles released (Fig. 7C–E), indicating that NUP153 is an important conserved host protein in the orthoflavivirus lifecycle.

NUP153 plays an important role in flavivirus infection by impacting the ratio of structural to nonstructural viral proteins
Orthoflaviviruses have a single-stranded RNA genome that encodes a polyprotein that is subsequently processed into 10 viral proteins. Many viruses employ mechanisms to regulate viral protein translation, increasing the translation of specific viral proteins while decreasing the translation of other proteins to ensure optimal replication. However, how orthoflavivirus protein is regulated; during translation or via post translational modification and whether all proteins are expressed equally during different stages of the viral amplification cycle is unknown43. Since we found that NUP153 interacts with numerous ribosomal proteins (Supplementary Table 1), is required early on during infection (Fig. 2I, J), and interacts with the viral mRNA at the end of the coding region of the structural proteins (Fig. 5C, E), we synthesized that it could play a major role in regulating viral RNA translation to increase the ratio of viral structural proteins compared to nonstructural proteins, leading to enhanced viral particle production during the early stages of infection. To test this hypothesis, we performed an in vitro translation assay of the TBEV genome in the presence or absence of purified NUP153 RBD. We used a mRNA construct that encodes for the 5’ untranslated region, the viral proteins C, prM, and E, together with luciferase (Fig. 8A and Supplementary Fig. 7A). As a control we also used a mRNA in which the binding peak in E was mutated, however as the terminal part of E containing the binding peak can be found duplicated after the luciferase (Mut-28G), we expect this mutated construct to still bind the RBD of NUP153 (Fig. 8A and Supplementary Fig. 7A). The binding of RBD to both these constructs was first confirmed by RNA EMSA, with the WT construct having a higher binding affinity compared to the Mut-28G mutant (Fig. 8B, C, and Supplementary Fig. 7B, C). These constructs were then used for in vitro translation in the presence of either purified His-eGFP-NUP153 or His-eGFP protein. We found that the addition of the NUP153 RBD resulted in a 50% reduction in the luciferase signal compared to addition of eGFP for the WT mRNA construct, while resulting in an increase in luciferase signal for the Mut-28G construct (Fig. 8D). To further investigate the translation products produced during these reactions we performed Western blot using anti-TBEV E antibody to detect in vitro translated proteins (Supplementary Fig. 7D). We confirm that the addition of NUP153 RBD results in a reduction of the WT full length luciferase construct, however we still observe translation of a lower molecular weight construct that could correspond to a translation product encoding for C-prM-E lacking the luciferase.

Interestingly, for the Mut-28G construct, we also observe enhanced production of the lower molecular weight product (C-prM-E) in the absence of NUP153 RBD, suggesting that this sequence might also be important for efficient polyprotein translation. However, addition of the NUP153 RBD to the Mut-28G mRNA stabilized translation of the full-length construct in line what we also observed with measurement of the luciferase (Supplementary Fig. 7D and Fig. 8D). Taken together our results indicate that the NUP153 RBD can regulate translation in vitro through binding of a specific site at the end of the E coding region of the viral mRNA.

To determine whether this translation regulation could also be observed in infected cells, we measured the levels of E and NS1 in TBEV-infected cells, and E and NS2B in WNV-infected cells using the AID-system and immunostaining. We observed that NUP153 depletion resulted in an approximately 20% reduction in E levels in E positive cells for both, TBEV and WNV (Fig. 8E, F). However, the expression of TBEV NS1 and WNV NS2B increased in the absence of NUP153 (Supplementary Fig. 8A, B), leading to higher NS1/E and NS2B/E ratios (Fig. 8G), whereas no difference in protein expression was detected in control DLD-1 cells (Fig. 8G). To further investigate the differences in the expression of the different viral proteins in the presence or absence of NUP153 we decided to measure the ratio of E to other TBEV viral proteins using Western blots (Supplementary Fig. 8C). We found that depletion of NUP153 resulted in 40–60% reduction of the different viral proteins (Supplementary Fig. 8D). Additionally, when looking at the ratios between E and the other viral proteins, we observed that similar to our immunofluorescence assay (Fig. 8G), depletion of NUP153 resulted in an increased ratio of nonstructural proteins (NS1, NS2B, NS3, and NS5) to E, while no difference was observed for the ratios of the other structural proteins (C and M) (Fig. 8H). To validate our findings using a different orthoflavivirus, we decided to also measure the ratios and viral protein levels in WNV infected cells, however, as we lack antibodies for this virus, we instead used mass spectrometry to detect these. Using mass spectrometry we were able to detect seven of the viral proteins (C, prM, E, NS1, NS3, NS4B, and NS5), with the levels of these significantly being reduced when NUP153 was depleted (Supplementary Fig. 8E). Next, we determined the protein ratio between structural and nonstructural proteins in the same samples. We found no difference in the protein ratio between the structural proteins (C/E and prM/E) and E in the presence or absence of NUP153 (Fig. 8I). However, similar to what we observed for TBEV, the protein ratio of NS1/E, NS3/E and NS5/E showed an increase after auxin treatment and depletion of NUP153 (Fig. 8I) supporting the immunofluorescence quantification of WNV NS2B/E ratio (Fig. 8G), indicating that this is a general trend of the E versus nonstructural proteins.

Furthermore, to exclude that the effects we observed was a result of decreased protein stability following NUP153 depletion, we performed a cycloheximide (CHX) assay on TBEV-infected cells and found no significant differences in the stability of the viral proteins E, NS2B and NS3 between auxin-treated and untreated samples over time (Fig. 8J and Supplementary Fig. 8F, G). However, we did observe a small change in NS1 expression 6h post CHX treatment (Supplementary Fig. 8F, G). Taken together, this indicated that NUP153 interacts with the RNA corresponding to the E terminal region, and thereby modulates the expression of downstream nonstructural proteins to favor the expression of the structural proteins.

Efficient production of progeny virions early in infection is crucial for viral spread and for preventing the cellular antiviral type 1 interferon signaling leading to enhanced infection44–46. Accordingly, we measured the efficiency with which the TBEV and WNV viruses spread to new cells in the presence or absence of NUP153. Brefeldin-A (BFA), which disassembles the Golgi complex and prevents virus release47, was used as a reference of initial infection, and was added at 16 hpi. In the presence of NUP153, both viruses spread to new cells between 16 hpi (BFA was added) and 24 hpi. However, in the absence of NUP153, no additional cells became infected in the first 24 hpi for both TBEV and WNV, compared to the BFA-treated cells (Fig. 8K and Supplementary Fig. 8H, I). At 48 hpi, many new cells become infected even in the absence of NUP153, indicating that NUP153 is more important early in infection.

Taken together, our results suggest that NUP153 binds to orthoflaviviral RNA at the end of the E coding region, thereby balancing the protein ratio in favor of the structural proteins, leading to increased virion production and release. In the absence of NUP153, the viral protein ratios changes, decreasing the structural protein levels in the cells and thereby delaying the virion release (Fig. 9).

Methods
Cell culture and reagents
A549 and HEK293T cells are both good models for TBEV infection, A549 cells are better for immunofluorescence, and HEK293T cells are better for transfection. They were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 5% fetal bovine serum (FBS; Gibco), 20 units/ml penicillin, and 20 µg/ml streptomycin (GE Healthcare) at 37 °C in 5% CO2. A549 MAVS−/− cells16 were cultured in DMEM supplemented with FBS, penicillin and streptomycin but also supplemented with 2 µg/ml puromycin (Gibco) at 37 °C in 5% CO2. VeroB4 cells were cultured in Hyclone Medium 199 with Earle´s Balanced Salts and L-Glutamine (Medium 199/EBSS; GE Healthcare) supplemented with 5% FBS, 20 units/ml penicillin, and 20 µg/ml streptomycin at 37 °C in 5% CO2. DLD-1 and NUP153(NG)AID cells were a kind gift from M. Dasso (Division of Molecular and Cellular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, USA) and were cultured in DMEM+GlutaMAX (Gibco) media supplemented with 10% FBS, 20 units/ml penicillin, and 20 µg/ml streptomycin at 37 °C in 5% CO218. DLD-1 parental cells were engineered to express Neongreen and AID tagged NUP153 (NUP153 (NG)AID), the DLD-1 control cells did not express AID tag18. Auxin (1 mM) was added to degrade NUP153 in NUP153(NG)AID cells. Cells were transfected using GeneJuice transfection reagent (Novagen) according to the manufacturer’s instructions. Brefeldin A (BFA) (Sigma) was used at a concentration of 5 µg/mL.

Viruses
TBEV-Torö or TBEV-Neudoerfl was produced in VeroB4 cells and harvested 72 hpi50. LGTV was produced in A549 MAVS−/− cells and harvested 72 hpi. Japanese encephalitis virus (JEV) (Nakayama strain), West Nile virus (WNV) (WNV_0304h_ISR00), yellow fever virus (YFV) (Asibi), and dengue virus (DENV) (serotype-2; PNG/New Guinea C) were kind gifts from S. Vene (the Public Health Agency of Sweden) and were grown in VeroB4 cells. ZIKV (MR766) was a kind gift from G. Dobler (Bundeswehr Institute of Microbiology, Munich, Germany) and was grown in VeroB4 cells. Viruses where titrated by focus forming unit (FFU) assay on VeroB4, and FFU was determined 48 hpi by counting E-stained (anti-TBEV E 1783.3 or anti-panFlaviE) foci50. Titration was performed on the different cell lines to ensure a correct MOI dose.

Plasmids
Mammalian expression plasmids (backbone pI.18) encoding C-terminally 3×FLAG-tagged TBEV-Hypr NS3; NS2B3 and NS550. eGFP-NUP153, eGFP-NUP153 1–650 and eGFP-NUP153 880–1475 were a gift from B. Fahrenkrog (Addgene plasmid # 64,268; http://n2t.net/addgene: 64268; RRID: Addgene_64,268; Addgene plasmid # 64,318; http://n2t.net/addgene: 64318; RRID: Addgene_64,318; Addgene plasmid # 64,317; http://n2t.net/addgene: 64317; RRID: Addgene_64,317). eGFP-NUP153 1–250, eGFP-NUP153 1–650-RBD and eGFP-NUP153 RBD were cloned using standard PCR cloning methods. Plasmids for protein expression of His-eGFP and His-eGFP-NUP153 RBD were cloned using standard PCR cloning methods into the pQE-30Xa (Qiagen) vector. Fragments used to clone plasmids for reverse transcription were ordered from Genewiz and cloned by standard methods into pcDNA3.1. WT construct used in the in vitro translation assay was cloned by standard cloning methods. Fragment to mutate peak 1 in the Mut-28G construct was ordered from Eurofins Genomics and cloned by standard cloning methods into the WT construct used for the in vitro translation assay. All plasmids were sequenced to verify their correctness.

Immunofluorescence
Cells were grown in 8-well chamber slides (Sarstedt), transfected and/or infected and incubated for the given amount of time. Samples for immunofluorescence were infected with a MOI 10 to ensure infection in all cells. Samples in which nucleoporin antibodies were used were permeabilized with 0.02% Triton X-100 in PBS for 30 s before fixation. They were fixed with 4% formaldehyde for 30 min and incubated in permeabilization buffer (PBS containing 2% goat serum and 0.3% Triton-X) containing primary or secondary antibodies for 1 h each at room temperature (Supplementary Table 1). Samples were washed with PBS-Tween in between the incubations and also after the secondary antibody before being mounted using mounting media. Samples were imaged and analyzed using a Leica SP8 Laser Scanning Confocal Microscope with a 63× oil objective (Leica) and Leica Application Suit X software (LASX, Leica). For SIM cells were imaged with Zeiss Elyra 7 microscope with Lattice-SIM2 with a Plan-Apochromat 63×/1.4 oil objective (Zeiss).

Fluorescent in situ hybridization (FISH)
A549 cells were grown in 8-well chamber slides (Sarstedt) and infected with TBEV MOI 10 for 24 h. Cells were fixed with 4% formaldehyde for 30 min and washed with PBS. They were then incubated with 70% ethanol overnight at 4 °C. Cells were then washed with wash buffer (2× saline-sodium citrate, 10% formamide) and incubated overnight with probes (poly-T 1:2000) and primary antibody (NS3 1:500) in hybridization buffer (2× saline-sodium citrate, 10% dextran sulfate, 10% formamide) at 37 °C. Cells were washed once and incubated 1 h in wash buffer containing secondary antibody and DAPI. Cells were washed once in wash buffer and mounted using mounting media. Samples were imaged and analyzed using a Leica SP8 Laser Scanning Confocal Microscope with a 63× oil objective (Leica) and Leica Application Suit X software (LASX, Leica).

Image analysis and quantification
Images obtained from the confocal microscope were processed using ImageJ62, and quantification were then done using CellProfiler63. The DAPI channel was used to identify the nuclei as primary objects, while GAPDH or expansion of the DAPI channel was used to identify the whole cell as secondary object. These two objects were then used to identify the cytoplasmic fraction as a tertiary object. “Measure Object Intensity” and “Measure Colocalization” were then used to analyze the stained proteins in the different fractions. Microsoft Excel was then used to process tables for the selection of infected or transfected cells. Final plots were then generated using GraphPad Prism.
SIM images were processed and quantified using Imaris software (version 10.2) with the ImarisColoc package used to determine the Manders’ coefficient in entire images.

RT-qPCR
Samples for RNA extraction were infected with MOI 10 to ensure infection in all cells and also obtain comparable results to the data obtained from immunofluorescence assays. In experiments performed on NUP153(NG)AID cells an MOI of 0.5 was used to be able to compare results with other infectious assay readouts. RNA was isolated using the NucleoSpin RNA Plus (Macherey Nagel) kit according to manufacturer’s instructions. cDNA was synthesized using high-capacity cDNA Reverse Transcription kit (Thermo Fisher). qPCRBIO SyGreen mix Hi-ROX (PCR Biosystems) was used to detect IFNB1 RNA (QT00225372, Qiagen) and NUP153 RNA (primers found in Supplementary Table 2). qPCRBIO probe mix Hi-ROX (PCR Biosystems) was used to detect LGTV and TBEV RNA (primers found in Supplementary Table 2). Samples were normalized to actin (QT01680476, Qiagen) that was used as reference gene. RT-qPCR experiments were run on a StepOnePlus real-time PCR system (Applied Biosystems).

Platereader immunofluorescence assay
Twenty-five thousand cells were seeded into CELLSTAR® 96-well (Greiner) plates and infected with different viruses using approximately MOI 0.5, titrated on the specific cell lines used, to reach approximately 50% infection at the end of the experiment. Cells were then fixed for 30 min using 4% formaldehyde and washed once with PBS. They were then permeabilized for 10 min with permeabilization buffer (0.5% Triton-X, 20 mM glycine in PBS) at room temperature. Cells were then incubated in primary antibody in dilution buffer (10% FBS, 0.05% Tween-80 in PBS) for 1 h. They were then washed three times with PBS-Tween and incubated 1 h with secondary antibody diluted in dilution buffer. Cells were washed three times with PBS and imaged and analyzed with Cytation 5 and BioTek Gen5 software (Agilent).

Affinity purification-mass spectrometry
HEK293T cells were seeded in T-175 flasks and transfected with 10 µg eGFP-NUP153 or eGFP. They were infected with TBEV-Neudörfl (MOI 5) for 1 h at 37 °C and 5% CO2. Then the inoculum was removed and replaced with fresh medium. After 24 h of infection cells were washed in PBS and then lysed in cold lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40 substitute (Sigma 74385), and 2x Protease inhibitor (Roche, cOmplete, Mini, EDTA-free, 4693159001) for 1 h. Lysates were then centrifuged at 16,000×g, 4 °C for 15 min. Supernatants were transferred to new tubes and were flash freezed and stored at −80 °C.
Total protein concentration in HEK293T cell lysates expressing eGFP or eGFP-NUP153, infected with TBEV-Neudörfl or mock infected was quantified using the DC assay (Bio-Rad). Three biological replicates per condition were prepared.
The lysates were diluted to 0.4 mg/ml protein concentration using dilution buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1x Protease inhibitors), and 1250 µl of this (0.5 mg protein) was used for each sample. The diluted cell lysate was incubated with 25 µl of GFP-trap® Dynabeads™ (Chromotek) at 4 °C for 1 h on a rotator. The beads were then washed three times with wash buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) and the binding proteins were eluted using 50 µl acidic elution buffer (200 mM glycine-HCl, pH 2.5) while vigorously pipetting. The eluate was neutralized with 10 µl neutralization buffer (1 M ammonium bicarbonate), then reduced with DTT and alkylated using IAA. The proteins were digested using trypsin on 37 °C overnight. The digested samples were then acidified using 6.5 µl acidifying solution (83.3% acetonitrile (AcN), 16.7% TFA) to pH <3. The peptides were desalted using in-house-made STageTips64,65. Briefly, the STageTip was constructed using 2 layers of C18 membrane (3M Empore) packed into a 200 µl pipette tip. The membrane was activated with methanol, then washed with 80% AcN, 0.1% formic acid once and twice with 0.1% formic acid. The acidified samples were loaded onto the tips, washed with 0.1% formic acid and eluted with 80% AcN, 0.1% formic acid. The eluate was then vacuum-dried and stored at −80 °C until use.
The instrumental setup consisted of an Easy-nLC 1000 nanoLC (Thermo Fisher Scientific) equipped with an Acclaim PepMap 100 pre-column (Thermo, 75 µm × 2 cm, 3 µm, 100 Å) and an EASY Spray PepMap RSLC C18 analytical column (Thermo Fisher Scientific, EASY spray, 75 µm × 15 cm, 2 µm, 100 Å), while the mass spectrometer (MS) used was a QExactive Plus instrument (Thermo Fisher Scientific). The EASY Spray source was operated on 40 °C, and the spray voltage was set to 1.9 kV. The liquid chromatography gradient method went from 4 to 76% acetonitrile in 79 min with both mobile phases containing 0.1% formic acid. The MS was used in the positive ion mode, the full scan having a resolution of 140,000 (400–1700 m/z) and an automatic gain control (AGC) target of 3 × 106, while the resolution and AGC target was 17,500 and 1 × 105 for MS/MS, respectively. The top 10 most abundant ions were fragmented and measured in MS/MS using data-dependent acquisition with dynamic exclusion of 30 s enabled.
The generated raw files were searched using MaxQuant66 (2.0.1.0) against the human FASTA file (Uniprot, Homo sapiens, reviewed, 20,360 entries, downloaded on 2022-02-21.) supplemented with TBEV (U27495.1, protein sequences downloaded from NCBI (AAA86870.1) on 2022-04-06.) and LGTV (strain TP21, protein sequences downloaded from NCBI (NC_003690.1) on 2022.04.06) protein sequences. Trypsin/P was used as the digestion enzyme with maximum 2 missed cleavages allowed. Carbamidomethylation of cysteines was selected as fixed modification, while methionine oxidation and N-terminal acetylation was used as variable modifications. Label-free quantification (MaxLFQ algorithm67) was selected with a minimum ratio count of 2. Peptide mass tolerances were 20 and 4.5 ppm for the first and main search, respectively.
PSM and protein FDR was set to 0.01. The minimum number of detected peptides required was 2, and the minimum number of unique peptides was 1 for identification.
As viral proteins are often short and have many basic residues, they are more challenging to identify in regular searches. For this reason, specific searches were carried out. First with MaxQuant (2.0.1.0) using only the specific viral sequences as the FASTA file and lowering the minimum peptide length for unspecific search to 4 amino acids (7 and 8 in the original search, respectively), and only 1 peptide and 1 unique peptide was required. These searches are, however, biased and can result in many false positives. The search was repeated in Proteome Discoverer (1.4.0.288) (Thermo Fisher Scientific) using SequestHT and Target Decoy PSM validator. The corresponding settings to an Orbitrap instrument were selected, and the same modifications used as above. The target FDR was 0.01 (strict) and 0.05 (relaxed). For protein identification, the high-confidence peptide fragment spectra were observed so that the sequence identity could be confirmed. The peptides were also compared to the human proteome using BLAST to validate that they are not human peptide hits.

Statistical analysis (AP-MS)
The generated LFQ intensity data from the MaxQuant output file “proteinGroups.txt” was first used to identify proteins found uniquely in NUP153 either TBEV, and mock-infected, and then further processed for statistical testing employing Perseus (2.0.3.0)68, selecting the LFQ intensity values as main. Proteins found in more than 1 out of 6 GFP samples were removed from the analysis. Potential contaminants, proteins only identified by site or reverse hits were removed, and the LFQ intensities transformed to a log2(x) base. The two sample groups were defined, then the proteins were filtered based on valid intensity values in the samples (minimum 2 valid values in both groups). Missing values were imputed using values from normal distribution (width 0.3, downshift 1.8, total matrix). To determine the significantly enriched proteins a two-sided t-test was used (permutation-based FDR: 0.05, S0: 0.1, 250 permutations).

Protein synthesis measurement using OPP
Cells were induced with auxin 2 h prior to infection with TBEV MOI 10. A high MOI was selected to ensure all cells were infected. Global translation was measured using the OPP assay (OPP, Thermo Fisher Scientific) following the manufacturer’s instructions. In brief, cells were treated with 5 µM OPP for 30 min at 37 °C in 5% CO2. The cells were then fixed for 30 min using 4% formaldehyde and permeabilized in 0.5% Triton X-100 for 15 min. OPP was then fluorescently labeled 30 min by Click-iT Plus OPP reaction cocktail with Alexa Fluor 647 picolyl azide. Cells were then analyzed by Cytation 5 (Agilent) by measuring the mean fluorescent intensity.

Trypan blue viability assay
Cells were induced with auxin 2 h prior to infection with TBEV (MOI 10). Following treatment cells were detached using 0.25% trypsin. Cells were mixed in a 1:1 ratio with Trypan Blue stain 0.4% (Invitrogen) and measured with the countess II automated cell counter (Invitrogen).

Co-immunoprecipitation
HEK293T cells were seeded in T-75 (Sarstedt) and transfected with 6 µg of the plasmids of interest and infected with TBEV or LGTV (MOI 1) for 48 h. Cells were washed once in PBS and lysed using lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1% Triton X-100 and protease inhibitor [1 tablet per 5 mL]). For RNase treatment lysis buffer was supplemented with 20 µg/mL RNase A (Thermo Fisher Scientific). Cell lysates were centrifuged at 15,000×g, 4 °C for 10 min, and the supernatants were precleared by the addition of 25 µL control beads (Chromotek) and rotated for 2 h at 4 °C. Beads were removed by centrifugation, and 60 µl of cell lysate was saved for the input control. GFP precipitation was performed on the remaining precleared sample using 25 µl GFP-trap beads (Chromotek) and rotated for 1 h at 4 °C. GFP-trap beads were washed four times in lysis buffer, and proteins were eluted by incubation at 95 °C 2× Laemmli sample buffer prior to Western blotting.

SDS-PAGE and Western blot
Cell lysate or co-immunoprecipitation samples were separated by SDS-PAGE using precast bolt 4%-to-12% Bis-Tris gels (Invitrogen). PageBlue protein staining solution (Thermo Scientific) was used according to manufacturer’s instructions to visualize proteins on SDS-PAGE. For Western blots, proteins were transferred onto an Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore), followed by incubation in blocking buffer (0.02% ECL Prime Blocking Reagent [Amersham] diluted in 0.05% PBS-Tween). The membrane was first incubated with primary antibody overnight at 4 °C, washed three times with PBS-Tween, and then incubated with fluorescent secondary antibody for 1 h at room temperature. After washing steps, band detection was performed using the Amersham Imager 680 (GE Life Sciences).

Cross-link immunoprecipitation (CLIP)
HEK293T cells were seeded in 6-well plates and let to settle overnight. The cells were then transfected with 1.5 µg plasmid of the proteins of interest for 24 h and infected with LGTV (MOI 1) for 48 h. Culture media was removed and cells were irradiated at 250 mJ/cm2 (254-nm wavelength) using a Spectro Linker XL-100 UV crosslinker (Spectronics Corporation) to cross-link the proteins with the RNA. They were then detached using ice-cold PBS and transferred to 1.5 mL tubes. Samples were centrifuged at 250×g for 10 min. Supernatant was removed and cells were resuspended and lysed in 400 µL lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1.5% Triton X-100, protease inhibitor cocktail [Roche], 5 U/µl RNase inhibitor SUPERase-In [Thermo Fisher Scientific]) for 30 min on ice. Samples were then centrifuged at 16,000×g at 4 °C for 15 min and supernatants were transferred to new Eppendorf tubes. Protein A agarose beads (Merck) (50 µl/sample) or control beads (Chromotek) (25 µl/sample) were washed three times in lysis buffer or dilution buffer (10 mM Tris [pH 7.5], 150 mM NaCl, 0.5 mM EDTA diluted in water), added to supernatants and incubated for 2 h to preclear the lysates. Samples were then centrifuged at 1000×g and supernatants were transferred to new tubes. Fifty microliters of cell lysate was saved as input control. The precleared lysates were then incubated either overnight with antibody (anti-eGFP) or for 1 h with GFP-trap beads (Chromotek). After overnight incubation samples were then incubated with Protein A agarose beads for 1 h at 4 °C. Following incubation with either Protein A agarose beads or GFP-trap beads, beads were washed four times with wash buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Triton X-100). The RNA was then eluted from the beads using QIAzol (Qiagen) according to manufacture’s instructions and purified and eluted in 40 µl RNA-free water using the NucleoSpin RNA kit (Macherey-Nagel) according to manufacture’s instructions. cDNA (High-Capacity cDNA Reverse Transcription kit; Thermo Fisher Scientific) was synthetized from the RNA according to manufactures instructions. To quantify the viral RNA, qPCR was performed on the StepOnePlus instrument using the KAPA probe MM (Techtum) and the LGTV NS3 probes and primers69. Ct values of IP samples were normalized against Ct values of the respective input collected prior to immunoprecipitation. Furthermore, values were further normalized to the control bead immunoprecipitation of each transfected construct.

Protein purification
Twenty milliliters of LB broth (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl) containing 100 µg/ml ampicillin and 25 µg/ml kanamycin were inoculated with either bacteria for His-eGFP or His-eGFP-NUP153 RBD production and grown overnight at 37 °C using a bacterial shaker. One liter of LB broth containing 100 µg/ml ampicillin and 25 µg/ml kanamycin was inoculated with the overnight culture and grown at 37 °C on a bacteria shaker until an OD600 of 0.6 was reached. Expression of protein was induced for 5 h with 1 mM IPTG. Cells were harvested by centrifugation at 4000×g for 20 min. Pellet was resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl and 10 mM Imidazole [pH 8]) supplemented with 1 mg/mL of lysozyme for 30 min on ice. Lysate was sonicated with a sonicator equipped with a microtip using 10 s bursts at 200–300 W with a 10 s cooling period between each burst. 10 µg/mL RNase A and 5 µg DNase was added to the lysate and incubated for 15 min on ice. Lysate was centrifuged at 10,000×g for 30 min at 4 °C to clear lysate. Fifty percent Ni-NTA (Qiagen) slurry was added to cleared lysate and mix gently by shaking on a rotary shaker at 4 °C for 60 min. Lysate-Ni-NTA mixture was loaded into a polypropylene column (Qiagen) and liquid was allowed to flow through. Ni-NTA was washed twice with wash buffer (50 mM NaH2PO4, 300 mM NaCl and 20 mM Imidazole [pH 8]) and protein was eluted using elution buffer (50 mM NaH2PO4, 300 mM NaCl and 250 mM Imidazole [pH 8]) in 0.5 mL fractions. Fractions were analyzed by SDS-PAGE. Fractions containing protein were pulled and a buffer exchange to PBS was performed using the Pur-A-Lyzer Maxi 3500 (Sigma). Following this, protein was concentrated using Amicon Ultra -4 10k columns (Millipore) according to manufacturer’s instructions.
The His and eGFP tags were added to the NUP153 RBD to obtain an overall more neutral isoelectric point of the protein, as the isoelectric point of the NUP153 RBD is ~12 making very positively charged which can be problematic for certain assays.

In vitro transcription
Reverse transcription was performed according to manufacturer’s instructions using the MEGAscript T7 transcription kit or MEGAscript SP6 transcription kit (Invitrogen). Following the reaction DNA was degraded with 1 µl/sample TURBO DNase (Invitrogen) and RNA was precipitated using Lithium chloride. RNA was dissolved in RNase-free water and size fragments were verified by gel electrophoresis. To label the RNA, UTP was replaced by Aminoallyl-UTP-Cy3 (Jena Bioscience) in in vitro transcription reaction.
For in vitro translation assays, in vitro transcription was performed using the mMESSAGE mMACHINE SP6 transcription kit (Thermo Fisher), to produce capped RNA.

RNA electrophoretic mobility shift assay (RNA EMSA)
His-eGFP-NUP153-RBD or His-eGFP protein in different concentrations was mixed with 80 ng of Cy3 tagged viral RNA fragments in 10 µL RNA binding buffer (25 mM Tris pH [7.6], 100 mM KCl, 3 mM MgCl2, 0.01% Tween 20, 1 mg/mL BSA and 1 mM dithiothreitol [DTT], dissolved in water) for 30 min at room temperature. Six microliters of 50% glycerol was added to each sample, loaded on a 0.8% Agarose gel and ran for 30 min at 100 V. Gels where imaged using Amersham Imager 680 (GE Lifesciences). The ImageJ62 software was used to quantify the amount of bound and unbound RNA. The data was then plotted using GraphPad Prism and the nonlinear fit one site specific binding function was used to approximate binding curves.

Cross-link immunoprecipitation sequencing (CLIP-seq)
HEK293T cells were seeded in TC100 dishes (Sarstedt) and transfected with 5 µg eGFP-NUP153 1–650 plasmid. Cells were then infected with TBEV (MOI 1) for 48 h. Culture media was removed, and cells were irradiated at 250 mJ/cm2 (254-nm wavelength) to cross-link the proteins with the RNA. They were then detached using ice-cold PBS and transferred to 1.5 mL tubes. Samples were centrifuged at 250×g for 10 min. Supernatant was removed, and cells were resuspended and lysed in 500 µL RIPA buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1.5% Triton X-100, 0.5% Sodium deoxycholate and 0.1% SDS supplemented with protease inhibitor cocktail [Roche], 5 U/µl RNase inhibitor SUPERase-In [Thermo Fisher Scientific]) for 1 h at 4 °C. Samples were then centrifuged at 16,000×g at 4 °C for 15 min and supernatants were transferred to new tubes. Control beads (Chromotek) (25 µl/sample) were washed three times in RIPA buffer and incubated for 2 h with sample at 4 °C on a rotor to pre-clear the lysates. Samples were then centrifuged at 1000×g, and supernatants were transferred to new tubes. The precleared lysates were then incubated 1 h with GFP-trap beads (Chromotek) at 4 °C on a rotor. Following this RNase A (Thermo Fisher Scientific) in a concentration of 1:2500 was added to the samples and incubated for 15 min at room temperature on a rotor. GFP-trap beads were then washed four times with RIPA buffer. The RNA was then eluted from the beads using QIAzol (Qiagen) and isopropanol RNA precipitation according to manufacture’s instructions. RNA fragment size and concentration were checked using the RNA 600 Pico kit (Agilent) and measured using the 2100 Bioanalyzer (Agilent).
cDNA library was generated with the NEBNext small RNA sequencing kit (New England Biolabs) according to manufacturer’s instructions and purified using the DNA clean & concentratorTM-5 (Zymo). Samples were then pooled, and Sera-Mag Speed beads (Cytiva) were used at a concentration of 3× to perform one-sided purification to remove longer sequences. DNA fragment size and concentration was then verified and measured by Bioanalyzer High sensitivity DNA kit (Agilent) and Qubit dsDNA HS assay kit (Invitrogen). The resulting cDNA libraries were sequenced using the Illumina MiSeq platform.
Prior to analysis, adapters and indexes were removed using cutadapt70 and the quality of reads was checked using FastQC. To be able to map the sequences that are unique to the virus genome, the human genome and the TBEV-Torö genome (DQ401140.3) were first concatenated using bowtie271. Sequences were then aligned to the concatenated genome using bowtie, allowing for up to 3 mismatches and a maximum of 10 identical reads with the best stratum72. Samtools73 was then used to sort and index the reads, and bedtools was then used to convert the files from BAM files to BED files. The galaxy platform was then used to identify significant peaks using PEAKachu74. Finally, Gviz75 was used to plot the data.

Multiple sequence alignment
Multiple sequence alignment was performed using AliView76. The sequences used for alignment are TBEV (DQ401140.3), LGTV (EU790644.1), POWV (NC_003687.1), JEV (NC_001437.1), WNV (NC_009942.1), YFV (NC_002031.1), DENV2 (NC_001474.2:97-10272), and ZIKV (NC 012532.1).

In vitro binding of RNA fragments to NUP153
HEK293T cells were seeded in T-75 flasks and transfected with 5 µg of eGFP-NUP153 1–650 plasmid using GeneJuice according to manufacturer’s instructions. Twenty-four hours after transfection, cells were lysed with RIPA buffer supplemented with RNase A in a concentration of 1:500. At this concentration of RNase A no RNA could be detected using the RNA 600 Pico kit (Agilent) and measured using the 2100 Bioanalyzer (Agilent). Samples were then pre-cleared using control beads, and proteins were purified using GFP-trap beads. Five microgram in vitro transcribed RNA was then added in 200 µl RNA-binding buffer (5 mM HEPES [pH 7.2], 3 mM MgCl2, 5% Glycerol, 1 mM DTT and 5 mM ATP in milli-Q water, supplemented with protease and 5 U/µl RNase inhibitor SUPERase-In) with or without 100 mM KCl and cross linked with 400,000 µJ/cm2 energy at 254 nM UV on ice. Samples were then incubated overnight at 4 °C on rotor and cross-linked again with 400,000 µJ/cm2 energy at 254 nM UV on ice. Samples were then washed four times in RIPA buffer, and RNA was isolated and precipitated with QIAzol and isopropanol according to manufacturer’s instructions. RNA concentration was then determined using Qubit RNA HS assay kit (Invitrogen) according to manufacturer’s instructions.

Circular dichroism spectroscopy (CD)
CD experiments were carried out in Jasco-J1700 CD spectrophotometer, having built-in Peltier cell holding carousel and temperature controller. The wild-type and mutant RNA sequences were diluted to 4 µM in the buffer containing 10 mM Tris (pH 7.0), supplemented with or without 100 mM KCl and annealed by heating at 70 °C for 5 min, followed by slowly cooling down to room temperature for 24 h. Samples were scanned within 320–210 nm wavelengths at 25 °C with a scanning speed of 100 nm/min. Data points were acquired at 0.5 nm interval and averaged over 3 accumulations. For each measurement, we used quartz cuvettes of 1 mm path length (Hellma) having a reaction volume of 200 µL. The digital integration time and the bandwidths were 2 s and 1 nm, respectively. Ellipticity values were converted to molar ellipticity or molar circular dichroism (MCD) using the following formula and had been corrected for buffer contributions.
MCD = θ / (32980 × c × l) θ is the ellipticity value in mdeg; while c and l denote the concentration of RNA (in g/L) and path-length of cuvette (in cm), respectively. To understand the effect of KCl on G4 stabilization, we conducted temperature-induced CD melting experiments in these samples using the parameters mentioned above. For thermal scans, we heated the samples from 25 to 95 °C, having temperature gradient of 5 °C/min and a delay time of 300 s. The MCD values of positive maxima were normalized to 25 °C (maximum folded G4) and 95 °C (maximum unfolded G4) using the following formula to calculate the melting temperatures (Tm):
Fraction of folded G4 at any temperature = (θ t − θ U) / (θ F − θ U) where θ t denotes MCD at any temperature, θ F and θ U represent the MCDs at 95 and 25 °C, respectively.

Mass spectrometry on NUP153(NG)AID cells infected with WNV
NUP153(NG)AID were seeded in 6-well plates and let to settle overnight. They were induced with 1 mM auxin 2 h prior to infection with WNV (MOI 0.5). At 48 hpi cells were lysed using RIPA buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate and 1% SDS, supplemented with protease inhibitor) for 1 h at 4 °C. Proteins were subsequently diluted with Ammonia-Bicarbonate-Buffer (100 mM NH4HCO3, pH 8, Thermo Fisher Cat#: 393212500), reduced with 10 mM DTT (Merck/Millipore, Cat#: 1114740001) and alkylated with 55 mM iodoacetamide (Thermo Fisher, Cat#: 122270050). Samples were precipitated using acetonitrile (Merck/Millipore, Cat#: 1000291000) and digested with trypsin (Pierce Trypsin Protease MS Grade, Cat#: 90305) at 37 °C o/n. Tryptic peptides were next acidified with 1% Formic Acid (Carl Roth, Cat#: 1EHK.1), desalted via reverse phased C18 Tips (Fisher Scientific, Cat#: 10615555), and separated via nano high-performance liquid chromatography (Vanquish Neo, Thermo Scientific, Waltham, MA, USA). Subsequently, a nano electrospray ion source interfaced with an Orbitrap Tribrid Mass Spectrometer (Eclipse, Thermo Scientific, Waltham, MA, USA) was used to ionize peptides. Briefly, the peptides were separated using an acetonitrile gradient over a 142-min total gradient on a C18 column. Survey full-scan MS spectra were recorded in data-dependent acquisition mode at a resolution of 60,000, and MS2 spectra at a resolution of 15,000. Fragmentation was performed using higher-energy collisional dissociation with a normalized collision energy of 30. Mass spectrometric data were analyzed via the Proteome Discoverer software (version 3.1, Thermo Scientific, Waltham, MA, USA) against the Uniprot human reference proteome database (EMBL-EBI, Cambridgeshire, UK) (last updated on 27/03/2024) using the SequestHT search engine (The University of Washington’s Proteomics Resource, Seattle, WA, USA) and against the full-length West Nile virus strain proteome. FDR was set to 1% on the peptide and protein level. Trypsin specificity allowing up to two missed cleavages, fixed carbamidomethyl modification on cysteine, and variable oxidation on methionine were set as parameters. Moreover, at least one unique peptide with a length of minimum six amino acids per protein group was demanded. Quantifications were performed using the Minora feature detector algorithms.

In vitro translation
Prior to vitro translation, mRNA was heated at 65 °C for 3 min and let cool immediately on ice, to denature RNA. In vitro translation was performed using the Flexi Rabbit Reticulocyte Lysate System (Promega) according to manufacturers’ instructions. Reactions where supplemented with 4 µM of either His-eGFP-NUP153 or His-eGFP protein. Following 90 min incubation at 30 °C, luciferase signal was immediately measured using the Luciferase Assay system (Promega) and the CLARIOstar Plus (BMG LABTECH) Luminometer. Five microliters of the reaction was then mixed with 20 µL of SDS sample buffer, heated for 10 min at 95 °C and analyzed by Western blot. Detection of translated proteins was performed with anti-TBEV_E (GTX642364) antibody using chemiluminescent detection with SuperSignal™ West Femto Maximum Sensitivity Substrate kit (Thermo Scientific) and imaged with the Amersham Imager 680 (GE Lifesciences).

Protein stability assay
500,000 NUP153(NG)AID cells were seeded into 6-well plates (Avantor, VWR). Cells were induced with auxin 2 h prior to infection with TBEV MOI 0.5. After 24 h of infection cells were treated with 50 µg/mL of cycloheximide and then lysed at 0, 3, and 6 h post cycloheximide treatment in cold lysis buffer (1.5% Triton-X100, 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% SDS and 0.5% sodium deoxycholate) and 2x Protease inhibitor (Roche, cOmplete, Mini, EDTA-free, 4693159001) for 1 h. Lysates were then centrifuged at 15,000×g, 4 °C for 10 min. Supernatants were transferred to new tubes and were then boiled with 1× NuPAGE™ LDS Sample Buffer (Thermo Scientific) and 2× NuPAGE™ Sample Reducing Agent (Thermo Scientific) for 10 min. Samples were then subjected to SDS-PAGE and Western blot.

Statistical analysis
Data from immunofluorescence, RT-qPCR, Qubit, RNA EMSA, infection assays and Western blot assays were analyzed using the GraphPad Prism 10.2.3 software.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Peters et al present evidence that the nuclear pore complex protein Nup153 is a proviral factor that promotes flavivirus infection. With a specific focus on tick-bore flaviviruses (TBEV and LGTV) the authors show that Nup153 is upregulated and re-localized to the cytosol during infection and that depletion of Nup153 leads to a decrease in the number of infected cells. Furthermore, they present evidence that Nup153 interacts with viral NS3 and NS5 proteins and suggest that these interactions facilitate Nup153 recruitment to the cytosol. The authors also show evidence that Nup153 interacts with viral RNA through an RNA binding domain at several G-rich regions of the viral Genome. Finally, they use light microscopy to evaluate the levels of viral structural and non-structural proteins in Nup153 depleted cells and report a change in the ratio between E and NS2B. Based on these data, the authors posit that Nup153 is involved in promoting the translation of structural proteins to facilitate virus assembly and spread.\nIn general, the study is well structured, using various techniques to comprehensively evaluate the role of Nup153 in flavivirus infection. My major concern with the current study is that the overall phenotype of Nup153 re-localization and the effects of Nup153 depletion on virus infection are modest. Considering that the authors also note a significant decrease in cell proliferation upon Nup153 depletion (Figure 3), it is possible or even likely that Nup153 may not have a direct role in virus infection. Rather, Nup153 may be required to maintain other cellular pathways that are needed for infection. Finally, the current data do not sufficiently support a role for Nup153 in viral translation. Substantial further experimentation is needed to support the author’s conclusions.\nSpecific points:\n1.Previous reports have shown that NUP153 is elevated in nuclear and cytosolic fractions during stress responses (PMID: 20041180). Therefore, the re-localization of Nup153 may not be virus-specific but is caused by the upregulation of Nup153 which leads to increased cytosolic protein levels that localize to the ER. Therefore, the authors need to provide sufficient controls and parallel validations to confirm the specificity of their results for virus infection. For example, ER general markers for microscopy experiments and ER resident specificity control proteins for IP experiments.\n2.There is some discrepancy between the systems for Nup153 depletion in Figure 2. For the KO cells, there is a 20% reduction in Nup153 leading to a 70% reduction in infected cells while a complete depletion of Nup153 using the AID system only reduces infection by 50%. Also, while depletion experiments are performed for both LGTV and TBEV using the AID system, the KO experiments are only done for LGTV and not TBEV.\n3. Throughout the manuscript, the timing and MOIs for infection experiments as well as the use of LGTV vs TBEV in specific experiments are not well justified. Considering that the authors are reporting an effect specifically at early timepoints after infection they should include detailed information on the kinetics of virus infection (RNA levels and virus titers) over the course of infection. They should also be consistent, when possible, with infection times and MOIs. Some specific examples are listed below.\na. The authors use 16hr, 24 hr or 48 hr for TBEV, 24 h or 48 h for LGTV and 48 hr for all other flaviviruses without justification. These differences make it difficult to compare results between experiments.\nb. There are significant differences in MOI used for experiments with no explanation as to why. For example, the virus spread experiment in Figure 6G was done with an MOI of 0.05 for TBEV and an MOI of 5 for WNV. For this example, it is not clear how a virus spread experiment can be performed with a starting MOI of 5 which should infect nearly all cells with the initial infection. Similar MOI discrepancies are seen throughout the manuscript and should be addressed.\nc.In Fig 2 the analysis of LGTV and TBEV is inconsistent. Time of addition experiments for LGTV should also be shown as well as FFU assays for all time points. These data would be useful for identifying the specific effects of Nup153 depletion timing.\n4. The authors state that Nup153 is localized to the “amplification site” and not “replication vesicles” based on images and quantification in Figure 1H-I. First, the differences between these two regions are not well defined. Second, since previous reports have shown that replication vesicles form within rough-ER sheets that also support translation (see PMID: 26073783, 28249158, 19380115) it is not clear how sites of replication and amplification can be resolved using confocal microscopy.\n5. The function of Nup153 in promoting structural protein translation is not well supported by the data. The authors see only a very modest change in the levels of 2 viral proteins (E and NS2B) which could be explained through changes in cell growth or protein stability. These findings should be validated by western blot and additional structural and non-structural proteins evaluated.\n6. Further to the previous point, the authors identify an interaction between Nup153 and viral RNA, but it is not clear if Nup153 binding is specific to viral RNA or just general RNA binding activity. Less than 1.4% of the reads in the CLIP-Seq were associated with viral RNA at a time point where the viral RNA should be highly abundant. If Nup153 is significantly impacting translation, the amount bound to viral RNA at this point in infection should be much higher.\n7. In Figure 4, the relevance of the Nup153 RBD for infection should be validated with RBD specific deletions in the full- length Nup153. Rescue experiments should also be done to show that expression of RBD containing Nup153 variants can rescue Nup153 depletion during TBEV or LGTV infection.\nMinor comments\n• The CLIP sequencing experiment in Figure 4 shows several other peaks including a strong peak in the 3’UTR that is not discussed.\n• The authors state that the interaction between Nup153 and C is RNA dependent (Fig3H) but there appears to be a loss of C in the input when RNase is added in all cases.\n• Several of the western blots show multiple species for Nup153 without explanation or molecular mass indicators.", "reviewer_2": "In this study, Peters and colleagues have studied the relationship between the nuclear pore protein NUP153 and flaviviruses (mostly tick-borne viruses (TBEV and LGTV)). They show that this host factor is overexpressed in infected cells and is relocalized to the cytoplasm although the latter must be analyzed further. The authors nicely show that NUP153 binds to the viral RNA and that its protein interactome is modulated upon infection. Since NUP153 is required for cell survival, the authors took advantage of an inducible system to rapidly degrade NUP153 and assess its role during viral infection. They further propose that NUP153 regulates flavivirus life cycle at the translational level by changing the expression ratio between structural and non-structural proteins. Unfortunately, as explained below such model is not supported by the data in my opinion since no translation assays was performed. Furthermore, the requirement of NUP153 for TBEV replication remains to be confirmed.\nMajor comments:\n1- The included data of Figure 1 were not entirely convincing regarding a relocalization of NUP153. Indeed, as shown in Fig 1D-E, NUP153 is overexpressed in LGTV and TBEV-infected A549 cells at both protein and mRNA level. Thus, the observed cytoplasmic signal in Fig 1A could simply be due to the fact that the sensitivity is higher. Actually, the nuclear signal increases as well in the same order of magnitude. To claim that there is a relocalization of NUP153 to the flavivirus replication compartment, the cytosol signal/nuclear signal ratio is expected to increase and should be determined in Fig 1A-C and 6A-B. According to the data of Fig 1C, it seems not to change much. Thus, the apparent cytoplasmic signal would be due to a global overexpression, and this would not be a relocalization process. In that case, it would only demonstrate that NUP153 colocalizes with NS3. To support their conclusion, the authors have performed sub-cellular fractionation, and the resulting quantification showed a significant increase of NUP153 in the endoplasmic reticulum (ER) fraction. However, the corresponding immunoblotting are importantly missing and must be shown. In addition, markers of the nucleus, mitochondria, ER and vesicles should be used to ensure that the fractionation process was actually efficient in separating the organelles.\nFig 1H clearly shows some colocalization between NUP153 and dsRNA. This is not consistent with the quantification shown in Fig 1I. Such colocalization was expected in my view given that NS3 mostly associates with replication complexes and that NUP153 associates with both the minus (-) and plus (+) strands of viral RNA (Fig 4B). This is not consistent. In the same line of idea, the authors conclude with Fig 4B that NUP153 preferentially associates with the (+) strand, but the data show that it also associates with the (-) strand. Such binding is not trivial given that there are many more copies of (+) strands than (-) strands in the replication compartment (which is not measured here). Considering this, Fig 4B would support the conclusion that NUP13 associates with replication complexes. In any case, the input (-) and (+) RNA levels (i.e., before IP) should be measured.\n2- The potential proviral role of NUP153 needs to be confirmed. First, all the replication assays were performed by measuring the % of infected cells. This is not the best readout as it measures the capacity of the virus to spread in cluture but not directly its replication efficiency. All the results in Fig 2H, 6C and 6G should be confirmed by plaque/FFU assays and/or RT-qPCR (ideally both) which provide a better dynamic window than an imaging/FACS approach. More importantly, Fig 2I is problematic since no reduction in particle production is observed, implying that NUP153 depleted cells are still able to resume a full life cycle. This was measured at 6 hours post-auxin addition, a time point at which the phenotype of the % of infected cells is not obvious (Fig 2H). This should be performed when the treatment is optimal (e.g. addition at 2 hours post-infection). Furthermore, Fig 3F shows that NUP153 depletion reduces cell proliferation, which may be detrimental for viral replication in general, that is, not only because of the lack of NUP153 in the replication compartment. Without a clear impact of NUP153 depletion on vRNA levels and/or viral particle production (which both rely on efficient viral translation) for same number of infected cells, it cannot be concluded at that stage that NUP153 modulate flavivirus life cycle.\n3- Mostly based on NUP153-dependent differences in NS3, NS2B and E protein expression levels, the authors conclude that viral RNA translation is regulated by NUP153. However, this was never addressed in this study in proper translation-based assays. Moreover, as the authors mention in the discussion, the 10 structural and nonstructural proteins are produced from the same open reading frame as a polyprotein and hence, at an equimolar ratio following processing. So, it is really hard to envision how a modulation of translation would explain the changes in NS1/E and NS2B/E protein ratios upon NUP153 depletion. Since E coding region is upstream that of NS proteins (more abundant in depleted cells), how would that work in the absence of a stop codon? Would there be an alternative translation initiation site upstream the NS1 coding region (which requires E amino-terminus to be inserted in the ER membrane). It is much more likely that viral protein expression level is modulated at the posttranslational level. Moreover, in that specific case, quantifying viral protein expression using microscopy is not appropriate since some protein population might be less accessible to the primary antibodies without allowing the monitoring of protein molecular weight/maturation. This must be done using immunoblotting. Without addressing translation per se, the model proposed in Fig 6H is not supported and really hard to conceptually comprehend, especially since it goes against the current knowledge of flaviviral polyprotein production. Using a reverse genetics system of TBEV could help characterize mutated genomes (which do not bind to NUP153) during translation and viral RNA replication in transfected cells.\nOther comments:\n- How is NUP153 overexpressed? Since these experiments were performed in interferon competent A549 cells, is this the consequence of the TBEV/LGTV-induced innate immune response? The authors mention MAVS knockout A549 cells in the Methods section. Is the infection-related induction of NUP153 expression also observed in these immune-deficient cells?\n- Are the observed phenotypes specific to NUP153? Other NUPs should be tested for colocalization with viral proteins and potential pro/antiviral roles.\n- Line 41: “, and virion assembly we define…”. Please rephrase\n- Line 51: “has is required”. This should be rephrased.\n- Line 52: To my knowledge, HCV does not induce convoluted membranes. Moreover, reference 15 relates to a study on HIV and not HCV.\n- Line 53: “similar but different…”. This reads strange.\n- Line 88: NS3 is not located in the the ER (i.e. in its lumen) but is anchored to its cytosolic side by interacting with its co-factor NS2B\n- Line 160: Examples of identified proteins involved in cell cycle regulation should be indicated.\n- Fig 3H: Why is there a shift in the size of C in the input upon RNase treatment?\n- Line 178 : The fact that RNase treatment did not disrupt the interaction between NUP153 and NS3 or NS5 does preclude that it is indirect (e.g., mediated by other proteins). This sentence must be rephrased.\n- Line 180: “flaviviruses” (typo)\n- Fig 4E: The input RNA level should be measured to determine whether the -strand/+strand ration is the same in the IP.\n- Fig 5: Is there a change in the binding of NUP153 to cellular mRNAs. Such analysis of the data acquired in the CLIP would significantly strengthen the study and would be consistent with the observed change in NUP153 protein interactome.\n- Line 370: This conclusion is not supported by the data as there is no experiment demonstrating the importance of the conserved G-rich region during replication. This statement must be tuned down.\n- Line 402: This statement must be rephrased as the observed phenotypes on viral protein abundance could be very well due to a posttranslational regulation.", "reviewer_3": "The manuscript submitted by Peters, et al. is an interesting study suggesting that a nuclear pore protein, NUP153, is a proviral factor for TBEV and several other members of the Orthoflavivirus genus. The authors suggest that NUP153 is co-opted during flavivirus infection and recruited from the nucleus to the ER, where it regulates viral polyprotein translation. The data presented convincingly demonstrates that NUP153 is upregulated in response to tick-borne flavivirus infection and suggests some relocalization from the nucleus to the ER upon infection. Further, the authors present evidence showing that the RNA binding domain of NUP153 is required for relocalization and that NUP153 interacts with flavivirus RNA at G-quadruplex structures. The authors go on to suggest that NUP153 regulate translation of the flavivirus polyprotein via the interaction with viral RNA at the C-terminal portion of the E protein coding sequence, resulting in a relative increase in the translation of the upstream structural proteins, as compared to the downstream nonstructural proteins. While this is a novel and compelling model, the data presented in the manuscript do not sufficiently support these claims. Further, the addition of controls and orthogonal approaches to bolster these data are required to support many of the other conclusions.\nMajor Comments\n1. In Figure 1A, the immunofluorescence images presented to suggest co-localization and re-localization require controls for proper interpretation. The overall signal of NUP153 appears to increase everywhere in the cell, which supports upregulation of this protein during infection. However, the evidence that abundance is increasing at viral replication sites is not convincing and requires further experimentation, including cellular fractionation with appropriate cellular component controls. Both an ER marker and another nuclear envelope protein should be included in these experiments and in IF experiments throughout the manuscript.\n2. Similarly, markers for all compartments should be included in fractionation experiments (Figure S1E). It is possible that the integrity of the nuclear envelope compromised during infection, resulting in a change in localization of nuclear proteins. Why are calnexin and GAPDH present in the pellet of all fractions in uninfected cells? This suggests the fractionation of specific compartments is not robust. Additional cellular markers, including mitochondrial and another nuclear marker should be included in this experiment. Further, the quantification of the IF data in Figure 1C suggests an increase in NUP153 in the nucleus but the quantitation of the fractionation data in Figure 1G does not reflect this. This discrepancy should be addressed.\n3. Figure 1D does demonstrate an increase in NUP153 RNA and but the increase in protein is rather modest and does not appear equivalent to the increase in signal shown in Figure 1A. Further, the authors claim that this data distinguishes between increased expression and increased stability of NUP153. As NUP153 protein stability assays have not been performed here, this claim is not supported.\n4. The transient knock-down of NUP153 is very inefficient, despite being performed twice and sorting on GFP positive (Cas9 positive) cells. While there is a remarkable decrease in infection in these cells, it is difficult to compare to the control (GBF1) as no knock-down data is provided here. Transfection of siRNA or shRNA targeting NUP153 would also provide transient depletion at increased efficiency. Further, this would also allow for additional infection assays, such as quantitation of FFU or PFU in the cells to further support a proviral role for NUP153 and viral life cycle assays to supplement the findings from the time of addition assays.\n5. The NUP153(NG)AID system appears very robust but the infection phenotype is less pronounced here than in the transient knock-down experiment (Figure 2B) despite the complete depletion in AID system. This suggests that the phenotype in Figure 2B is not solely dependent on NUP153. The expression of NUP153 post-auxin addition should be shown for all time points. After the addition of auxin, at what point does expression of NUP153 recover? Additional viral infection assays, including the FFU assay should be included for all time points, not just the 6h timepoint at which there is no effect on infection.\n6. In general, the description of experimental design, assays, normalizations and reagents used is insufficient. For several experiments, it is not clear what is being used as a negative control nor what the assays are precisely measuring. For example, in Figure 2A, data is normalized to CAS9. Is this Cas9 alone or with the non-targeting guide RNAs? Similarly, a description of the NUP153(NG)AID and control cell lines used needs a clearer description to be able to adequately interpret the results.\n7. In Figure 3, the differences in NUP153 interactors in mock vs. infected cells is interesting and potentially informative for function. However, there are no experiments validating these differential interaction results.\n8. In Figures 4C-E, another unrelated RNA binding protein should be used as a control. Additionally, a non-viral RNA should be quantified in 4C-4E to show specificity of interaction with viral RNA.\n9. The results in Figure 5 are well-done and suggest that NUP153 is binding to G quadraplex regions. However, the findings from the CLIP-seq data showing an interaction between NUP153 and viral RNA would be strengthened by performing an orthogonal assay, such as a gel-shift assay with viral RNA regions in question.\n10. The hypothesis that NUP153 can modulate relative amounts of viral protein translation is intriguing. However, there is not sufficient evidence provided to make this claim and there is no data that support the proposed mechanism by which NUP153 might regulate translation. Further, changes in relative levels of E/NS1/NS2b are quite modest and representative IF images for TBEV NS1 and E are not provided. Additional experimentation, including a viral protein translation and/or a minimal system with reporters adjacent to the putative NUP153 binding site to demonstrate an effect on translation is required. Evidence that a truncated polyprotein is produced, dependent on the presence of NUP153, would also be more convincing.\nMinor Comments\n1. In Figure S2F, why does the addition of auxin at 6 hpi lead to an increase in the percentage of infected cells? This should be addressed.\n2. Several different cell types are used throughout the study, which may partially explain differences in the magnitude of phenotypes. The addition of more relevant cell types would also be informative.\n3. The authors do not provide a justification as to why the aa 1-650 construct was used for the CLIP-seq experiment.\n4. Line 178: the authors claim the interaction between Nup153 and NS3/NS5 upon RNase treatment is evidence that the interaction between these proteins is direct. This experiment does not support that conclusion, only that the interaction between Nup153 and NS3/NS5 is not dependent on RNA. The authors more accurately describe this result in lines 195-196 but should clarify in the previous statement."}, "author_response": "Point by point response to reviewer, answers are marked in red.\nReviewers' comments:\nReviewer #1 (Remarks to the Author):\nPeters et al present evidence that the nuclear pore complex protein Nup153 is a proviral\nfactor that promotes flavivirus infection. With a specific focus on tick-bore flaviviruses (TBEV\nand LGTV) the authors show that Nup153 is upregulated and re-localized to the cytosol\nduring infection and that depletion of Nup153 leads to a decrease in the number of infected\ncells. Furthermore, they present evidence that Nup153 interacts with viral NS3 and NS5\nproteins and suggest that these interactions facilitate Nup153 recruitment to the cytosol. The\nauthors also show evidence that Nup153 interacts with viral RNA through an RNA binding\ndomain at several G-rich regions of the viral Genome. Finally, they use light microscopy to\nevaluate the levels of viral structural and non-structural proteins in Nup153 depleted cells\nand report a change in the ratio between E and NS2B. Based on these data, the authors\nposit that Nup153 is involved in promoting the translation of structural proteins to facilitate\nvirus assembly and spread. In general, the study is well structured, using various techniques\nto comprehensively evaluate the role of Nup153 in flavivirus infection.\nWe thank the reviewer for the positive feedback and for appreciating the study..\nMy major concern with the current study is that the overall phenotype of Nup153 re-\nlocalization and the effects of Nup153 depletion on virus infection are modest. Considering\nthat the authors also note a significant decrease in cell proliferation upon Nup153 depletion\n(Figure 3), it is possible or even likely that Nup153 may not have a direct role in virus\ninfection. Rather, Nup153 may be required to maintain other cellular pathways that are\nneeded for infection. Finally, the current data do not sufficiently support a role for Nup153 in\nviral translation. Substantial further experimentation is needed to support the author’s\nconclusions.\nWe have addressed the raised questions in a point-by-point manner and added experimental\nresults that further support the conclusion.\nSpecific points:\n1.Previous reports have shown that NUP153 is elevated in nuclear and cytosolic fractions\nduring stress responses (PMID: 20041180). Therefore, the re-localization of Nup153 may not\nbe virus-specific but is caused by the upregulation of Nup153 which leads to increased\ncytosolic protein levels that localize to the ER. Therefore, the authors need to provide\nsufficient controls and parallel validations to confirm the specificity of their results for virus\ninfection. For example, ER general markers for microscopy experiments and ER resident\nspecificity control proteins for IP experiments.\nWe thank the reviewer for the suggestions.\nWe have restructured figure 1 and added super resolution microscopy Structural illumination\nmicroscopy (SIM) to show that NUP153 localizes together with the ER marker Calnexin\nduring infection (see new figure 1D) together with quantification of the Manders coefficient\n(new Fig 1F), which answers the question. Moreover, we have also analyzed the\nupregulation of NUP153 in MAVS-/- ko A549 cells which are unable to upregulate IFNb and\nshowed that NUP153 is upregulated after infection independent of type I IFN response (new\nfigure 1H-J).\n2.There is some discrepancy between the systems for Nup153 depletion in Figure 2. For the\nKO cells, there is a 20% reduction in Nup153 leading to a 70% reduction in infected cells\nwhile a complete depletion of Nup153 using the AID system only reduces infection by 50%.\nAlso, while depletion experiments are performed for both LGTV and TBEV using the AID\nsystem, the KO experiments are only done for LGTV and not TBEV.\nWe thank the reviewer for pointing out this discrepancy. To avoid confusion, we have\nremoved the KO cells and only use the results of the much cleaner AID system, which makes\nthe manuscript clearer. We have further focused figure 2 on TBEV.\n3. Throughout the manuscript, the timing and MOIs for infection experiments as well as the\nuse of LGTV vs TBEV in specific experiments are not well justified. Considering that the\nauthors are reporting an effect specifically at early timepoints after infection they should\ninclude detailed information on the kinetics of virus infection (RNA levels and virus titers) over\nthe course of infection. They should also be consistent, when possible, with infection times\nand MOIs. Some specific examples are listed below.\na. The authors use 16hr, 24 hr or 48 hr for TBEV, 24 h or 48 h for LGTV and 48 hr for all\nother flaviviruses without justification. These differences make it difficult to compare results\nbetween experiments.\nThank you for pointing this out. We have now reduced the number of cell types used in the\nstudy to make the message clearer. The MOI stated was calculated based on virus tritiated\non VeroB4, however the kinetics of virus infection is different in different cell types, so we\nhave recalculated the MOI corresponding to the cell type in the specific experiments. The\ndifferent time points correspond to 50 % infection rate and since the different viruses have\ndifferent kinetics we needed to use different infection times. We have clarified these points in\nthe material and method and also in the figure legends.\nb. There are significant differences in MOI used for experiments with no explanation as to\nwhy. For example, the virus spread experiment in Figure 6G was done with an MOI of 0.05\nfor TBEV and an MOI of 5 for WNV. For this example, it is not clear how a virus spread\nexperiment can be performed with a starting MOI of 5 which should infect nearly all cells with\nthe initial infection. Similar MOI discrepancies are seen throughout the manuscript and\nshould be addressed.\nThank you for pointing this out originally the titers of the stock was calculated on the VeroB4\ncells, however as mention in the previous point we have recalculated the actual infectious\ndose by titrating the virus on the specific cell lines.\nc. In Fig 2 the analysis of LGTV and TBEV is inconsistent. Time of addition experiments for\nLGTV should also be shown as well as FFU assays for all time points. These data would be\nuseful for identifying the specific effects of Nup153 depletion timing.\nWe agree. We have added FFU for both TBEV and LGTV in figure 2. However, as we now\nfocus the manuscript on TBEV we are not adding time of addition of LGTV.\n4. The authors state that Nup153 is localized to the “amplification site” and not “replication\nvesicles” based on images and quantification in Figure 1H-I. First, the differences between\nthese two regions are not well defined. Second, since previous reports have shown that\nreplication vesicles form within rough-ER sheets that also support translation (see PMID:\n26073783, 28249158, 19380115) it is not clear how sites of replication and amplification can\nbe resolved using confocal microscopy.\nWe have performed super resolution microscopy SIM to clarify if NUP153 co-localize with\ndsRNA which is a marker for the replication vesicles. Most of the dsRNA colocalize with\nNUP153, although not all NUP153 localize to the dsRNA (New figure 1D-G). This indicate\nthat NUP153 does not target the replication vesicle specifically but rather is in the ER, which\nwe also show through the colocalization with the ER marker Calnexin. We choose to use\n“amplification site” as a more general term instead of “replication vesicles”, as the resolution\nof the SIM is not high enough to show that NUP153 is inside the vesicles.\n5. The function of Nup153 in promoting structural protein translation is not well supported by\nthe data. The authors see only a very modest change in the levels of 2 viral proteins (E and\nNS2B) which could be explained through changes in cell growth or protein stability. These\nfindings should be validated by western blot and additional structural and non-structural\nproteins evaluated.\nThank you for this comment. We have worked extensively on this point and generated\nseveral figure panels (see Figure 8). Regarding the immunofluorescence data on the ratios of\nE against NS1/NS2B, we opted to use microscopy so that we could calculate protein levels\nexclusively in infected cells (Figure 8E-G), and we clarified this in the materials and methods\nsection. Additionally, we have added WB of AID cells infected with TBEV (Figure 8H and\nSupp Figure 8C). Using the available Ab for TBEV we could show that ratio C/E and M/E\ndoes not change but NS1/E, NS2B/E, NS3/E and NS5/E increases (Figure 8H). Also as seen\nby the tubulin staining in these Western blots when cells are seeded to be confluent as done\nfor all infection assays NUP153 depletion has minimal to no effect on number of cells (Supp\nFigure 8C). Since we do not have the required set of antibodies against WNV we choose to\ndo mass spectrometry on the WNV infected cells and found that C/E, prM/E does not change\nbut the ration NS1/E, NS3/E and NS5/E do increase (New supp Fig 8E and Fig 8I). These\ndata support our original claim. We have also added in vitro translation in the presence of the\nRNA binding domain and found that by adding NUP153-RBD the downstream protein\nproduction is reduced new figure 8D.\n6. Further to the previous point, the authors identify an interaction between Nup153 and viral\nRNA, but it is not clear if Nup153 binding is specific to viral RNA or just general RNA binding\nactivity. Less than 1.4% of the reads in the CLIP-Seq were associated with viral RNA at a\ntime point where the viral RNA should be highly abundant. If Nup153 is significantly\nimpacting translation, the amount bound to viral RNA at this point in infection should be much\nhigher.\nNUP153 is an RNA binding protein that is involved in the transport of RNA (and proteins)\nthought the nuclear pore, and still has its normal function during infection, in addition to\nbinding to viral RNA.\nThe virus is not affecting the RNA transcription of cellular RNA.\nConsistent with other studies (PMID: 31384002; PMID: 40161615) we find that only a few %\nof the binding RNA is viral RNA during infection. We further show that NUP153 as expected\nbind to cellular RNA, as well as very specifically to the motif identified in the virus genome.\nThe suggested binding site in the viral genome is very specific. To support this, we have now\nadded RNA EMSA experiments of NUP153-RBD and different RNA constructs that clearly\nshow that the RBD binds with higher affinity to the peak identified with clip-seq as compared\nto mutated version of the peak (Figure 5E-H).\n7. In Figure 4, the relevance of the Nup153 RBD for infection should be validated with RBD\nspecific deletions in the full-length Nup153. Rescue experiments should also be done to\nshow that expression of RBD containing Nup153 variants can rescue Nup153 depletion\nduring TBEV or LGTV infection.\nThank you for this helpful suggestion. We have added the a truncated constructed of Nup153\nlacking RBD and found that it does not bind to the viral RNA in the Clip assay, and it can also\nnot rescue the infection (see new Figure 4C and F).\nMinor comments\n• The CLIP sequencing experiment in Figure 4 shows several other peaks including a strong\npeak in the 3’UTR that is not discussed.\nThis peak is not significantly enriched in all three replicas when using the PEAKachu\nalgorithm and therefore they are not evaluated further.\n• The authors state that the interaction between Nup153 and C is RNA dependent (Fig3H)\nbut there appears to be a loss of C in the input when RNase is added in all cases.\nThis is true, C protein of flaviviruses is known to be extremely positively charged which\nmeans it can bind to negatively charged RNA through electrostatic interactions. Using RNAse\nto degrade the RNA could therefore alter the C proteins stability or ability to efficiently migrate\nin a gel.\n• Several of the western blots show multiple species for Nup153 without explanation or\nmolecular mass indicators.\nYes we do acknowledge this, and the band with higher molecular weight corresponds to full\nlength NUP153-NG-AID construct, however NUP153 does have other isoforms that could\npotentially be detected with our antibody and be more pronounced in the NUP153 [NG]AID\ncells compared to their parental DLD cell line.\nReviewer #2 (Remarks to the Author):\nIn this study, Peters and colleagues have studied the relationship between the nuclear pore\nprotein NUP153 and flaviviruses (mostly tick-borne viruses (TBEV and LGTV)). They show\nthat this host factor is overexpressed in infected cells and is relocalized to the cytoplasm\nalthough the latter must be analyzed further. The authors nicely show that NUP153 binds\nto the viral RNA and that its protein interactome is modulated upon infection. Since NUP153\nis required for cell survival, the authors took advantage of an inducible system to rapidly\ndegrade NUP153 and assess its role during viral infection. They further propose that\nNUP153 regulates flavivirus life cycle at the translational level by changing the expression\nratio between structural and non-structural proteins. Unfortunately, as explained below such\nmodel is not supported by the data in my opinion since no translation assays was performed.\nFurthermore, the requirement of NUP153 for TBEV replication remains to be confirmed.\nMajor comments:\n1- The included data of Figure 1 were not entirely convincing regarding a relocalization of\nNUP153. Indeed, as shown in Fig 1D-E, NUP153 is overexpressed in LGTV and TBEV-\ninfected A549 cells at both protein and mRNA level. Thus, the observed cytoplasmic signal in\nFig 1A could simply be due to the fact that the sensitivity is higher. Actually, the nuclear signal\nincreases as well in the same order of magnitude. To claim that there is a relocalization of\nNUP153 to the flavivirus replication compartment, the cytosol signal/nuclear signal ratio is\nexpected to increase and should be determined in Fig 1A-C and 6A-B. According to the data\nof Fig 1C, it seems not to change much. Thus, the apparent cytoplasmic signal would be due\nto a global overexpression, and this would not be a relocalization process. In that case, it\nwould only demonstrate that NUP153 colocalizes with NS3. To support their conclusion, the\nauthors have performed sub-cellular fractionation, and the resulting quantification showed a\nsignificant increase of NUP153 in the endoplasmic reticulum (ER) fraction. However, the\ncorresponding immunoblotting are importantly missing and must be shown. In addition,\nmarkers of the nucleus, mitochondria, ER and vesicles should be used to ensure that the\nfractionation process was actually efficient in separating the organelles.\nWe thank the reviewer for the comment and have decided to remove the fractionation.\nWe instead added super resolution microscopy to show the relation between the viral proteins,\nNUP153 and ER marker Calnexin. Here we show that the staining of NUP153 and Calnexin\noverlaps almost completely. We have calculated the ratio between the signal in the nucleus\nand cytoplasm and there is no difference between uninfected and infected cells this data has\nbeen added as Supp Fig 1A.\nFig 1H clearly shows some colocalization between NUP153 and dsRNA. This is not\nconsistent with the quantification shown in Fig 1I. Such colocalization was expected in my\nview given that NS3 mostly associates with replication complexes and that NUP153\nassociates with both the minus (-) and plus (+) strands of viral RNA (Fig 4B). This is not\nconsistent. In the same line of idea, the authors conclude with Fig 4B that NUP153\npreferentially associates with the (+) strand, but the data show that it also associates with the\n(-) strand. Such binding is not trivial given that there are many more copies of (+) strands\nthan (-) strands in the replication compartment (which is not measured here). Considering\nthis, Fig 4B would support the conclusion that NUP13 associates with replication complexes.\nIn any case, the input (-) and (+) RNA levels (i.e., before IP) should be measured.\nWe thank the reviewer for this comment, and agree that this is important to clarify if NUP153\nbinds to positive or negative RNA strands, or both. We have chosen to remove the confocal\nmicroscopy image quantification in figure 1I and replaced them with super resolution\nmicroscopy and quantification of that new figure 1D-G. Here we show that most of the\ndsRNA colocalize with NUP153 but only a fraction of the NUP153 colocalize with dsRNA.\nNUP153 is rather localized to the ER as shown with the overlap in the calnexin staining.\nFor the CLIP assay the values are already normalized to the input for the negative and\npositive, we have now clarified this in the figure legend. No negative stranded RNA was\ndetected in the CLIP Seq, suggesting that NUP153 does not bind to negative RNA. We\npotentially observe some binding to the negative strand RNA in the CLIP assay that could be\na result of it indirectly being pulldown in the form of partial dsRNA, however based on our\nCLIP-seq assay we believe the interaction occurs on the positive strand RNA.\n2- The potential proviral role of NUP153 needs to be confirmed. First, all the replication\nassays were performed by measuring the % of infected cells. This is not the best readout as\nit measures the capacity of the virus to spread in culture but not directly its replication\nefficiency. All the results in Fig 2H, 6C and 6G should be confirmed by plaque/FFU assays\nand/or RT-qPCR (ideally both) which provide a better dynamic window than an\nimaging/FACS approach. More importantly, Fig 2I is problematic since no reduction in particle\nproduction is observed, implying that NUP153 depleted cells are still able to resume a full life\ncycle. This was measured at 6 hours post-auxin addition, a time point at which the phenotype\nof the % of infected cells is not obvious (Fig 2H). This should be performed when the\ntreatment is optimal (e.g. addition at 2 hours post-infection). Furthermore, Fig 3F shows that\nNUP153 depletion reduces cell proliferation, which may be detrimental for viral replication in\ngeneral, that is, not only because of the lack of NUP153 in the replication compartment.\nWithout a clear impact of NUP153 depletion on vRNA levels and/or viral particle production\n(which both rely on efficient viral translation) for same number of infected cells, it cannot be\nconcluded at that stage that NUP153 modulate flavivirus life cycle.\nThank you for suggesting. We have now the FFU for all these panels and time points, we\nhave also transfected AID cells with NUP153 to rescue the infection, to confirm that NUP153\nis important for infection Fig 2N and Fig 4F. Q-PCR measurements has been added Fig 2M.\nWe have removed the cell proliferation assay. In contrast to the proliferation assay,\ninfection assays were seeded to obtain confluent wells, meaning that at end point similar number of\ncells can be found in the wells. Additionally, we show that NUP153 depletion does not affect\nviability, however as NUP153 is required for assembly of the nuclear pore complex it is\nessential for the replication of the cells.\n3- Mostly based on NUP153-dependent differences in NS3, NS2B and E protein expression\nlevels, the authors conclude that viral RNA translation is regulated by NUP153. However, this\nwas never addressed in this study in proper translation-based assays. Moreover, as the\nauthors mention in the discussion, the 10 structural and nonstructural proteins are produced\nfrom the same open reading frame as a polyprotein and hence, at an equimolar ratio\nfollowing processing. So, it is really hard to envision how a modulation of translation would\nexplain the changes in NS1/E and NS2B/E protein ratios upon NUP153 depletion. Since E\ncoding region is upstream that of NS proteins (more abundant in depleted cells), how would\nthat work in the absence of a stop codon? Would there be an alternative translation initiation\nsite upstream the NS1 coding region (which requires E amino-terminus to be inserted in the\nER membrane). It is much more likely that viral protein expression level is modulated at the\nposttranslational level. Moreover, in that specific case, quantifying viral protein expression\nusing microscopy is not appropriate since some protein population might be less accessible\nto the primary antibodies without allowing the monitoring of protein molecular\nweight/maturation. This must be done using immunoblotting. Without addressing translation\nper se, the model proposed in Fig 6H is not supported and really hard to conceptually\ncomprehend, especially since it goes against the current knowledge of flaviviral polyprotein\nproduction. Using a reverse genetics system of TBEV could help characterize mutated\ngenomes (which do not bind to NUP153) during translation and viral RNA replication in\ntransfected cells.\nWe have invested significant efforts towards understanding the experimental observations.\nWe have added WB of TBEV infected AID cells and calculated the ratio between C/E, M/E,\nNS1/E, NS2B/E, NS3/E and NS5/E (limited by the availability of antibodies). We did not see\na difference in the ratio between C/E and M/E in the absence of NUP513 but the ratio of\nNS1, NS2B, NS3 and NS5 to E increased. In parallel we used WNV infection, and since the\navailability of Abs are limited, we used mass spectrometry instead (peptides were detected\nfor C, prM, E, NS1, NS3, NS4B, NS5). Data were very similar between WNV and TBEV\nexperiments supporting our original claim. We have also expressed the RBD from NUP153\nand tested its effect on in vitro translation. Here we show that adding NUP153-RBD reduces\nluciferase (Luc) expression downstream of the RNA binding site, and increases the Luc\nsignal if the RNA motif is downstream the Luc ORF. Additionally, WB analysis of the in vitro\ntranslation assay in the presence of RBD results in production of lower molecular weight\nprotein that corresponds to C-prM-E.\nFinally, we note that the RNA motif identified to bind to NUP153 is highly conserved among\nall flaviviruses. We constructed mutant viruses lacking the conserved Gs, however they were\nnot viable even after adding E in trans.\nOther comments:\n- How is NUP153 overexpressed? Since these experiments were performed in interferon\ncompetent A549 cells, is this the consequence of the TBEV/LGTV-induced innate immune\nresponse? The authors mention MAVS knockout A549 cells in the Methods section. Is the\ninfection-related induction of NUP153 expression also observed in these immune-deficient\ncells?\nThank you for this question. We have added a new panel fig 1H-J where we analyze if\nNUP153 upregulation after infection is dependent on IFN upregulation and we can show that\nit is not. We also show that overexpression of NS3 and NS5 expression plasmids results in\nincreased expression of NUP153 (Supp fig 3B-C).\n- Are the observed phenotypes specific to NUP153? Other NUPs should be tested for\ncolocalization with viral proteins and potential pro/antiviral roles.\nWe agree. However, there are many NUPs and it is out of the scope of this paper to test all of\nthe NUPs for their function during Flavivirus infection. We are working on some other NUPs\nand their function during TBEV infection.\n- Line 41: “, and virion assembly we define…”. Please rephrase Ok\n- Line 51: “has is required”. This should be rephrased. Ok\n- Line 52: To my knowledge, HCV does not induce convoluted membranes. Moreover,\nreference 15 relates to a study on HIV and not HCV. We have changed it to membranous\nweb and removed the reference\n- Line 53: “similar but different…”. This reads strange. Rephrased\n- Line 88: NS3 is not located in the the ER (i.e. in its lumen) but is anchored to its cytosolic\nside by interacting with its co-factor NS2B. Has been clarified.\n- Line 160: Examples of identified proteins involved in cell cycle regulation should be\nindicated. Ok\n- Fig 3H: Why is there a shift in the size of C in the input upon RNase treatment?\nC protein of flaviviruses is known to be extremely positively charged and bind to RNA in an\nelectrostatic dependent manner. Treating samples with RNAse could alter the protein stability\nor ability to efficiently migrate through a gel.\n- Line 178 : The fact that RNase treatment did not disrupt the interaction between NUP153\nand NS3 or NS5 does preclude that it is indirect (e.g., mediated by other proteins). This\nsentence must be rephrased. done\n- Line 180: “flaviviruses” (typo) ok\n- Fig 4E: The input RNA level should be measured to determine whether the -strand/+strand\nration is the same in the IP. It is normalized to the input has been added to the figure legend.\n- Fig 5: Is there a change in the binding of NUP153 to cellular mRNAs. Such analysis of the\ndata acquired in the CLIP would significantly strengthen the study and would be consistent\nwith the observed change in NUP153 protein interactome.\nThis is a good suggestion, however, the Clip-seq data does not have enough depth to do this\nanalysis. We looked at the binding of NUP153 to Actin RNA with Clip assay and found that\nduring infection NUP153 bind actin RNA Supp fig 4A.\n- Line 370: This conclusion is not supported by the data as there is no experiment\ndemonstrating the importance of the conserved G-rich region during replication. This\nstatement must be tuned down. Rephrased\n- Line 402: This statement must be rephrased as the observed phenotypes on viral protein\nabundance could be very well due to a posttranslational regulation.\nWe respectfully disagree. We have provided additional data to support it, see the previous\ncomments.\nReviewer #3 (Remarks to the Author):\nThe manuscript submitted by Peters, et al. is an interesting study suggesting that a nuclear\npore protein, NUP153, is a proviral factor for TBEV and several other members of the\nOrthoflavivirus genus. The authors suggest that NUP153 is co-opted during flavivirus\ninfection and recruited from the nucleus to the ER, where it regulates viral polyprotein\ntranslation. The data presented convincingly demonstrates that NUP153 is upregulated in\nresponse to tick-borne flavivirus infection and suggests some relocalization from the nucleus\nto the ER upon infection. Further, the authors present evidence showing that the RNA\nbinding domain of NUP153 is required for relocalization and that NUP153 interacts with\nflavivirus RNA at G-quadruplex structures. The authors go on to suggest that NUP153\nregulate translation of the flavivirus polyprotein via the interaction with viral RNA at the C-\nterminal portion of the E protein coding sequence, resulting in a relative increase in the\ntranslation of the upstream structural proteins, as compared to the downstream nonstructural\nproteins. While this is a novel and compelling model, the data presented in the manuscript do\nnot sufficiently support these claims. Further, the addition of controls and orthogonal\napproaches to bolster these data are required to support many of the other conclusions.\nThank you for your analysis, we have now added several new orthogonal assays to support\nour claims.\nMajor Comments\n1. In Figure 1A, the immunofluorescence images presented to suggest co-localization and re-\nlocalization require controls for proper interpretation. The overall signal of NUP153 appears\nto increase everywhere in the cell, which supports upregulation of this protein during\ninfection. However, the evidence that abundance is increasing at viral replication sites is not\nconvincing and requires further experimentation, including cellular fractionation with\nappropriate cellular component controls. Both an ER marker and another nuclear envelope\nprotein should be included in these experiments and in IF experiments throughout the\nmanuscript.\nThank you for the suggestion. We show that NUP153 increases both in the nucleus and the\ncytoplasm after infection but that the ratio stays the same (see new Supp Fig 1A). As it is\nvery difficult to fractionate cells without contamination between the different organelles, we\nremoved the fractionation data and analyzed the localization of NUP153 with super\nresolution microscopy. In these experiments we used calnexin as an ER marker, NS3,\nand\ndsRNA for virus and calculated the colocalization between the different staining (see New\nfigure 1D-G). The integrity of the nuclear envelope after infection was analyzed by FG\nbinding Ab Mab 414 (see Supp fig 1 C-D).\n2. Similarly, markers for all compartments should be included in fractionation experiments\n(Figure S1E). It is possible that the integrity of the nuclear envelope compromised during\ninfection, resulting in a change in localization of nuclear proteins. Why are calnexin and\nGAPDH present in the pellet of all fractions in uninfected cells? This suggests the\nfractionation of specific compartments is not robust. Additional cellular markers, including\nmitochondrial and another nuclear marker should be included in this experiment. Further, the\nquantification of the IF data in Figure 1C suggests an increase in NUP153 in the nucleus but\nthe quantitation of the fractionation data in Figure 1G does not reflect this. This discrepancy\nshould be addressed.\nWe agree with the reviewer. It is very difficult to fractionate cells and separate different\ncellular compartments, as nucleus and ER are connected, and mitochondria and ER are\nconnected, we decided to remove this set of data. We instead chose to add super resolution\nmicroscopy to have a clearer picture of the NUP153 protein localization.\n3. Figure 1D does demonstrate an increase in NUP153 RNA and but the increase in protein\nis rather modest and does not appear equivalent to the increase in signal shown in Figure\n1A. Further, the authors claim that this data distinguishes between increased expression and\nincreased stability of NUP153. As NUP153 protein stability assays have not been performed\nhere, this claim is not supported.\nThank you for pointing this out. We have now rephrased our findings to only state that we\nobserve an increase in NUP153 expression on RNA and protein levels. The NUP153 RNA\nlevels after infection in 293 cells are greater, and this information has been added to the\nsupplementary (Supp Figure 1B). We have also added a reference about the stability of\nNUPs (PMID: 23993091 and PMID: 35412236).\n4. The transient knock-down of NUP153 is very inefficient, despite being performed twice and\nsorting on GFP positive (Cas9 positive) cells. While there is a remarkable decrease in\ninfection in these cells, it is difficult to compare to the control (GBF1) as no knock-down data\nis provided here. Transfection of siRNA or shRNA targeting NUP153 would also provide\ntransient depletion at increased efficiency. Further, this would also allow for additional\ninfection assays, such as quantitation of FFU or PFU in the cells to further support a proviral\nrole for NUP153 and viral life cycle assays to supplement the findings from the time of\naddition assays.\nAs the NUP153 protein half-life is long (PMID: 35412236) it affects all systems of transient\nknockdown that only reduces the newly synthesized NUP153. We have removed the\nknockdown data and focused on the more clean AID degradation system. We have also\nshown that NUP153 overexpression can rescue the effect of NUP153 degradation.\n5. The NUP153(NG)AID system appears very robust but the infection phenotype is less\npronounced here than in the transient knock-down experiment (Figure 2B) despite the\ncomplete depletion in AID system. This suggests that the phenotype in Figure 2B is not solely\ndependent on NUP153. The expression of NUP153 post-auxin addition should be shown for\nall time points. After the addition of auxin, at what point does expression of NUP153 recover?\nAdditional viral infection assays, including the FFU assay should be included for all time\npoints, not just the 6h timepoint at which there is no effect on infection.\nThank you for your comments, we have added FFU for all time points. In the experiments\nusing the AID cells the Auxin is present during the whole infection time so there is no\nrecovery of NUP153. We have also seen that the effect of NUP153 degradation on progeny\nparticle production is greater compared to number of infected cells.\n6. In general, the description of experimental design, assays, normalizations and reagents\nused is insufficient. For several experiments, it is not clear what is being used as a negative\ncontrol nor what the assays are precisely measuring. For example, in Figure 2A, data is\nnormalized to CAS9. Is this Cas9 alone or with the non-targeting guide RNAs? Similarly, a\ndescription of the NUP153(NG)AID and control cell lines used needs a clearer description to\nbe able to adequately interpret the results.\nThis is a valid point. We have removed the CAS9 data in favor of the more robust AID\nsystem. We have also added a description of the AID cells in the results section.\n7. In Figure 3, the differences in NUP153 interactors in mock vs. infected cells is interesting\nand potentially informative for function. However, there are no experiments validating these\ndifferential interaction results.\nWe agree that this is interesting however, this is out of the scope of this manuscript.\n8. In Figures 4C-E, another unrelated RNA binding protein should be used as a control.\nAdditionally, a non-viral RNA should be quantified in 4C-4E to show specificity of interaction\nwith viral RNA.\nWe understand the reviewer’s concern. However, as to our knowledge, it is not known what\nRNA binding proteins of the cell are not binding flaviviral RNA, therefore we are unable to do\nthis control. If the reviewer has a suggestion of an unrelated cellular RNA binding protein\nknown not to bind flavivirus RNA we would be happy to use it as control. Furthermore, most\nRNA binding proteins have RNA binding domains that are positively charged, and RNA being\nnegatively charged in general will always result in some level of binding. However, the added\nRNA-EMSA assay show that the RNA binding domain of NUP153 binds better to the\nidentified peak than the mutated constructs. We have also added RNA-EMSA using purified\nGFP protein to show that the shift that we observe is specific to the RNA binding domain of\nNUP153. In addition, it is difficult to choose a cellular RNA that NUP153 does not bind to, as\none of the functions of NUP153 is to bind to cellular mRNA. We also show now that NUP153\nbind to Actin mRNA in a Clip assay new Supp figure 4A.\n9. The results in Figure 5 are well-done and suggest that NUP153 is binding to G quadraplex\nregions. However, the findings from the CLIP-seq data showing an interaction between\nNUP153 and viral RNA would be strengthened by performing an orthogonal assay, such as a\ngel-shift assay with viral RNA regions in question.\nThank you for this suggestion. We have now done RNA EMSA experiments on full length\nviral RNA, different fragments and mutants. Our new data strongly show that NUP153 RBD\nspecifically bind the conserved G in Peak 1.\n10. The hypothesis that NUP153 can modulate relative amounts of viral protein translation is\nintriguing. However, there is not sufficient evidence provided to make this claim and there is\nno data that support the proposed mechanism by which NUP153 might regulate translation.\nFurther, changes in relative levels of E/NS1/NS2b are quite modest and representative IF\nimages for TBEV NS1 and E are not provided. Additional experimentation, including a viral\nprotein translation and/or a minimal system with reporters adjacent to the putative NUP153\nbinding site to demonstrate an effect on translation is required. Evidence that a truncated\npolyprotein is produced, dependent on the presence of NUP153, would also be more\nconvincing.\nThank you for these suggestions. We have now added in vitro translation experiments adding\nGFP or GFP-NUP153-RBD and find that translation (luciferase activity) after Peak 1\ndecreases after addition of the RBD and that luciferase activity increases when peak 1 is\nafter the Luc ORF, new fig 8 A-D. Additionally, we show that the addition of the RBD does\nnot result in no translation, resulting instead in production of a lower molecular weight\nproduct positive for E, further supporting our claims.\nWe have also run WB of several structural proteins and non-structural protein of TBEV and\nshown that the ratio of C/E, prM/E stays the same whereas NS1/E, NS2B/E, NS3/E and\nNS5/E ratios increases in the absence of NUP153. For WNV as we do not have antibodies\nwe instead used mass spectrometry and found similar results new figure 8H-I, supp fig 8C-E.\nMinor Comments\n1. In Figure S2F, why does the addition of auxin at 6 hpi lead to an increase in the\npercentage of infected cells? This should be addressed.\nWe do not know why there is an increase after 6 h, however, all the figures after this Auxin\nwas added 2 hours prior to infection, so it is not influencing the main findings. We also feel\nthat this observation is out of scope of this manuscript which focuses on NUP153.\n2. Several different cell types are used throughout the study, which may partially explain\ndifferences in the magnitude of phenotypes. The addition of more relevant cell types would\nalso be informative.\nWe see the same phenotype in A549 cells, A549 MAVS-/-, 293T cells and DLD1 cells, with all\nflaviviruses tested.\n3. The authors do not provide a justification as to why the aa 1-650 construct was used for\nthe CLIP-seq experiment.\nThank you for the comment. As the FG region of NUP153 is known to bind many different\ncellular proteins, we used the truncated version 1-650 lacking this unstructured protein\nbinding hub region to increase the specificity of the CLIP assay. Line 260 “We opted to use\nthe eGFP-NUP153 instead of the full-length construct to minimize indirect RNA binding\n1–650\narising from proteins bound to the FG-domain of NUP153 (Supp Fig 4C).” Additionally, we\nshow in Fig 4F that this construct is able to be found at the amplification site, bind RNA and\nrescue infection.\n4. Line 178: the authors claim the interaction between Nup153 and NS3/NS5 upon RNase\ntreatment is evidence that the interaction between these proteins is direct. This experiment\ndoes not support that conclusion, only that the interaction between Nup153 and NS3/NS5 is\nnot dependent on RNA. The authors more accurately describe this result in lines 195-196 but\nshould clarify in the previous statement.\nWe have clarified it in the text.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn their updated manuscript, the authors have provided significant additional data to\nsupport their claims. This has addressed many of my concerns and increased overall\nconfidence in the findings. However, there is still some uncertainty as to the specific role of\nNup153 in flavivirus infection. Although the authors provide compelling evidence that\nNup153 binds to viral RNA through its RBD and that Nup153 binding to viral RNA alters in\nvitro translation, the role of Nup153 within infected cells is less clear. Based on their data,\nthe authors conclude that Nup153 is altering the ratio of viral structural and non-structural\nproteins, leading to increased assembly. Additional evidence is needed to support a role for\nNup153 in virus assembly and to conclude that this effect is due to translational changes in\nthe polyprotein. Specifically, when Nup153 is depleted starting at 6 hr after infection\n(should be completely degraded by 8 hr post-infection), no effect of infected cells or virus\ntiters was observed. On the other hand, when BFA is added at 16 hr post infection there is a\ncomplete loss of virus production. If the effect of Nup153 depletion is specifically on virus\nassembly, there should still be a strong effect even when Auxin is added after 6hr, similar to\nBFA. Rather, these data suggest that Nup153 may act upstream of assembly. An effect on\ngenome replication or general polyprotein translation efficiency could also lead to\ndecreased spread observed in Figure 8. To make the claim that Nup153 is acting to\nspecifically promote assembly, the authors should rule out an effect on virus replication.\nWithout this direct evidence, it is challenging to determine if the minimal changes in\nprotein ratios observed in Figure 8 are leading to a significant change in assembly. In the\nabsence of additional experiments, their conclusions should be adjusted. Several specific\npoints are indicated below.\nThank you for the insightful suggestions and comments. We agree that it is hard to pinpoint\nthe specific effect of NUP153 on viral infection. We have therefore generated more data to\nclarify this. With the new data we see that early viral replication is not affected by depletion\nof NUP153 up to 12 h post infection. These results support our claim that NUP153 is not\ninvolved in replication per se.\nNew data Fig 2L and lines 130-135, “Because we detected differences between the\nreduction in number of infected cells compared to the released viral particles following\ndepletion of NUP153 we measured the amount of TBEV RNA at 2, 8, 12 and 16 h post\ninfection in cells induced with auxin 1hpi (Fig 2L). We found that depletion of NUP153 led to\napproximately 50% reduction in TBEV viral RNA at 16 hpi, however, no reduction at 8 or 12\nhpi (Fig 2L), indicating that NUP153 depletion does not affect early replication, but more\nlikely affects virus assembly and release.”\nWe also acknowledge that the direct effect of BFA on particle release has a different\nphenotype compared to NUP153 depletion. This might be due to BFAs effect on viral\nreplication if administered early in infection (DOI: 10.3390/v9010005). We choose to\nadminister BFA after the viral replication complexes has formed to isolate the effect to\nparticle release, however, the BFA remains in the supernatant until 24 hpi thus blocking\ninfection of virions released into the supernatant before administration.\n-What is the level of virus spread at 16hr in Nup153(NG)aid cells? In fig. 2, the authors show\nthat Nup153 depletion leads to a decrease in the number of infected cells, viral RNA and\ntiters. It is not clear if this effect is due to a decrease in virus spread or a decrease in virus\nreplication. The observation that the impact on virus infection is completely lost when you\ndeplete Nup153 6 hr after infection suggests that virus replication and not infection is\naffected.\nThank you for this suggestion. The level of infection in the NUP153 (NG) at 16hpi in Fig 2 is\napproximately 50%. To clarify in more detail in what stage the NUP153 is important for viral\ninfection, we have performed additional infection experiments. NUP153 depleted cells and\ncontrol cells were infected and TBEV RNA levels in the cells were quantified at different\ntimepoint post infection (2, 8, 12 and 16 h post infection) and normalized to the 2 hpi time\npoint. The 8 and 12 hpi time-point corresponding to when virus is replicating but not\nsecreting new virions, and the 16 h post infection (corresponding to virus release). These\ndata now replace the old Fig 2M. The results indicate that NUP153 depletion does not\naffect viral replication as measured at 8 and 12 h post infection, Lines 130-134.\n-To rule out an impact on virus replication, the authors should evaluate virus replication\nusing subgenomic replicons or in a system where there is limited virus spread.\nThank you for the suggestion, as these DLD NUP153 AID cells are very difficult to transfect\nwith subgenomic replicon we opted to analyze viral replication at early timepoints when\nvirion release has not yet been initiated. These data are now in the Fig 2L, we did not see\nany effects of NUP153 depletion up to 12 hours post infection.\n-Does Nup153 overexpression increase the structural to non-structural protein ratio in\ncells? Considering the proposed model, overexpression of Nup153 in infected cells should\nshift this ratio in the opposite direction from the depletion of Nup153.\nThank you for this suggestion we have performed overexpression and analysis of protein\nratio in transfected 293 cells and we do not see any changes when overexpressing NUP153.\nThe reason for this is not clear, but one hypothesis could be that the expression level of\nNUP153 is already increased during infection Fig 1J, thus the additional overexpression of\nNUP153 with plasmids might not affect viral replication. In support of this, we show that\nthe viral infection does not increase upon transfection of NUP153-eGFP in the no Auxin\ntreated DLD NUP153 AID cells (Fig 2M).\nReviewer #2 (Remarks to the Author):\nIn this revised version, Peters and colleagues have included many experiments that answer\nseveral important concerns I raised on the first version. Notably, the new data based on\nsuper-resolution microscopy as well as the titration assays convincingly demonstrate that\nNUP153 accumulate in the replication compartment and is a proviral factor. In that sense,\nthe manuscript has been much improved. A lot of nice work has been done.\nHowever, the main mechanistic conclusion regarding the regulation of viral translation by\nNUP153 depending on the coding sequence remains not convincing in my opinion. It\nseems indeed that there is a mild change in the abundance of E compared to NS proteins\nas now shown by mass spectrometry and immunoblotting. Nevertheless, the in vitro\ntranslation assays are not enough to explain the phenotypes. Even if this set of data\nsuggest that NUP153 RBD impact translation in a E sequence dependent manner in vitro,\nit remains very hard to comprehend how the rate of translation would be changed when the\nribosomes translate the sequences encoding non-structural proteins as part of the same\nopen reading frame. In line 64 and 475, the authors mention that the results “indicate\nNUP153 involvement in translation elongation” but this not addressed in this study per se.\nRibosome profiling would be the gold standard if the authors want to make such a\nconclusion. So far, the data only indicated that site 1 in E impact translation in vitro when\nNUP153 RBD is added. It might also be more complex in cells, when the ER is present and\nremodelled by the virus. Without additional experiments addressing translation elongation,\nthe conclusion is not supported by the data in my view. It is more likely that NUP153 impact\nthe viral protein abundance at the posttranslational level, which was not addressed. The\nhalf-life the viral proteins could be tested by western blotting in wt and NUP153-depleted\ncells. This should not change according to the model elaborated by the authors.\nThank you for your insightful comments and suggestions, we have now removed translation\n“elongation” throughout the manuscript. As we cannot pinpoint that elongation is the\ntarget and precise mechanism of NUP153s proviral function. We have also analyzed the\nhalf-life of the different viral proteins after cycloheximide treatment at different timepoints\npost treatment. Here we do not find a difference in stability of the viral proteins between wt\nand NUP153 depleted cells, this data is now new panels in Fig 8J and Supp fig 8. We have\nmodified the results section to include the data line 394-397.\n“Furthermore, to exclude that the effects we observed are a result of decreased protein\nstability following NUP153 depletion, we performed a cycloheximide (CHX) assay on TBEV\ninfected cells and found no significant differences in the stability of the viral proteins E,\nNS1 and NS2B between auxin treated and untreated samples over time (Fig 8J and Supp Fig\n8F,G).\nOther comments:\n-In Fig 4D, even if the data have been normalized to the input, these experiments tend to\nshow that NUP153 associate with the viral negative strand (actually in a similar extent as\nthe positive strand if the upper data point is not considered in the far right series). Even if\nthe negative strand was not detected in the CLIP-seq data, the conclusion about the\nexclusive binding to the positive strand should be tuned down.\nThank you for your suggestion we have now adjusted the text to better reflect our results,\nline 223 and 445.\n- The quality of Fig S7D is not optimal, and the results are hard to appreciate for the reader.\nWe agree that the fig S7D blot is a bit hard to interpret, but it is particularly difficult to\ndetect in vitro translated proteins using WB, that is why we opted to use a luciferase system\nto have a more sensitive and quantitative method for our assay and include the WB as a\nsupporting data piece. However, we do find that the provided WB reflects and supports the\nfindings in the luciferase assay.\n-Fig S8C is a bit confusing. What is the difference between the multiple “+” lanes? Are they\nbiological replicates?\nThank you for pointing this out, the + lanes are TBEV infected samples and we show all the\nbiological replicas in each blots, to be able to quantify and compare. We have now clarified\nit in the figure legend line 1449.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed the majority of my concerns and added significant new\nexperimentation and compelling evidence to support their claims. There are some\noutstanding questions/comments to address:\n1. There is not a significant explanation as to why auxin addition after 4 hpi would have no\nimpact. Why would this mechanism not be effective later in the infection cycle, closer to\nwhen C-prME are required for virion assembly?\nThank you for the comment. Auxin addition at 4 h post infection (NUP153 depletion at 6 h)\ndo actually give an effect on infection Fig 2I-J, but upon addition of Auxin at 6 hpi (depletion\nat 8 h ; Fig 2 K and Supp Fig 2E) we no longer see an effect. To better understand the\nkinetics when NUP153 is required for virus infection we have performed additional\nexperiments. We analyze the viral replication by Q-PCR assay at different timepoint post\ninfection (2, 8, 12 and 16) in cells expressing NUP153 or AID cells depleted of NUP153. The\nRNA levels were normalized to the input timepoint 2 h, and we show that there is no effect\non viral replication 8 and 12 h post infection (New Fig 2L). We believe that this is because\nNUP153 is important early in infection for the first round of viral particle release to promote\nviral propagation. We further validate this in Fig 8K with the BFA assay where we observe\nthe delay in infection.\n2. The data suggests a mechanism controlling translation; however, there are no data\nexamining differences in protein stability. This caveat could be addressed in the text.\nThank you for this suggestion. To address protein stability, we have performed an additional\nexperiment where we treated infected cells with cycloheximide to block protein translation,\nand analyzed protein amount in the cell using WB. Auxin was added 2 h prior infection and\ncycloheximide was added 24 h post infection. This is now new figure panels Fig 8J and Fig\nS8F-G, lines 393-398.\n“ Furthermore, to exclude that the effects we observed was a result of decreased protein\nstability following NUP153 depletion, we performed a cycloheximide (CHX) assay on TBEV\ninfected cells and found no significant differences in the stability of the viral proteins E,\nNS2B and NS3 between auxin treated and untreated samples over time (Fig 8J and Supp Fig\n8F,G). However, we did observe a small change in NS1 expression 6 hours post CHX\ntreatment (Supp Fig 8F,G). Taken together, .”\n3. In Figure 4G, RNA binding may be somewhat dependent on the protein expression levels\nof the various truncated constructs. Expression of eGFP seems to be variable by IF in Figure\n4B. Is RNA binding normalized to protein expression? A western blot showing relative\nprotein expression of the constructs should be included.\nThank you for this suggestion. Assuming that the comment relates to figure 4C, where we\ndo clip assay on different truncated constructs, this is a valid point. The RNA levels are not\nnormalized to protein levels in the cell, however, we do not believe that the protein amount\nof the mutants play a major role in the results obtained with the clip assay. The reason\nbeing: When we analyze the protein expression levels of the different transfected\nconstructs, they are somewhat different as indicated in the comments. However, the\nexpression levels cannot explain the difference detected in the clip assay. NUP153wt, 1-\n650 and 1-650-RBD show similar protein levels; NUP153wt and 1-650 bind RNA, whereas\n1-650-RBD does not bind viral RNA; similarly 880-1475 and 1-250 show comparable protein\nexpression 880-1475 bind but 1-250 does not bind viral RNA.\nWe have added a western blot showing this as new panel A in supplementary fig 4.\nLine 211 “First protein expression of the NUP153-GFP constructs were verified by WB (Supp\nFig 4A). “\n4. The paragraph explaining the binding peaks and the mutants generated is difficult to\nunderstand (lines 251-267). A more clear explanation would be helpful.\nThank you for this suggestion we have modified the text to be more clearly describe the\nexperiments and results.\n“The RNA-binding domain of NUP153 has previously been shown to associate with G- and\nGC-rich regions of RNA molecule12,42. G-rich sequences can fold into G-quadruplex (G4)\nstructures, a type of four-stranded nucleic acids secondary structure that can fold in\ncertain G-rich sequences (43). Peak 1 contains two predicted G4 structures (Fig 5D boxes\nand Supp Fig 6A). The second of which is highly conserved among orthoflaviviruses\nsequences (Supp Fig 4E) (44,45). We used in vitro transcription to generate RNA fragments\ncontaining the wildtype (WT) peak 1, a mutated construct (11G1C) lacking the second\nhighly conserved G4 motif and a construct lacking all orthoflavivirus conserved Gs (28G)\n(Fig 5D). We also in vitro transcribed three versions of peak 2, the WT peak, a mutated\nconstruct lacking the GA pattern that we identified to be conserved among\northoflaviviruses (8G8A) and a construct lacking all conserved Gs (19G) (Supp Fig 5A).\nRNA-EMSA was used to determine the interaction of the NUP153 RBD with the different\nRNA fragments corresponding to peak 1 and 2 and their mutants. We found that WT\nfragments of peaks 1 and 2 bound better to the NUP153 RBD compared to the 28G and 19G\nfragments respectively that lacked all conserved Gs (Fig 5E-H and Supp Fig 5B-C),\nindicating that Gs in these regions are important for the interaction with NUP153.”\n5. Figure 2G – while the interaction between NUP153 and Capsid is diminished with RNase\ntreatment, it is not abolished as stated in the manuscript (Lines 179-181). Further, it\nappears that RNase treatment results in an overall decrease in capsid expression in the\nlysate.\nThank you for this comment. We have now changed the description of our results to better\nrepresent the data shown. The reason for the lower C levels during RNase A treatment in\nthe input might be due to decreased stability of the C protein in the absence of RNA. We\nhave modified the text accordingly line 182-186.\nLine 178-183: “All three viral proteins co-immunoprecipitated with NUP153 in the presence\nof RNA, but the interaction between NUP153 and C was reduced after RNase A treatment,\nindicating that NS3 and NS5 bind to NUP153 independent of RNA whereas the C\ninteraction is likely is indirect via RNA dependent. Furthermore, we also find that RNase A\ntreatment resulted in decreased C protein in the input (Fig 3G), suggesting that RNA could\nbe important for this protein stability.”\nResponse to Reviewers\nReviewer #1 (Remarks to the Author):\nThe additional data provided by the authors significantly enhance confidence in their\nconclusions, and the revisions to the manuscript satisfactorily address my major\nconcerns.\nThank you so much.\nReviewer #2 (Remarks to the Author):\nIn this revised version of the manuscript, Peters and colleague have notably\ncomplemented the study with data showing stability of viral proteins over 6 hours and\nreplication kinetics in wt and NUP153-deficient cells. Unfortunately, while these data\nare informative, they do not contribute to firmly conclude that NUP153 regulates the\ntranslation of structural proteins to favor viral particle production, the main claim of the\nwork. Although all aspects related to elongation have been removed from the text, a\npotential role of NUP153 on translation still only relies on in vitro assays and reporter\nconstructs far for the reality of a flaviviral genome. Thus, my previous concern remains,\nespecially since the new results from stability assays as such (Fig 8 and Suppl Fig 8) do\nnot allow to measure the half-life of the viral proteins as they look quite stable over the 6\nhour time frame. Again, direct evidence should support this claim such as for instance:\n1- evaluating vRNA distribution change in the fraction of a polysome sucrose gradient, or\n2- viral protein/reporter expression following the transfection of a replication-deficient\nfull-length viral genome (which does not require high transfection efficiency).\nThank you for your suggestions we have now adjusted the text throughout to not\noverstate our claims regarding NUP153s ability to modulate protein translation.\nReviewer #3 (Remarks to the Author):\nThe authors have provided sufficient experimentation to address the majority of reviewer\nconcerns. Additional studies, including replicon-based translation assays and more in-\ndepth work to quantify viral protein half-life in the context of infection would further\nsupport a role for NUP153 in specifically regulating translation. However, this is an\ninformative study that convincingly shows that NUP153 is a pro-viral factor in flavivirus\ninfection, which impacts the ratio of structural to nonstructural proteins and posits an\nintriguing model for translational control in flavivirus infection.\nThank you for your kind words."}]

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