Zika virus NS1 drives tunneling nanotube formation for mitochondrial transfer and stealth transmission in trophoblasts

Michita, Rafael T.; Tran, Long B.; Bark, Steven J.; Kumar, Deepak; Toner, Shay A.; Jose, Joyce; Mysorekar, Indira U.; Narayanan, Anoop

doi:10.1038/s41467-025-56927-2

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Published: 20 February 2025

Zika virus NS1 drives tunneling nanotube formation for mitochondrial transfer and stealth transmission in trophoblasts

Nature Communications volume 16, Article number: 1803 (2025) Cite this article

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Abstract

Zika virus (ZIKV) is unique among orthoflaviviruses in its vertical transmission capacity in humans, yet the underlying mechanisms remain incompletely understood. Here, we show that ZIKV induces tunneling nanotubes (TNTs) in placental trophoblasts which facilitate transfer of viral particles, proteins, mitochondria, and RNA to neighboring uninfected cells. TNT formation is driven exclusively via ZIKV non-structural protein 1 (NS1). Specifically, the N-terminal 1-50 amino acids of membrane-bound ZIKV NS1 are necessary for triggering TNT formation in host cells. Trophoblasts infected with TNT-deficient ZIKV^ΔTNT mutant virus elicited a robust antiviral IFN-λ 1/2/3 response relative to WT ZIKV, suggesting TNT-mediated trafficking allows ZIKV cell-to-cell transmission camouflaged from host defenses. Using affinity purification-mass spectrometry of cells expressing wild-type NS1 or non-TNT forming NS1, we found mitochondrial proteins are dominant NS1-interacting partners. We demonstrate that ZIKV infection or NS1 expression induces elevated mitochondria levels in trophoblasts and that mitochondria are siphoned via TNTs from healthy to ZIKV-infected cells. Together our findings identify a stealth mechanism that ZIKV employs for intercellular spread among placental trophoblasts, evasion of antiviral interferon response, and the hijacking of mitochondria to augment its propagation and survival and offers a basis for novel therapeutic developments targeting these interactions to limit ZIKV dissemination.

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Introduction

Zika virus (ZIKV) is a mosquito-borne positive-strand RNA virus within the Flaviviridae family. Following a period of sporadic infections in Africa and Asia, ZIKV began rapidly spreading in the Pacific islands (2007-2013)^1,2 before reaching epidemic proportions in the Americas (2015-2018)³, with estimates as high as 130 million total cases by 2018⁴. ZIKV infection can lead to neurological disorders and fetal abnormalities such as microcephaly and fetal demise, collectively known as congenital Zika syndrome^5,6. The propensity for horizontal and vertical transmission^7,8,9,10, and the ability to traverse blood-tissue barriers, including the blood-placental barrier^11,12,13, of ZIKV are unique among Flaviviridae^6,14. Murine studies from our group and others have shown that ZIKV can infect fetal trophoblasts and endothelial cells of the placenta, which form the primary barrier between the maternal and fetal circulations, leading to ZIKV entry into fetal circulation¹¹. Placental infections and vertical transmission can occur whether ZIKV is introduced via vaginal route or intrauterine route^15,16. Studies further show ZIKV has broad tropism in the human placenta^{17,18,19,20,21,22}, infecting cytotrophoblasts (CTBs), syncytiotrophoblasts (STBs), extravillous trophoblasts (EVTs), endothelial cells, and fetal macrophages in the intervillous space, possibly allowing the virus to cross the protective barrier. However, different trophoblasts show differential permissiveness for ZIKV. Whereas STBs derived from primary human trophoblasts (PHTs) are more resistant due to interferon (IFN)-λ response¹⁸, CTBs and EVTs, represented by cell lines JEG-3 and HTR-8/SVneo (HTR-8), respectively, appear susceptible to ZIKV infection^11,18. Antiviral IFN response, mainly mediated by type-III (IFN-λ), has been shown to confer protection against ZIKV^18,23,24.

The orthoflavivirus positive-strand RNA genome encodes three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Orthoflavivirus nonstructural proteins participate in viral replication, assembly, and hijacking of host pathways for the completion of the virus life cycle^{25,26,27,28,29}. The orthoflavivirus NS1 is a glycosylated, membrane-associated nonstructural protein existing as a dimer in the endoplasmic reticulum (ER)^30,31 where it is essential for viral RNA replication³¹, and as a hexamer secreted from the infected cells with diverse roles in immune evasion and pathogenesis³². ZIKV NS1 shares structural similarities with NS1 proteins of other orthoflaviviruses such as dengue virus (DENV), West Nile virus (WNV), and JEV (Japanese encephalitis virus); however, amino acid sequences vary^33,34. NS1 appears to be necessary for ZIKV pathogenesis in the placenta, as a neutralizing antibody to NS1 has been shown to limit placental/fetal damage³⁵. However, the functional role and mechanism of NS1 in ZIKV pathogenesis remains incompletely understood.

Tunneling nanotubes (TNTs) are intercellular conduits that hover over the substratum and directly connect to the plasma membrane of neighboring cells, measuring up to 700 µm in length³⁶. As TNTs allow cytoplasmic continuity between connected cells and transfer cargo, they differ from other cellular protrusions such as filopodia, intercellular bridges, and cytonemes. TNTs are composed of either F-actin or microtubules^37,38 and enable the long-range exchange of nucleic acids, proteins, electrical signals (Ca²⁺), lipids, organelles including mitochondria, and infectious particles between connected cells^{36,39,40,41,42,43}. Viruses such as HIV^44,45, influenza⁴⁶, porcine reproductive and respiratory syndrome virus (PRRSV)⁴⁷, WNV⁴⁸, and SARS-CoV-2^49,50 have been shown to induce TNTs in infected cells and use these structures to spread to naïve cells. The TNT-associated mode of intercellular transmission likely provides protection for the virus from the extracellular immune response, including neutralizing antibodies and/or immune cells (as reviewed elsewhere^43,51).

Here, we report that ZIKV induces the formation of TNTs in multiple cell types, including placental trophoblasts. Among ZIKV structural and non-structural proteins, we demonstrate that NS1 is necessary to induce TNT formation. Notably, TNTs were found to be conduits to spread virions, dsRNA, and proteins from infected to neighboring cells and transfer mitochondria from healthy to ZIKV-NS1-expressing cells. Interactome analysis revealed NS1 is directly or indirectly associated with mitochondrial proteins and pathways leading to mitochondrial transfer. The observed accumulation of mitochondria may provide an energetic boost to virus-infected cells to promote viral replication. Accordingly, disruption of mitochondrial respiration and mobility limits virus replication in trophoblast cells. We also show that ZIKV uses TNTs to disseminate to neighboring cells directly, potentially avoiding the activation of large-scale antiviral interferon defenses. Mutant ZIKV that do not make TNTs induce robust antiviral IFN-lambda response, while inhibiting mitochondrial motility suppresses this response. Thus, together, our findings identify ZIKV-NS1 as a critical mediator for TNT formation and uncover a previously unrecognized mechanism harnessed by ZIKV to promote intercellular trafficking of the virus and its proteins among placental cells while simultaneously using TNTs to hijack mitochondria from uninfected neighboring cells and evade antiviral responses.

Results

ZIKV induces TNT formation in trophoblast cells and transfer viral proteins

We engineered ZIKV MR-766 cDNA as previously described^52,53 to express mCherry using reverse genetics to track real-time infection in live cells (Fig. 1A). ZIKV-mCherry (MR-766) was used to infect HTR-8 [extravillous trophoblasts (EVTs)], JEG-3 [cytotrophoblasts (CTBs)], and primary human trophoblast cells (PHTs) isolated from term placentas [multiplicity of infection (MOI) = 0.1-3, 24 hours (hrs)] (Fig. 1B). Confocal imaging revealed that ZIKV MR-766 induces the formation of thin and long actin-based filaments that hover over the substrate and connect neighboring cells in trophoblast cells (Supplementary Fig. 1A). These filaments are distinct and longer than filopodia (close-ended protrusions of F-actin reaching up to 1-5 μm length), intercellular bridges, and cytonemes in length (0,2-2 μm in intercellular bridges), function (close-ended extremities) and formation, in addition to being more diverse in composition (actin, anillin, and tubulin)^54,55. For this study, we considered the structures >15 μm long connecting two cells as TNTs. Next, we asked whether TNT induction was a common feature of ZIKV strains and other orthoflaviviruses in the Flaviviridae family. Our results show that in addition to MR-766, the new world ZIKV strain, PRVABC-59 was also able to generate TNTs in trophoblast cells (Fig. 1B). However, no TNTs were observed in trophoblasts infected with DENV2 and yellow fever virus (YFV) (Supplementary Fig. 1B), suggesting that TNT formation is not a common feature to all orthoflaviviruses. Together, our findings demonstrate that JEG-3 and HTR-8 (representing placental CTBs and EVTs) and PHTs form TNTs in response to ZIKV infection.

**Fig. 1: Multiple ZIKV strains induce TNT formation in human placental trophoblast cells and transfer viral proteins.**

Because TNTs are known to transfer a variety of cargo³⁶, we reasoned that ZIKV may co-opt TNTs for viral dissemination. To test this, we infected cells with ZIKV MR-766 (MOI = 1) and probed with antibodies specific to the ZIKV structural proteins envelope (E) and capsid (C) in immunofluorescence assays. In the TNTs formed between neighboring cells, we observed areas of colocalization of capsid and envelope proteins, suggesting that the TNTs might act as a cell-associated conduit for the assembled virions and viral proteins to be trafficked from one cell to another (Fig. 1C).

We next sought to determine if viral genomic RNA can also be transported through TNTs as a mode of viral transmission. We generated a ZIKV MR-766 replicon construct from the cDNA clone, where the virus structural protein genes were replaced with the fluorescent protein mEmerald (Fig. 1D). A549 lung epithelial cells transfected with the replicon construct showed mEmerald expression and formed TNTs. At 48 hrs post-transfection (hpt), the neighboring untransfected cells connected via TNTs to transfected cells and showed mEmerald fluorescence, suggesting TNTs allow cell-to-cell transfer of the viral replicon RNA. To further determine viral RNA transfer via TNTs in trophoblast cells, we infected HTR-8 cells with ZIKV MR-766 and stained for viral RNA using anti-dsRNA antibody. Our data supports the notion that ZIKV-induced TNTs can transport ZIKV double-stranded RNA (Fig. 1E). This suggests that ZIKV might use TNTs to spread viral genomic RNA from one cell to another, even in the absence of intact virions. Together, our findings show that ZIKV-induced TNTs serve as conduits to transfer virions and viral material to uninfected cells.

The NS1 protein of ZIKV uniquely induces TNT formation

To determine which ZIKV protein is responsible for inducing TNTs, we cloned the three structural proteins (C, prM, E) and the seven nonstructural proteins (NS1, NS2A, NS2B-NS3, NS4A, NS4B, and NS5) of ZIKV MR-766 with a C-terminal mCherry tag into mammalian expression plasmids. Transfection of cells with individual ZIKV expression constructs revealed that among the viral proteins, NS1 expression uniquely induces TNT formation (Fig. 2A). Despite orthoflavivirus NS1 being structurally well-conserved and being the principal target of positive selection during flavivirus speciation^30,56, we found that ZIKV-NS1 induces multiple TNTs in A549 and HTR-8 cells compared with NS1 proteins of DENV1, DENV2, DENV3, DENV4, DTV, WNV, and JEV which produce close to 0 TNTs (Fig. 2B and D). We did observe that WNV NS1 has some limited capacity to form TNTs, as described elsewhere⁴⁸. Importantly, the similar expression levels of orthoflavivirus NS1 support the specificity of ZIKV NS1 (MR-766 and PRVABC-59) in inducing TNT formation (normalized fluorescence (NF) intensity of NS1 expression depicted in Fig. 2C and E). Confocal microscopy imaging of HTR-8 cells transfected with NS1 (red) from ZIKV (MR-766 and PRVABC-59), DENV1-4, DTV, WNV, and JEV further demonstrate that only ZIKV robustly induces TNTs (with limited TNT formation induced by WNV NS1) (2F-O). Finally, we show that ZIKV NS1 induced TNTs in trophoblast cell lines HTR-8, JEG-3, and BeWo, and multiple non-trophoblast cell types including A549, U-87 MG, SH-SY5Y with the exception of Vero E6 cells. HTR-8 cells were most efficient in TNT formation while Vero E6 cells produced very few short (< 15 μm) structures (Fig. 2P, Q).

**Fig. 2: The non-structural protein 1 (NS1) of ZIKV is unique and necessary for inducing TNT formation in multiple cell types.**

The N-terminus of ZIKV NS1 is important for TNT formation

The NS1 protein consists of three domains: a β-roll dimerization domain (amino acids 1-29) at the N-terminus, a wing domain (amino acids 30-180) with glycosylation sites and subdomains, and a β-ladder domain (amino acids 181-352)^30,34. To delineate which structural domain of ZIKV-NS1 plays a role in TNT formation, we generated NS1 mutant clones using overlap PCR cloning where multiple domains of ZIKV NS1 were substituted with corresponding domains from DENV2-NS1, which did not induce TNTs (Fig. 3A). Subsequent transfection and expression of NS1 mutants revealed that the TNT-forming ability of ZIKV NS1 is determined by the N-terminal 50 amino acids (NS1 mutant pNS1^ΔTNT), which spans the β-roll dimerization domain and part of the wing domain of NS1 (Fig. 3B, C). Of note, the N-terminal domain of NS1 displays variation across various orthoflaviviruses, with an amino acid identity ranging from 42% to 44% when compared to ZIKV-NS1. Changing the N-terminal 50 amino acids of ZIKV to the corresponding domain of DENV4, which is genetically closer to ZIKV, or swapping the ZIKV N-terminal 50 amino acids in DENV-2 NS1 also abolished TNT formation (Supplementary Fig. 2). Next, to determine whether the amino acid changes introduced in the N-terminal region of ZIKV NS1 affect TNT formation without affecting viral replication and assembly, we introduced mutations in the NS1 N–terminal region of the ZIKV cDNA clone using site-directed mutagenesis. Mutations were not tolerated in the N-terminal 1-39 amino acids, resulting in a lethal phenotype. We obtained an infectious ZIKV^ΔTNT mutant virus with amino acids changed to DENV2 sequence in the 40-52 region, which did not produce TNTs upon infection (Fig. 3E) and protein expression (Fig. 3F). To investigate whether the NS1 mutation in ZIKV^ΔTNT affects viral replication and release, we assessed the growth kinetics of ZIKV in three distinct cell types: Vero E6 cells, which we show do not generate TNTs in response to NS1; JEG-3 cells, which show moderate TNT formation; and HTR-8 cells, which exhibit extensive TNT formation upon infection. Our findings indicate that both wild-type and ZIKV^ΔTNT have comparable growth at all time points suggesting virus replication and assembly is not affected in the TNT mutant virus in Vero E6 cells. In contrast, ZIKV^ΔTNT showed approximately 1 log lower virus titer than wild-type ZIKV in both HTR-8, and JEG-3 cells at all time points, indicating TNT formation is advantageous to the cell-to-cell spread of the virus in the monolayers of trophoblast cells (Fig. 3D).

**Fig. 3: The N-terminus of ZIKV-NS1 is necessary to induce TNT formation.**

Recent work has suggested that NS1 secreted into the extracellular environment influences host responses⁵⁷ and compromises the integrity of the human placenta and endothelial barrier^57,58. We thus tested whether the secreted NS1 protein could also induce TNTs by treating HTR-8 trophoblast cells with secreted His-mCherry NS1 protein purified from culture supernatants of HEK 293-T cells as previously described⁵⁹ (Fig. 3G). Confocal images of the His-mCherry NS1- treated cells show accumulation of NS1 in the endosomal vesicles, but no TNTs were formed (Fig. 3H). Together, our data suggests that ZIKV NS1 expression on the plasma membrane, likely as membrane-associated dimers, is necessary for inducing TNTs rather than the secreted hexameric NS1.

ZIKV NS1 colocalizes with actin and tubulin filaments in TNT structures

Next, we investigated whether ZIKV NS1 associates with cytoskeletal filaments. We found that intracellular pNS1-ZIKV colocalizes with actin filaments predominantly on the plasma membrane (Fig. 4A, B), which is not observed with pNS1^ΔTNT expressing cells (Fig. 4C). To confirm that the TNTs require actin polymerization, we treated the cells with a known inhibitor of actin polymerization, cytochalasin-D. Our results show that cells with inhibited actin polymerization do not form TNTs (Fig. 4D). Microtubules and actin containing-TNTs are thicker than thin actin-restricted TNTs which confers increased resistance and capacity for transferring large organelles³⁶. Interestingly, some ZIKV pNS1-induced TNTs have both NS1, actin, and tubulin filaments, with long TNTs showing tubulin and short cellular protrusions enriched for actin (Fig. 4E). Despite the association of ZIKV NS1 with cytoskeleton filaments, we found that expression levels of CDC42 (Cell division cycle 42) and RAC1 (Ras-related C3 botulinum toxin substrate 1) small Rho GTPases that may be involved in TNT formation^60,61, are not affected by NS1 (Fig. 4F). Similarly, no difference was observed in the expression of motor proteins DNML1 (Dynamin 1-like), MYO10 (Myosin X), which are both involved in TNT function, or expression of TNFAIP2 (Tumor necrosis factor-alpha induced protein 2), a key TNT-inducing factor⁶². ZIKV pNS1 and pNS1^ΔTNT were found to downregulate MYO5 (Myosin V), a motor protein involved in the formation of TNT-like structures from double filopodial bridges⁶³. Together, these findings suggest that TNTs are associated with actin cytoskeleton.

**Fig. 4: ZIKV NS1 colocalization with cytoskeletal components and regulation of TNT-related genes.**

TNT-forming NS1 interacts with mitochondrial proteins

To dissect protein-protein interactions crucial for NS1-mediated TNT formation, we performed affinity-purification mass-spectrometry (AP-MS)^64,65 from cells expressing pNS1-ZIKV and pNS1^ΔTNT with C-terminal His-tags. We tested the model that any binding partners exclusive to the full-length membrane-bound wild-type NS1 would be necessary for supporting TNT formation. Proteomics data consisting of protein identification and spectral counts were analyzed to estimate relative protein abundance levels⁶⁶ and subsequent identification of unique proteins interacting with TNT-forming ZIKV NS1. Out of the 326 protein-protein interactions with a fold change of >2 and P < 0.05, 178 protein-protein interactions were upregulated in NS1-ZIKV (Fig. 5A). By querying NS1 protein interactions with the Uniprot database for subcellular compartments, we found that the most represented interactions in trophoblasts with functional TNTs were associated with mitochondrial functions (32%) (Fig. 5B and Supplementary Data 1). Next, we investigated the specific protein-protein interactions only enriched in TNT-forming ZIKV NS1 (n = 50). Consistent with our previous results, we observed that 44% (n = 22/50) of the identified interactions were related to mitochondrial proteins (Fig. 5C), which are highly enriched in ZIKV-NS1 compared to pNS1^ΔTNT (Fig. 5D).

**Fig. 5: NS1-induced TNTs are associated with mitochondrial proteins.**

ZIKV-NS1 induces mitochondrial accumulation via TNTs

The prevalence of mitochondrial proteins in the NS1 interactome prompted us to investigate their associations with ZIKV infection, NS1, TNT formation, and mitochondrial transfer in trophoblast cells. To address this, we transfected HTR-8 cells (shown to vigorously form TNTs) with ZIKV-NS1 (acceptor cells) and co-cultured with untransfected HTR-8 cells pre-stained with Mitotracker green to mark their mitochondria (donor cells). Following a 16-hour period of co-culture and NS1 transfection, we show that the NS1-transfected HTR-8 cells had acquired mitochondria from the untransfected donor cells. Furthermore, we demonstrate that NS1 colocalizes with mitochondria (Fig. 5E).

Next, we investigated how ZIKV infection affects mitochondrial content in JEG-3 cells, a cell-based model of CTBs that is also permissive to ZIKV infection (Fig. 3D) and shown in infection assays using human placental explants to be more susceptible to ZIKV MR-766 than extravillous trophoblasts⁶⁷. We infected JEG-3 cells (MR-766, MOI = 0.1) and analyzed mitochondrial content 16 hpi. We observed a significant increase in mitochondria content in ZIKV-infected (Fig. 5F, G) cells, but also ZIKV NS1-transfected cells (Fig. 5H, I).

To determine whether TNTs are involved in mitochondrial acquisition by NS1 expressing trophoblast cells, we transfected HTR-8 cells with ZIKV-NS1 (acceptor cells) and co-cultured with homotypic or heterotypic untransfected cells which were pre-stained with Mitotracker green to mark their mitochondria (donor cells). Following a 16-hour period of co-culture and NS1 transfection, live-cell imaging revealed that NS1-expressing cells acquired mitochondria from donor cells via TNTs (Fig. 6A). Consistent with this observation, we further showed that cells expressing pNS1^ΔTNT that lack the ability to form TNTs have limited ability to accumulate mitochondria from neighboring donor cells (Fig. 6B). These findings suggest that ZIKV-NS1-mediated TNT formation triggers the transfer of mitochondria from bystander cells to NS1-expressing cells via TNTs (Supplementary Movie 1-2).

**Fig. 6: ZIKV NS1 induces mitochondrial transfer between cells via TNTs.**

To quantify the relationship between NS1 expression, TNT formation, and mitochondria accumulation via TNTs, we performed flow cytometry on co-cultured cells (Fig. 6C). Acceptor HTR-8 cells transfected with NS1 from ZIKV MR-766 or PRVABC-59 strains were co-cultured with untransfected homotypic (HTR-8), or heterotypic donor cells (JEG-3, THP-1) labeled with Celltrace Violet and Mitotracker Green. Because secreted NS1 can be taken up by untransfected cells (see Fig. 3H), we focused on quantifying the impact of ZIKV-NS1 in the donor-to-acceptor transfer of mitochondria. Further, by using THP-1 monocytes differentiated into resting macrophages (M0) and JEG-3 (cytotrophoblasts), we tested the hypothesis that TNTs induced by NS1-expressing cells (HTR-8, extravillous trophoblasts) would mediate mitochondria transfer between different but related cells in the placenta. We next co-cultured donor and acceptor cells in a Boyden chamber, physically separated by a membrane with 400 nm pores allowing secreted signaling molecules to pass through but preventing physical cell-cell contact via TNTs⁶⁸.

We observed no mitochondria transfer to NS1-expressing cells in all co-culture conditions when physically separated by Boyden chambers (P < 0.0001) (Fig. 6D, F, H). In homotypic HTR-8 cell co-cultures, over 99% of Celltrace violet-negative acceptor cells (both NS1-positive and NS1-negative) acquired mitochondria from donor cells within 24 hrs of co-culture. Although HTR-8 transfected with pNS1^ΔTNT also received mitochondria from donor cells, the transfer was significantly lower compared to ZIKV-NS1 transfected cells (Fig. 6D, E). We quantified the mitochondria signal intensity (Mitochondria MFI ratio) to measure mitochondria transfer from untransfected donor to acceptor HTR-8 cells (ZIKV-NS1 transfected). Our results indicate a significant increase of mitochondria transfer to ZIKV-NS1 transfected HTR-8 cells compared to TNT-deficient pNS1^ΔTNT transfected cells (Fig. 6E).

We next determined whether ZIKV-NS1-induced TNTs facilitate mitochondria transfer in heterotypic co-cultures of transfected HTR-8 acceptor cells with untransfected donor JEG-3 cells. Our findings suggest that JEG-3 cells are efficient mitochondria donors and HTR-8 ZIKV-NS1-expressing cells are more effective in siphoning mitochondria (Fig. 6F, G). This finding supports our data showing that HTR-8 makes more TNTs compared with JEG-3 cells, which may enable them to transfer mitochondria and viral proteins more effectively. We next tested heterotypic transfer between ZIKV-NS1 transfected HTR-8 and untransfected THP-1 macrophages (MΦ) and found that HTR-8 could also siphon mitochondria from macrophages (Fig. 6H) albeit at reduced levels relative to those from other trophoblast cells (Fig. 6I). The negative mitochondria MFI ratio suggests that mitochondrial transfer from macrophages to HTR-8 is higher in NS1-negative cells compared with NS1-positive HTR-8 cells. Together, these findings underscore that ZIKV NS1-induced TNTs, specifically EVTs, form cell-to-cell conduits and siphon mitochondria homotypically and heterotypically. This mitochondrial transfer strongly supports the hypothesis of NS1-mediated mitochondrial uptake and direct interaction between NS1 and mitochondrial components, which further corroborate our proteomic findings.

TNTs are functionally important for dampening IFN response

Our results suggest that ZIKV infection promotes TNT formation via the NS1 protein, allowing cell-to-cell transport of virus particles, viral RNA, and proteins. Studies have established that ZIKV infection of trophoblasts is restricted in part due to rapid induction of a robust antiviral response, particularly type III interferons (IFN-lambda or IFN-λ)^18,23,24. To test the model that ZIKV uses TNTs to traffic to other cells and propagate without triggering an immune response, we performed multiplex assays on supernatants from trophoblast cells infected with wild type MR-766, PRVABC-59, or ZIKV^ΔTNT (MOI = 0.1). Remarkably, we found that while the anticipated IFN-β and IFN-λ responses were induced following infection with the MR-766 and PRVABC-59 strains (Fig. 7A–E), the ZIKV^ΔTNT mutant incited a remarkable 30-60-fold surge in IFN-λ1, 2, and 3 levels in JEG-3 cells (Fig. 7D-E). Interestingly, we found that HTR-8, which exhibits features of EVTs, appear to have a significantly lower IFN-λ response to ZIKV (MR-766 and PRVABC-59) relative to JEG-3 cells (Fig. 7F) despite having functional RNA-sensing innate immunity. In addition, JEG-3 and HTR-8 cells exhibit distinct patterns of innate immune gene expression in response to wild-type ZIKV and ZIKV^ΔTNT infections (Fig. 7G-L). These differences underscore the cell type-specific antiviral responses that may influence the permissiveness of trophoblasts to ZIKV infection^18,67. In JEG-3 cells, the heightened IFN-λ response upon ZIKV^ΔTNT infection may be partly attributed to the lack of TNT formation, and the downregulation of IRF1 (interferon regulatory factor 1) and NFKB (nuclear factor-kappa B) (Fig. 7I and L).

**Fig. 7: The TNT-forming ability of ZIKV affects IFN response in trophoblasts.**

Inhibition of mitochondrial function and mobility affects ZIKV growth and IFN response

Viruses are known to manipulate mitochondrial dynamics in infected cells, fostering conditions to facilitate their replication and evade host immune defenses^69,70. Having determined that ZIKV infected or ZIKV NS1 expressing cells accumulate mitochondria from neighboring cells, we investigated the role of mitochondria function in ZIKV replication and IFN response. We treated JEG-3 cells with Rotenone, an inhibitor of mitochondrial complex I (Fig. 8A) and evaluated its effect on ZIKV replication. Using an LDH assay, we identified that non-cytotoxic concentrations ranged between 0.001 to 0.1 µM for JEG-3 cells (Fig. 8B). Of note, Rotenone was found to be more cytotoxic for HTR-8 cells, suggesting that they are more sensitive to mitochondrial dysfunction than JEG-3 cells (Supplementary Fig. 3). Rotenone treatment of infected JEG-3 cells shows that disruption of mitochondrial respiration negatively affects ZIKV replication (Fig. 8C).

**Fig. 8: Disruption of mitochondrial function and mobility impairs ZIKV replication and modulates the IFN response.**

Next, we evaluated whether blocking mitochondria mobility would impact viral replication. We treated ZIKV-infected cells with Miro-1 reducer at non-cytotoxic concentrations (Fig. 8D-E). Miro1 Reducer has been shown to facilitate the proteasomal degradation of MIRO1^71,72, which acts as an adaptor protein connecting mitochondria to motor proteins, facilitating their transport through the cytoskeleton. Further, loss of MIRO1 has been shown to reduce mitochondria transfer via TNTs^68,73. By treating ZIKV-infected cells with Miro1 reducer, we found that limiting mitochondria mobility affects ZIKV replication (Fig. 8F). Next, we tested whether mitochondrial function or mobility impacts the innate immune response to ZIKV (Fig. 8G-K). Remarkably, we found that Miro1 reducer affects IFN response by significantly reducing IFN lambda (λ) (Fig. 8J, K) but increasing IFN alpha (α) (Fig. 8G). These findings underscore a novel dynamic in ZIKV pathogenesis in trophoblast cells, where both preserved mitochondrial function and mitochondrial motility are critical for viral replication.

Discussion

Our investigation reveals a previously unknown mechanism of intercellular transmission exploited by ZIKV, setting it apart from other orthoflaviviruses such as WNV, DTV, YFV, JEV, and DENV as it possesses the capacity to induce TNT formation via its NS1 protein in multiple cell types, including trophoblasts. A limited number of viruses have been reported to induce the formation of TNTs in infected cells, such as HIV, herpes simplex virus (HSV), and influenza A virus (IAV) (reviewed in ref. ⁴³). TNTs have also been shown to transfer various cargo, including virus particles, viral RNA, and replication complexes between connected cells^36,43,49. Our results indicate that by inducing the formation of TNTs, ZIKV gains direct entry into neighboring cells, leading to a rapid cell-associated spread of ZIKV, potentially increasing the pathogenicity. Notably, TNTs provide a conduit for higher ZIKV transmissibility through cell-to-cell interactions compared to cell-free virus transmission, as neutralizing antibodies are ineffective in inhibiting viral spread in vitro⁷⁴. Other positive-strand RNA viruses such as SARS-CoV-2 and chikungunya virus (CHIKV) have been shown to exploit cell-to-cell transmission to infect non-permissive cells that lack viral entry factors and bypass the effect of neutralizing antibodies and important blood tissue barriers^49,75.

Previous studies have shown that the N-terminal residues forming the β-roll and connector subdomain of the wing are important for membrane binding of NS1⁷⁶, but vary between orthoflaviviruses. We speculate that the unique property of ZIKV NS1 to induce TNT formation is linked to this amino acid sequence and the resulting variation in charge distribution and membrane binding properties. ZIKV NS1 forms homodimers, associates with the ER and Golgi membranes, and has been shown to play a role in viral maturation and membrane remodeling⁷⁷. The membrane-associated, intracellular NS1 (dimeric form) induces TNT formation, whereas secreted NS1 (hexameric form) does not. This suggests NS1 acts as a molecular scaffold, facilitating interactions with actin and other membrane-associated proteins. Further studies are needed to explore the mechanisms by which NS1 may influence membrane curvature. Interestingly, WNV-NS1 has been shown to induce formation of TNT-like structures, while secreted WNV-NS1 promotes remodeling of the cytoskeleton suggestive of F-actin depolymerization in a cell type specific manner⁴⁸. Given that high levels of the extracellular NS1 hexamers circulate in the bloodstream of flavivirus-infected patients⁷⁸, it is unlikely that secreted ZIKV NS1 induces TNTs but instead leads to tissue permeability and endothelial damage^57,58,79,80. Interestingly, anti-NS1 monoclonal antibodies (mAbs) targeting cell-surface NS1 (presumably expressed as a dimer³⁰) or the N-terminal region of NS1 have been shown to limit ZIKV infection in animal models^35,81,82,83. While the protective mechanisms of non-neutralizing ZIKV NS1-targeted mAbs have yet to be elucidated, a growing body of evidence shows that NS1-based vaccines confer protection against ZIKV infection in animal models^{84,85,86,87,88}. Further investigation is needed to determine the molecular mechanism by which ZIKV NS1 induces TNTs, and whether mAbs or NS1-based vaccines target TNT formation in ZIKV-infected cells, potentially limiting viral infection and spread.

We found that the N-terminal region of NS1 (1-50 amino acids) confers the unique ability of ZIKV to induce TNTs through a mechanism that has yet to be determined. Many proteins have been implicated in triggering signaling pathways leading to TNT formation^36,89,90. However, ZIKV NS1 does not affect the transcriptional levels of key actin cytoskeletal (i.e., CDC42, RAC1, and TNFAIP2)^60,61,62 and motor proteins (MYO10, DNML1)^91,92 involved in TNT formation and function. Myosin 5 (MYO5) helps to form TNTs by twisting filopodial bridges, driving the helical deformation needed for their transformation into stable TNTs⁶³. The low expression levels of MYO5A induced by pNS1-ZIKV and pNS1^ΔTNT suggest that NS1 domains other than the NS1 N-terminal region affect MYO5A expression levels. Of note, MYO5A knockdown has been shown to reduce helical double filopodia bridges (DFBs); however, MYO5 inhibition does not affect TNT formation as other myosins (i.e., Myosin II) participate in the transition of DFBs to TNTs⁶³.

We report that TNTs triggered by ZIKV infection transport viral material and help dampen host IFN response. The host immune defense against ZIKV initially involves the recognition of viral RNA by receptors like RIG-I, which interact with the mitochondrial antiviral signaling protein (MAVS) leading to the production of type I and III interferons, such as IFN lambda, which activate antiviral genes⁹³. IFN-lambda is constitutively expressed by primary human trophoblasts and is known to limit ZIKV infection¹⁸. Infection of JEG-3 cells, which are representative of CTBs, with ZIKV^ΔTNT which does not drive TNT formation, elicits a multi-fold increase in IFN response compared to ZIKV wild-type infection (TNT-competent). Previous studies have shown that ZIKV dampens host IFN and RIG-I-dependent innate immunity and manipulates mitochondrial dynamics^25,29,94,95. Importantly, since mutant ZIKV^ΔTNT is replication-competent, and infectious, compared to wild-type ZIKV, we propose that the TNT forming capacity is the key differential resulting in immune IFN response evasion by ZIKV. While we cannot rule out the influence of DENV-NS1 N-terminal region on IFN signaling, the surge of IFN response to ZIKV^ΔTNT suggests that TNTs are shielding the virus from the immune system. Our observation that TNTs are co-opted by ZIKV to dampen trophoblast IFN response, facilitating the transfer of viral particles and mitochondria, highlights a new function for NS1. We speculate that inhibition of TNT formation and halting mitochondria siphoning from neighboring cells could result in more robust, IRF1 and NFkB-independent antiviral signaling to limit viral transmission. It is noteworthy that lack of TNTs affects mitochondria transfer and type III IFN response. One possible mechanism could be via peroxisome signaling⁹⁶. Despite the fact that ZIKV^ΔTNT is a chimera, downregulation of IRF1 and NFKB suggest alternative pathways for induction of IFN type I and III which could be determined by its unique N-terminal, such as the cyclic GMP-AMP synthetase (cGAS)-stimulator of interferon genes (STING) signaling pathway⁹⁷.

Our work sheds light on the specificity of the N-terminal region of ZIKV-NS1 in inducing TNTs and mitochondria accumulation via TNTs. While mitochondria transfer between cells through TNTs has been documented in various physiological and pathological conditions, its role in viral infections remains incompletely understood^43,98,99. Nonetheless, PRRSV has been shown to promote mitochondria transfer from uninfected to infected cells in allogeneic and xenogeneic co-cultures to rescue infected cells from apoptosis/necrosis⁹⁹. Also, PRRSV proteins were found to colocalize with mitochondria in TNTs, suggesting that the virus hitchhikes with mitochondria for intercellular transportation^47,99. This is consistent with our findings that ZIKV NS1 protein-protein interactions were highly enriched for mitochondrial proteins, and the observation that NS1 colocalizes with mitochondria acquired from neighboring cells. TNT formation has been reported to be associated with ROS expression levels and the release of ‘call-for-help signals’ such as S100 proteins^99,100,101. The intercellular exchange of mitochondria through TNTs has been shown to rescue stressed cells^102,103,104 by providing metabolic support and delaying cell death^68,99. Our observation of mitochondrial accumulation in ZIKV-infected trophoblasts, facilitated by the transfer of mitochondria via TNTs from neighboring naïve cells, suggests that ZIKV exploits this physiological stress response to enhance its transmission and survival. Other pathways such as cell-free mitochondrial uptake or transfer via extracellular vesicles containing mitochondria may also contribute to this accumulation¹⁰⁵. Irrespective of the mode of transfer, however, we discovered that Rotenone and Miro1 reducer treatment inhibits viral replication, suggesting that ZIKV requires functional mitochondria to siphon and support their replication. Interestingly, we found that HTR-8 cells are more permissive to Rotenone cytotoxicity compared with JEG-3 cells. The lower tolerance to mitochondrial damage in HTR-8 cells could potentially translate to a greater need for mitochondria, thus promoting increased formation of TNTs to siphon mitochondria from different cell types.

ZIKV has been noted to infect multiple placental cell compartments including fetal endothelial cells, cytotrophoblasts, and fetal macrophages. However, ZIKV infection has not been clearly demonstrated in STBs^{17,18,20,106,107}, the trophoblast cell layer contacting the maternal blood that protects the fetus from blood-borne infections and constitutively secrets type III IFN^18,108. Furthermore, the maternal decidual compartment contains placental EVTs expressing the tolerogenic human leukocyte antigen (HLA)-G+ molecules¹⁰⁹ that are important for mediating immune tolerance of the semi-allogeneic fetus and typically produce basal levels of IFN^110,111, yet these cells remain susceptible to ZIKV infection^107,112. Given that cell-to-cell interactions between fetal and maternal cells occur at the anchoring villi, which attach and anchor the placenta to the decidua, the formation of TNTs by ZIKV in EVTs could contribute, at least in part, to its dissemination from the decidua to the placenta. Accordingly, we found that EVT-like HTR-8 trophoblast cells are more responsive to TNT formation upon ZIKV infection and ZIKV-NS1 expression compared to other trophoblast cells (CTBs and STBs/PHTs), while secreting limited levels of IFN. The non-TNT forming ZIKV^ΔTNT exhibit slower growth than wild-type ZIKV in JEG-3 and HTR-8 cells and comparable growth in Vero E6 cells, suggesting TNTs are important for efficient ZIKV spread in TNT-responsive trophoblast cells. In contrast, in TNT-lacking cells such as Vero E6, viral growth remains unaffected, suggesting that transmission may rely on exocytosis, attachment factors, or receptor-mediated entry^113,114. Our data suggest that in HTR-8 cells, a lower lFN response, enhanced TNT formation, and increased cargo transfer between homotypic and heterotypic cells may facilitate more efficient ZIKV spread to less permissive cells.

Our study has some limitations. We did not explore whether ZIKV^ΔTNT lost the ability to antagonize type I and type III IFN signaling. Further investigation is required to clarify the interplay between IRF1, NFKB, type III IFN production, and IFN-β upregulation by the ZIKV^ΔTNT mutant. Further, the mechanism by which ZIKV-mediated TNT formation attenuates the IFN response via RNA-sensing innate immunity remains to be fully elucidated. The connection between NS1, mitochondrial dysfunction, and IFN production also requires deeper examination, as it represents a significant and promising avenue for future research. Another limitation of our study is the lack of robust in vivo models to determine the role of TNTs in facilitating ZIKV vertical transmission and evading placental immune responses. Investigating these processes in relation to mitochondrial metabolism and dynamics in vivo remains an important next step. Nevertheless, recent studies provide promising evidence of TNT formation in human placenta. Volume electron microscopy identified TNTs extending up to 7 μm connecting endothelial cells in human term placenta^115,116. Further, TNT-like structures have been observed in co-cultures of primary human decidual immune cells and trophoblast cells in placental organ-on-chip models¹¹⁷. The functional relevance of TNTs in maternal and fetal cell-to-cell interactions during infection has been further demonstrated by the decidual NK cell immune response to Listeria monocytogenes infection of trophoblast cells¹¹⁸. Further investigation of other viruses known to infect the placenta and induce TNT formation, such as SARS-CoV-2 and HIV, is warranted^{45,49,119,120}. It is intriguing to suppose that TNT formation may serve as a defining feature of placenta-infecting pathogens.

Our study offers a new mechanism for ZIKV to infect placental cells involving NS1-mitochondria interactions. These findings offer vital insights that could be used to develop therapeutic strategies targeted against this stealth transmission mode.

Methods

Cell lines

Human trophoblasts cells BeWo (ATCC, #CCL-98), JEG-3 (ATCC, #HTB-36), HTR-8 (ATCC, #CRL-3271), and PHTs were cultured with Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12, (DMEM/F-12 GIBCO, #11330032) supplemented with 10% fetal bovine serum (FBS) (Gibco, #16140071) and maintained at 37 °C with 5% CO2. THP-1 (ATCC, #TIB-202) was cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco, #A1049101) supplemented with 10% FBS and 0.05 mM 2-Mercaptoethanol (Gibco, #21985023). THP-1 differentiation was performed as previously described¹²¹. PHTs were isolated from term placentas from deidentified uncomplicated pregnancies at Barnes-Jewish Hospital Labor and Delivery Service, St. Louis, MO (IRB approval # 201012734)¹²². The purification of cytotrophoblasts were performed by percoll centrifugation gradient¹²³. PHTs were thawed and cultured in a 6-well plate (Corning, 353046) at 2 ×10⁶ cells/well using cell culture media described previously¹²⁴. Following cell attachment, PHTs were washed with Iscove’s Modified Dulbecco’s Medium (IMDM; Gibco, #12440053) with 10% FBS, 100 U/mL Penicillin-Streptomycin (Gibco, #15070063) 10 μM Y-27632 (Selleckchem, #S1049), 0.05 mM 2-Mercaptoethanol (Gibco, #21985023), and 30 ng/mL mouse EGF Recombinant Protein (Gibco, #PMG8041). Cells were maintained in IMDM media for three days, subcultured at a 1:2 split ratio, and maintained for 5 days until virus infection in IMDM medium without Y-27632. HEK-293T (ATCC, #CRL-1573), Vero E6 (ATCC, #CRL-1586), A549 (ATCC, #CCL-185), Huh 7.5 (a kind gift from Dr. Charles M. Rice, Rockefeller University), BHK-15 (a kind gift from Dr. Richard J. Kuhn, Purdue University), U-87 MG (ATCC, #HTB-14), and SH-SY5Y (ATCC, #CRL-2266) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, 12800-082) supplemented with 10% FBS, non-essential amino acids (NEAA, HyClone, #SH30238.01), and Penicillin-Streptomycin (PS, Corning, #30-002-CI) and maintained at 37 °C with 5% CO₂. C636 cells were maintained in Minimum Essential Medium (MEM, GIBCO, #41500-018) supplemented with 10% FBS and Penicillin-Streptomycin (PS, Corning, #30-002-CI) and maintained at 30 °C with 5% CO₂.

ZIKV strains, expression plasmids, and cDNAs

The prototypical African ZIKV MR-766 Uganda strain, and PRVABC-59 were obtained from BEI resources. The cDNA clone derived from the 1947 Uganda MR-766 ZIKV genome placed at the transcriptional initiation site of the cytomegalovirus (CMV) promoter⁵³ was modified to replace Venus tag with mCherry for this study. Plasmids expressing NS1 protein from ZIKV MR-766 (pNS1-ZIKV) was generated by RT-PCR using OneTaq® One-Step RT-PCR Kit (NEB# E5315S) and cloning into pCDNA3.1 or Ligation Independent Cloning into pcDNA3 mCherry LIC cloning vector (Addgene, #30125). Plasmids expressing Dengue Virus-1 (pNS1-DENV1), West Nile Virus NY99 strain (pNS1-WNV), Powassan virus lineage II (Deer Tick Virus) (pNS1-DTV) were generated by PCR from cDNA clones available in the laboratory and were kind gifts from Dr Richard Kuhn and Dr. Aron Brault and cloning into pCDNA3.1 or Ligation Independent Cloning into pcDNA3 mCherry LIC cloning vector (Addgene, #30125). NS1 protein from Japanese Encephalitis Virus NSW strain (pNS1-JEV), Dengue Virus-1 (pNS1-DENV2, GenBank: OK469344.1), Dengue Virus-3 (pNS1-DENV3, GenBank: DQ675533.1), Dengue Virus-4 (pNS1-DENV4, GenBank: M14931.2), and chimeric ZIKV NS1 with DENV4 NTD (pNS1-ZIKV-DENV4), DENV2 NS1 with ZIKV NTD (pNS1-DENV2-ZIKV) genes were synthesized (Twist Bioscience, CA) and cloned into pCDNA3 mCherry LIC cloning vector. Amino acid substitutions into ZIKV cDNA clone to generate ZIKV (∆TNT), generation of ZIKV replicon, and His-tagged expression plasmids were constructed by site-directed mutagenesis using Phusion DNA polymerase (NEB, #E0553S), followed by DpnI digestion and transformation into NEB Stable Competent E. coli (NEB# C3040H). For mutagenesis of cDNA clone and for creating replicon plasmid construct, we used a ZIKV cDNA clone expressing mEmerald fluorescent protein. Plasmids were obtained from overnight cultures of E. coli colonies grown in Luria Bertani medium using the Qiagen miniprep kit (Qiagen, #27104) or Qiagen midiprep kit (Qiagen, #12143), and sequences of the resulting clones were confirmed via Sanger sequencing at the Sequencing Core Facility at The Pennsylvania State University. DNAs were quantified using NanoDrop™ One (ThermoFisher Scientific, Waltham, MA, USA), and aliquots stored at -20 °C.

To generate mutant ZIKV, cDNAs were transfected into HEK 293 T cells using Lipofectamine™ 3000 (Invitrogen, #L3000001). After 12 hrs, the media were replaced and incubated at 37 °C in the presence of 5% CO₂. Cell culture supernatants were collected after 4 days of growth, filtered with a 0.45 μm mixed cellulose membrane (MCE) filter, added HEPES at a final concentration of 10 mM, and stored at −80 °C. For expression of individual ZIKV proteins and orthoflavivirus NS1 proteins, 1 μg plasmids were transfected into cell lines of interest using Lipofectamine™ 3000. All experiments were performed under biosafety level 2 (BSL2) conditions.

ZIKA virus titration and propagation

ZIKV was propagated in African green monkey kidney (Vero E6) cells. Confluent cells were infected with ZIKV for five days, the supernatant containing viruses was harvested, and aliquots were stored at -80 °C. Virus titers for infection assays were determined by plaque assay of serial dilutions on Vero E6 monolayers¹¹. Plaque assay was also used for determining virus growth and replication and expressed as PFU/mL. To assess the kinetics of virus growth, fluorescence dilution assays were performed¹²⁵. In brief, monolayers of Vero E6 cells grown to confluency in 96-well plates (Greiner bio-one, #655180) were infected with supernatants from JEG-3, HTR-8, and Vero E6 infected with wild-type ZIKV and mutant ZIKV^∆TNT expressing mEmerald reporter. The culture supernatants were serially diluted in phosphate buffered saline (PBS) supplemented with 1% FBS and 1 mM each of CaCl2 and MgCl2. Plates were incubated at 37 °C in the presence of 5% CO2 for 3 days, and fluorescent cell clusters were counted under a fluorescent microscope and expressed as focus-forming units (FFU)/mL.

Affinity purification-mass spectrometry

The affinity purification-mass spectrometry method was employed to purify His-tagged NS1 proteins using Ni-NTA resin, as previously reported^64,65. We generated two plasmids, one expressing C-terminal Octa-histidine-tagged wild type NS1 and the mutant pNS1-ZIKV^ΔTNT by site-directed mutagenesis. Plasmids were purified using a Qiagen Midiprep kit, sanger sequenced and used to transfect JEG-3 cells grown in 150 mm dishes using Lipofectamine™ 3000, n = 3 all groups. At 48 hpt, cells were washed and harvested in 1 ml PBS supplemented with Protease Inhibitor Cocktail (Millipore Sigma, #P8340). The cells were lysed by sonication using a microtip attached to a sonicator (Branson), and membrane fractions were purified by ultracentrifugation at 100,000 x g for 90 minutes using a TLA 120.2 rotor and Optima TLX centrifuge (Beckman Coulter). The membrane pelleted obtained after centrifugation was re-suspended in 1 ml PBS supplemented with Protease Inhibitor Cocktail and 1% Fos-choline-12 and incubated at 4 °C with gentle rocking for 2 h. The extracted proteins were separated from insoluble fraction by a second round of ultracentrifugation and allowed to bind to NiNTA resin for 30 min at 4 °C with gentle rocking. The resin was washed with 10 column volumes of PBS with 0.01% Foscholine-12 (Anatrace, #F308) followed by 2 column volumes of PBS buffer and subjected to mass spectrometry analysis at the Indiana University Proteomics Core, Indianapolis, USA. Beads were briefly resuspended in 8 M Urea, 100 mM Tris pH 8.5. Cysteines were then reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and alkylated with 10 mM chloroacetamide (CAM). Samples were diluted to less than 2 M Urea with 50 mM Tris pH 8.5 and digested overnight at 37 °C with 0.5 μg Mass Spectrometry Grade Trypsin/Lys-C Mix (Promega, #V5072). Samples were filtered and acidified with formic acid (FA) before LC/MS/MS.

Nano-LC-MS/MS analysis

Nano-LC-MS/MS analyses were performed on a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer coupled to an UltiMate 3000 UHPLC RSLCnano System (Thermo Fisher Scientific, Hanna-Bremen, Germany). Approximately 10 μL from each sample was loaded and concentrated using an Acclaim^TM PepMap^TM 100 trap column (Thermo Fisher Scientific, #164535) at 3 µL/min for 5 mins in 100% Buffer A (Water + 0.1% FA). Chromatographic separation was done on an Easy-Spray PepMap column (Thermo Fisher Scientific, #ES901, ID 75 µm, 15 cm length, 3 µm particles with 100 Å pore sizes). The LC gradient consisted of holding at 3% Buffer B (Acetonitrile + 0.1% FA) for 5 minutes, followed by a gradient from 3-35% Buffer B over 75 minutes, followed by an increase to 95% Buffer B for 2 minutes, a decrease to 3% Buffer B for 2 minutes and hold at 3% Buffer B for 2 minutes.

The mass spectrometer was operated in positive mode, with a spray voltage of 1.8 kV and ion transfer capillary temperature of 250 °C. Data-dependent acquisition with the top 15 most intense ions for MS/MS. Full MS scan parameters were, Resolution 70k, AGC target 3E6, m/z range 200-2000; MS2 parameters were: Resolution 17.5k; AGC target 1E5; Maximum IT 50 ms; Isolation window 4.0 m/z; Fixed first mass 100 m/z; NCE 30.0.

Mass spectrometry data analysis

The resulting RAW files from mass spectrometry experiments were analyzed using Proteome Discover™ 2.5 (Thermo Fisher Scientific). The MS/MS spectra were searched against a database containing reviewed Homo sapiens proteins (downloaded from the UniProt on 10/04/2019) plus common contaminants. SEQUEST HT search engine was used with trypsin as the proteolytic enzyme, including two allowed missed cleavages, precursor mass tolerance of 10 ppm; and a fragment mass tolerance of 0.2 Da. Static modification of carbamidomethylation on cysteine residues was included, as well as dynamic or variable modifications of oxidation on methionine, peptide N-terminal methionine loss, acetylation, and methionine loss+N-terminal acetylation. Fixed PSM Validator was used as an FDR filter, and results were loaded into Scaffold^TM 4 (Proteome Software) to visualize data and calculate Fisher’s Exact test p-values with Benjamini-Hochburg correction. Relative quantitation of proteins comparing NS1 binding experiments used either Scaffold quantitation functions or Normalized Spectral Abundance Factor calculations and accounted for related isoform identifications^66,126. The STRING application (Version 2.1.0)¹²⁷ within Cytoscape (version 3.9.1)¹²⁸ was used for visualization of functional enrichment based on protein interaction networks from observed proteins. Subcellular localization was determined from the UniProt database (Release 2023_3).

Immunofluorescence assay

Cells were grown on glass coverslips in 24 well plates and were infected with ZIKV at an MOI of 0.1. At 36 hrs post-infection, cells were fixed with 3.7% paraformaldehyde in PBS for 15 minutes and further permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature. Cells were blocked with 10 mg/ml bovine serum albumin (BSA, Sigma-Aldrich, #A7906) in PBS overnight at 4 °C. Subsequently, the blocking buffer was removed, and cells were treated with primary antibodies (1:50 dilution) against envelope protein (4G2, a kind gift from Theodore C. Pearson) and capsid protein (GeneTex, #GTX134186) followed by treatment with fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothiocyanate (TRITC). ZIKV NS1 protein was stained with anti-NS1 (GeneTex, #GTX133323, 1:1000 dilution) followed by Alexa Fluor 594 dye-conjugated secondary antibodies (1:1000 dilution) for 2 hrs. Nuclei were stained using Hoechst-33342 (Invitrogen, #H3570) according to the manufacturer’s instructions. Cells were washed 3 times with phosphate-buffered saline (PBS), and coverslips were mounted onto microscope slides with FluorSave Reagent (Calbiochem, #3457) and confocal images were acquired using a Nikon A1R-MP confocal microscope fitted with a 60x oil objective lens with 1.4 numerical aperture (NA) and processed using the NIS Elements software (Nikon). Brightness and contrast were adjusted using look up tables (LUTs).

For imaging of dsRNA and microtubule, trophoblast cells were grown in 6 well glass bottom plates (Cellvis, #P06-1.5H-N) to a sub-confluency. After overnight culture, cells were infected/transfected. At 24 hpi, cells were fixed with cold 3.5% paraformaldehyde (PFA) (ThermoFisher Scientific, #47392) and 0.5% glutaraldehyde (Polysciences, #BLI1909) in PBS for 15 minutes and permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature. Samples were blocked with 10 mg/ml bovine serum albumin in PBS for 30 minutes at room temperature and treated with J2 monoclonal antibody against dsRNA (Scions, #10010500) or alpha/beta-tubulin (Cell Signaling Technology, #2148) diluted 1:1000 in PBS containing 1% BSA overnight at 4 C. Samples were then washed three times in PBS and incubated with goat anti-mouse Alexa fluor 488 (Invitrogen, #A-11001) for 1 hour at room temperature. Samples were subsequently stained with Hoechst 33342 (Invitrogen, #H3570) diluted 1:10,000 and 500 nM SiR700-Actin Kit (Cytoskeleton; #CY-SC013). Samples were mounted using Aqua-Poly/Mount (Polyscience, #18606-100) and imaged using Nikon A1R-MP.

Live-cell imaging

For screening TNT formation, cells were grown in 4-well chamber slides (IBIDI, #80427) and transfected with expression plasmids, or infected with ZIKV and ZIKV TNT- and incubated for 48 hrs. For live imaging of mitochondria transfer via TNTs, cells expressing mito-GFP mitochondria marker (Addgene, #49151) were used. Live imaging was performed in a Nikon A1R-MP confocal microscope, using a heated 60× oil immersion objective (1.4 NA) in a live imaging chamber (Tokai Hit, Fujinomiya, Shizuoka Prefecture, Japan) supplied with 5% CO2 at 37 °C. Images and videos were acquired using NIS-Elements software.

Quantification of mitochondria transfer

Transfer of mitochondria transfer via TNTs was quantified by flow cytometry. HTR-8 or JEG-3 cells were seeded in a 6-well plate at a density of 500,000 cells/well (Corning, #353046). Donor cells were stained 4 hrs before co-culture with 150 nM MitoTracker Green FM (Invitrogen, #M7514) and 8 μM CellTrace Violet (Invitrogen, #C34557). Cells were washed twice with complete medium after staining and again before co-culture to remove any potential unbound dye. HTR-8 acceptor cells were transfected with 1 μg of pNS1 ZIKV and pNS1-ZIKV^∆TNT plasmids using Lipofectamine™ 3000. At 4 hrs of post-transfection (hpt), 250,000 transfected and untransfected cells were harvested and co-cultured at a 1:1 ratio with or without Boyden chambers (Falcon, #353090). After 24 hrs post-co-culture, cells were harvested, stained with viability dye (Invitrogen, #L10119), and analyzed in the BD LSRFortessa (Becton Dickinson). For THP-1 co-culture, 250,000 cells/well (Thermo Scientific, #174901) were seeded and primed with 150 nM Phorbol 12-myristate 13-acetate (PMA, Sigma, #P8139) for 24 hrs and maintained in culture for three days until fully adherent and differentiated before co-culture with transfected HTR-8. Positive and negative gates were set based on FMO controls. Cells were gated for singlet, followed by live cells (Live/Dead stain) and CellTrace (Supplementary Fig. 4). The resulting cells were evaluated for NS1-mCherry and Mitotracker expression—total events = 30,000 cells. Mitochondria MFI ratio (%) = 100*((NS1 + /Mitotracker + - NS1-/Mitotracker + ) / NS1 + /Mitotracker +).

Quantification of mitochondria accumulation

Mitochondria accumulation was quantified by flow cytometry. JEG-3 was seeded at 250,000 cells/well and cultured overnight. On the next day, uninfected cells were stained 4 hrs before co-culture with 150 nM MitoTracker Green FM (Invitrogen, #M7514) and 8 μM CellTrace Violet (Invitrogen, #C34557). JEG-3 was either inoculated with ZIKV-mCherry (MOI = 0.1) or mock infected for 2 hrs and immediately co-cultured with uninfected cells at 1:1 ratio for 24 hrs. For mitochondria accumulation in transfected cells, JEG-3 were either transfected with 1 μg of pNS1-ZIKV or no plasmid using Lipofectamine™ 3000 Transfection Reagent (Invitrogen, #L3000001). At 4 hpt, transfected cells were co-cultured with untransfected cells at 1:1 ratio for 24 hrs. At 24 hrs post-co-culture, cells were harvested, stained with viability dye (Invitrogen, #L10119), and analyzed on BD LSRFortessa (Becton Dickinson). Cells were gated for singlet, followed by live (Live/Dead stain) and CellTrace (CellTrace Violet). The resulting cells were evaluated for median fluorescence intensity of MitoTracker Green to quantify accumulation of mitochondria in infected/ transfected cells. Total events = 30,000 live cells.

Inhibition of mitochondrial respiration and transport

ZIKV-infected trophoblast cells were treated with either Rotenone (Sigma, #R8875), an inhibitor of mitochondrial respiratory chain complex I, or Miro1 Reducer (Tocris Bioscience, #7091), a compound that promotes the degradation of RHOT1/Miro1 (PMID: 31564441). Dosage of the drugs were determined using lactate dehydrogenase cytotoxicity assay (described below).

Lactate dehydrogenase (LDH) cytotoxicity assay

LDH release was measured using CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, #G1780) according to the manufacturer’s instructions. Briefly, cells were seeded in a 96-well plate at density of 1 × 10⁴ cells/well. After overnight culture, cells were treated with Rotenone ranging from 0.001 to 100 μM or Miro1 Reducer ranging from 10 to 40 μM for 24 hrs. Cell culture media were collected and used for measuring LDH release. LDH levels were determined by recording absorbance at 490 nm using BioTek Epoch Microplate Spectrophotometer and the percent cytotoxicity was calculated per manufacturer’s instructions: Percent cytotoxicity = 100 × (Experimental LDH Release/ Maximum LDH Release).

Interferon level analysis

JEG-3 and HTR-8 cells were infected with ZIKV including MR-766, PRVABC-59, and ZIKV^ΔTNT (MOI = 0.1) for 48 hrs. LEGENDplex™ Human Type 1/2/3 Interferon Panel (5-plex) (BioLegend, #740350) was used to measure IFN-α2, IFN-β, IFN-γ, IFN-λ1, and IFN-λ2/3 levels simultaneously on the infected cells’ supernatant according to the manufacturer’s instructions. Bead-bound cytokines were measured using High Throughput Sampler (HTS) on BD LSR III (Becton Dickinson) and concentrations were calculated using the LEGENDplex™ Data Analysis Software (BioLegend). For mitochondrial respiration or transport inhibition, infected cells were treated with 0.01 µM Rotenone or 40 µM Miro1 Reducer, respectively. The dosages were determined using the drug concentration that reduces ZIKV replication by 1 log and does not cause cell cytotoxicity. Determination of IFN-λ2/3 levels in mouse plasma was performed by ELISA (RnD systems, #DY1789B-05) according to the manufacturer’s instructions.

Gene expression analyses

JEG-3 and HTR-8/SVneo (HTR-8) cells (0.4×10⁶ cells) were seeded a day prior to infection in a 6-well plate and transfected with 1ug of pNS1-ZIKV and pNS1^ΔTNT using Lipofectamine^TM 3000 according to the manufacturer instructions. At 24 hpt or 48 hpi cells were harvested in Trizol^TM (Invitrogen, #15596026) for RNA extraction. RNA was quantified using NanoDrop™ One (ThermoFisher Scientific, Waltham, MA, USA) and cDNA was synthesized using iScript™ gDNA Clear cDNA Synthesis Kit (Bio-Rad, #1725035) according to the manufacturer instructions. Quantitative PCR was performed using SsoAdvanced Universal SYBR green supermix (Bio-Rad, #1725274) on a QuantStudio 3 real-time PCR instrument (Applied Biosystems, Waltham, MA, USA). Relative gene expression differences between groups were assessed by comparing delta Ct values or using the 2^-ΔΔCT method. The primer list is available in Supplementary Table 1.

Statistical analysis and reproducibility

All analyses were carried out using GraphPad Prism 9. No statistical methods were used to predetermine sample sizes. The normal distribution of continuous variables was assessed by the Shapiro-Wilk test and the statistical significance of pairwise comparisons were assessed by the Student’s t-test or the Mann-Whitney test when appropriate. Comparisons of three of more groups were performed by the one-way ANOVA followed by the Tukey’s test to determine differences between groups. Where data showed a nonparametric distribution, a Kruskal–Wallis test was used followed by post-hoc Dunn’s test. Descriptive statistics, statistical tests, and post-hoc tests for multiple comparisons are reported in each figure legend and the accompanying source data. The number of independent experiments is described in the figure legend. For comparison of gene expression, we performed outlier analyses to identify data that deviates significantly from the rest of data at P < 0.05 using Grubb’s test. For comparison of mitochondria transfer, five independent experiments were used. For quantification of TNTs, five randomly chosen microscope fields containing ZIKV-NS1 expressing cells from three biologically independent replicates were used for statistical analysis. For all comparisons, a two-sided P value < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The AP-MS data generated in this study have been deposited in the MassIVE (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) under the accession code MSV000093683 and ProteomeXchange (https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD047909) databases. All data supporting the findings of this study are available within the paper, supplementary material, and Source data files. All microscopy image files, and biological materials generated are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

This work was supported in part by NIH/NIAID R01AI176505 to J.J. and I.U.M., NIH/NICHD grant, R01HD091218 to I.U.M., and Pennsylvania State University startup funds to J.J. This project was supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the CPRIT Core Facility Support Award (CPRIT-RP180672), the NIH (CA125123 and RR024574). We thank the Indiana University School of Medicine Mass spectrometry core for their services. We thank Drs. Jason Mills and Robert Lawrence for their valuable comments and editing.

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Authors and Affiliations

Department of Medicine, Section of Infectious Diseases, Baylor College of Medicine, Houston, TX, 77030, USA
Rafael T. Michita, Long B. Tran, Steven J. Bark, Deepak Kumar & Indira U. Mysorekar
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, PA, 16802, USA
Shay A. Toner, Joyce Jose & Anoop Narayanan
The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania, PA, 16802, USA
Joyce Jose
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, 77030, USA
Indira U. Mysorekar
Huffington Centre on Aging, Baylor College of Medicine, Houston, TX, 77030, USA
Indira U. Mysorekar

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Rafael T. Michita
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Long B. Tran
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Steven J. Bark
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Deepak Kumar
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Shay A. Toner
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Joyce Jose
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Indira U. Mysorekar
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Anoop Narayanan
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Contributions

R.T.M., A.N., and I.U.M. conceived the experimental plan; R.T.M., A.N., L.B.T., performed the majority of experiments and were assisted by D.K., S.A.T. S.J.B. analyzed the proteomics data. R.T.M., A.N., L.B.T., J.J. and I.U.M. wrote the manuscript, and all authors approved the final draft.

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Correspondence to Indira U. Mysorekar or Anoop Narayanan.

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Competing interests

I.U.M. serves on the scientific advisory board of Seed Health. The remaining authors declare no competing interests.

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Nature Communications thanks Loyda Meléndez, R. Michael Roberts, Christel Vérollet and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Michita, R.T., Tran, L.B., Bark, S.J. et al. Zika virus NS1 drives tunneling nanotube formation for mitochondrial transfer and stealth transmission in trophoblasts. Nat Commun 16, 1803 (2025). https://doi.org/10.1038/s41467-025-56927-2

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Received: 27 November 2023
Accepted: 05 February 2025
Published: 20 February 2025
Version of record: 20 February 2025
DOI: https://doi.org/10.1038/s41467-025-56927-2

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