O-GlcNAcylation of AMFR stabilizes TSPAN4 to regulate migrasome formation for viral release

Domain: chemistry
Published: 2026-01-07
Editorial outcome: major_revision

Abstract
Migrasomes are migration-dependent organelles, serving as delivery packets to mediate release of cytoplasmic contents. Tetraspanin 4 (TSPAN4) acts as a marker for migrasomes and is essential for their formation. However, the regulatory mechanism(s) underlying TSPAN4-mediated migrasome biogenesis and its physiological functions remain to be elucidated. Here, we identified AMFR, an ER-resident E3 ligase, regulates migrasome formation through catalyzing the K48-linked polyubiquitination of TSPAN4 for degradation. Further, O-GlcNAcylation of AMFR by OGT at threonine 643 disrupts AMFR-TSPAN4 interactions, thereby stabilizing TSPAN4 and promoting migrasome formation. Additionally, viruses dynamically regulate migrasome formation by modulating AMFR O-GlcNAcylation and TSPAN4 ubiquitination. During the early stages of VSV or HSV-1 infection, viruses enhance migrasome formation and exploit these structures to spread among neighboring cells, whereas abolish migrasome formation during the late stages of infection. Our findings reveal a negatively regulatory mechanism governing migrasome biogenesis, and highlight how VSV and HSV-1 manipulate this process to facilitate their release.

Introduction
Migrasomes are newly discovered organelles in migrating cells that mediating release of cytoplasmic contents, including mRNA, proteins, and mitochondria. When cells migrate on extracellular substrates, retraction fibers (RFs) are pulled out of the plasma membrane and then migrasomes subsequently form on the RFs, and are left behind when cells migrate away and the RFs break. The discarded migrasomes can be taken up by surrounding cells, facilitating cell-to-cell communication. Recent studies have demonstrated that migrasomes play important roles in organ morphogenesis and angiogenesis during embryonic development. Additionally, migrasomes also contribute to various pathological conditions, including ischemic brain damage, proliferative vitreoretinopathy, cerebral amyloid angiopathy. As one of the extracellular vesicles, migrasomes are found to play a role in virus replication. But the molecular mechanisms remain largely unknown.

Tetraspanin 4 (TSPAN4) was previously found to be abundant in the migrasome membrane and act as a marker for migrasomes. TSPAN4 belongs to the tetraspanin family, which has 33 members and all contain four transmembrane domains and are found in every cell type. Tetraspanin family can separate in the membrane plane into tetraspanin-enriched microdomains. Using live-cell system in vivo and reconstituted membrane system in vitro, recent study proposes that TSPAN4, together with cholesterol, are necessary and sufficient for migrasome formation. During migrasome formation, TSPAN4 was dynamically recruited to migrasomes and form discrete fast-moving puncta that concentrate on the migrasome membrane. Notably, overexpression of TSPAN4 can induce migrasome formation, while depletion of TSPAN4 impairs migrasome formation in MGC-803 cells and NRK cells. It has been known that TSPAN4 remains stable on the migrasome membrane. However, it remains to be explored whether there is a mechanism targeted TSPAN4 for degradation in the cytoplasm, which could negatively regulate migrasome formation. Members of the tetraspanin family are known to play important roles in viral entry, as TSPAN9 has been implicated in alphavirus entry, TSPAN8 is essential for coronavirus entry, TSPAN27 is involved in HTLV-1 entry, and CD81 serves as the receptor for HCV. However, the role of TSPAN4 in viral release has not been explored.

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation, also known as O-GlcNAcylation, is a dynamic post-translational modification that involves the attachment of single O-GlcNAc moieties to serine (Ser) and threonine (Thr) residues of various proteins to respond promptly to internal and external cues through direct and dynamic regulation of protein functions. O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) function together and control the dynamic cycling of this modification in a nutrient or stress-responsive manner. A previous study indicated that TSPAN4-GFP appears as a smeared double band on Western blots (WB), which was suggested to be a result of glycosylation, although this claim was not supported by further experimental investigation.

In this study, we report that O-GlcNAcylation of AMFR-mediated by OGT serves as a regulatory mechanism for AMFR-mediated TSPAN4 degradation and migrasome formation. Additionally, migrasomes can facilitate viral release and spread among neighboring cells. By leveraging the AMFR-TSPAN4 axis, Vesicular stomatitis virus (VSV) or HSV-1 infection initially promotes migrasome formation to facilitate viral release during the early stage of infection, when only a limited number of progeny viruses are produced, and subsequently suppresses migrasome formation during the late stage of infection, when a substantial quantity of viral particles is generated.

Results
Viruses use migrasomes for release and spread
Given that the primary function of migrasomes is their ability to serve as packets of delivery, we proposed that viruses may utilize migrasomes for their release and spread. We first overexpressed the mammalian tetraspanins TSPAN1, TSPAN2, or TSPAN4, which have been identified for their ability to induce migrasome formation. Overexpression of TSPAN1, TSPAN2, or TSPAN4 enhanced the replication of RNA virus VSV, while the expression of TSPAN8 or TSPAN19 neither promoted migrasome formation nor regulated virus replication.

Since TSPAN4 is one of the most effective tetraspanins for both migrasome induction and virus replication, we chose it for in-depth study. To study the role of TSPAN4 in viral release, we first confirmed that overexpression or knockdown of TSPAN4 had minor effect on IFNβ production. When infected with the RNA virus VSV or the DNA virus Herpes simplex virus 1 (HSV-1) at a low multiplicity of infection (MOI) of 0.01, extracellular viral production (virions released into the supernatant) was significantly enhanced in TSPAN4-overexpressing cells, whereas it was reduced in Tspan4 knockout cells. Through attachment and internalization assays, we further demonstrated that neither TSPAN4 overexpression nor Tspan4 knockout affected the entry of VSV or HSV-1. Additionally, we disrupted migrasome formation using previously reported strategies, such as the knockdown of Cert, Ceramide synthases 5 (CerS5), Sgms2, PIP5K1A, or Rab35, and the data showed that the impairment of migrasome formation led to decreased viral release. Thus, these data support the conclusion that migrasomes facilitate viral release.

Next, we aim to investigate how migrasomes function in viral release. We first purified migrasomes from VSV-infected cells and found that the viral protein M was detectable in the migrasome fraction. We further used these purified migrasomes to infect mock cells, and found that these migrasomes were infectious. Notably, the VSV-GFP signal was observed inside the migrasomes in VSV-infected cells. Similar results were obtained by detecting the HSV-1 virion signal using a viral N antibody. Furthermore, using transmission electron microscopy (TEM), we directly visualized hexagonal virus particles within the migrasomes from VSV-infected cells. These results indicate that viral particles were enriched in migrasomes.

Interestingly, using live imaging, we observed that when a non-infected cell migrated, it can uptake the virions contained-migrasomes left by neighboring VSV-infected cell, subsequently leading to infection. VSV-G protein is known to mediate membrane fusion during the infection process. We subsequently investigated whether the VSV-G protein could translocate to the migrasome membrane to facilitate the membrane fusion between virus-containing migrasomes and cellular membrane, thereby enabling viral transmission. Using proteinase K protection assay and confocal microscopy, we confirmed that VSV-G protein was partly enriched on the out membrane of migrasomes. We subsequently treated the purified migrasomes isolated from VSV-infected cells with proteinase K to degrade the migrasome outer membrane-localized VSV-G. These treated migrasomes were then incubated with control cells. Compared to the PBS-treated control, migrasomes treated with proteinase K alone exhibited a slightly reduction in infectivity, whereas co-treatment with proteinase K and Triton ×-100 abolished migrasome-mediated infectivity, indicating that the primary mechanism of migrasome-mediated viral transmission is mediated by VSV virions contained inside the migrasomes, whereas the VSV-G protein localized on the outer membrane of migrasomes contributes only minimally to this process. Collectively, these data indicate an interesting way for viral spread mediated by migrasomes.

TSPAN4 is degraded via ubiquitin-proteasome pathway
Strikingly, we observed a cumulative decrease in migrasome formation as the infection progressed. TSPAN4 is known to be critical for migrasome formation, as its gain and loss lead to a corresponding increase and decrease in migrasome formation. We then carefully examined the protein levels of TSPAN4 in virus-infected cells. The amount of TSPAN4 initially increased and then dramatically decreased at increasing intervals of time following VSV or HSV-1 infection. Hence, virus infection dynamically regulates the protein levels of TSPAN4.

To investigate how viruses regulate TSPAN4 expression, we first treated TSPAN4-Flag stably expressing cells with Cycloheximide (CHX) to inhibit protein synthesis, and found that TSPAN4-Flag was remarkably degraded following CHX treatment, even after just 30 min. To better understand the mechanism underlying TSPAN4 degradation, we inhibited the ubiquitin-proteasome pathway using MG132 and the autophagosome-lysosome pathway using Chloroquine (CQ). MG132 treatment significantly while CQ treatment only slightly blocked TSPAN4-Flag degradation, indicating that TSPAN4 can be degraded via the ubiquitin-proteasome pathway. Additionally, virus infection-induced TSPAN4 degradation was also blocked by MG132 treatment. We further found that TSPAN4 undergoes K48-linked polyubiquitination, and the mutant, which lacks amino acids 106–238, completely lost its ubiquitination capability. Six lysine residues are located within the 106–238 region of TSPAN4. Our observations indicated that all six lysine residues are essential for TSPAN4 ubiquitination, as the point mutant 6K/R (abbreviated as TSPAN46KR) completely abolished ubiquitin modification. TSPAN46KR exhibited greater stability than the wild-type in the CHX assay. Together, these data indicate that TSPAN4 is subject to ubiquitination and subsequent degradable.

TSPAN4 is degraded on ER by AMFR-catalyzed K48-linked polyubiquitination
To identify the E3 ligase responsible for the ubiquitination of TSPAN4, we conducted immunoprecipitation-mass spectrometry (IP/MS) using TSPAN4-Flag as the bait. Several candidates related to ubiquitin were identified. We then performed a small-scale RNA interference (RNAi) screening targeting these candidates, and found that knockdown of autocrine motility factor receptor (AMFR, also known as GP78 or RNF45) significantly inhibited the degradation of TSPAN4-Flag in the CHX assay. AMFR is an ER transmembrane protein that functions as a ubiquitin E3 ligase in ER-associated protein degradation. Using a co-immunoprecipitation (co-IP) assay, we confirmed that TSPAN4 interacts with AMFR. The direct interaction between TSPAN4 and AMFR was further validated through an in vitro GST pull-down assay. Colocalization of TSPAN4 and AMFR was observed in cells treated with or without fibronectin. Overexpression of wild-type AMFR, but not the catalytically inactive mutant AMFR C341/356/378S (abbreviated as AMFR3CS), increased the ubiquitination level of TSPAN4. The kinetics of AMFR expression correlated with the increase in wild-type TSPAN4 degradation, but not with TSPAN46KR. Overexpression of AMFR3CS failed to induce TSPAN4 degradation. Next, we generated Amfr knockout (KO) cells and found that, compared to wild-type cells, TSPAN4 was more stable in Amfr KO cells in the CHX assay. Furthermore, we conducted the rescue experiments and found that wild-type AMFR, but not AMFR3CS, restored the degradation of TSPAN4 in Amfr KO cells. Thus, AMFR is the E3 ligase responsible for TSPAN4 degradation.

Given that AMFR is an ER-resident E3 ligase, and TSPAN4 was partially localized on the ER. We first quantified the proportion of TSPAN4 localized in the ER relative to its total cellular pool, and found that 32.12 ± 2.31% of total TSPAN4 was enrich in the ER. To further explore the mechanism by which AMFR degrades TSPAN4, we analyzed the TSPAN4 stability in the ER fraction and non-ER fraction from cells overexpressing either AMFR or AMFR3CS. Only the ER-localized TSPAN4 was degraded upon AMFR overexpression, while the non-ER TSPAN4 remained stable. Furthermore, AMFR exhibited no co-localization with cell membrane-localized TSPAN4. Thus, these findings suggest that TSPAN4 can be degraded in the ER through AMFR-mediated ubiquitin-proteasome pathways.

Interestingly, we noticed that TSPAN4 degradation induced by AMFR can be significantly blocked by MG132 treatment, and also partly blocked by CQ treatment, indicating that beside ubiquitin-proteasome, autophagosome-lysosome may function as alternative pathways to AMFR-mediated degradation contribute to TSPAN4 turnover.

AMFR regulates migrasome formation by modulating TSPAN4 stability
Next, we investigated whether AMFR can modulate migrasome formation by inducing the degradation of ER-localized TSPAN4. We first confirmed that AMFR expression does not affect the speed of cell migration. ITGA5-mCherry was used as the migrasome marker, and confocal microscopy data revealed that overexpression of AMFR-GFP, but not AMFR3CS-GFP or GFP, resulted in a decreased number of migrasomes. We observed that AMFR was not localized on migrasomes, further confirming that AMFR does not mediate the degradation of migrasome-localized TSPAN4. Additionally, we conducted the rescue experiments in Tspan4 KO cells, and found that overexpression of AMFR, but not AMFR3CS-GFP, induced TSPAN4 degradation and abolished the migrasome formation that was rescued by wild-type TSPAN4 overexpression. In contrast, overexpression of either AMFR or AMFR3CS had a minor effect on the TSPAN4 stability and migrasome formation rescued by TSPAN46KR expression. Together, these results suggest that AMFR functions as the E3 ligase that mediates the degradation of ER-localized TSPAN4, thereby preventing the formation of migrasomes.

O-GlcNAcylation regulates TSPAN4 stability and migrasome formation
Next, we investigated the regulatory mechanism by which TSPAN4 is degraded by AMFR. A previous study indicated that TSPAN4 may undergo O-GlcNAcylation. We then aimed to determine whether O-GlcNAcylation regulate migrasome formation. Treatment with the OGA inhibitor Thiamet G (TMG) slightly increased the number of migrasomes, while treatment with the OGT inhibitor OSMI-1 obviously impaired migrasome formation. Consistently, knockdown of O-GlcNAc transferase OGT obviously inhibited migrasome formation. We further conducted rescue experiments in OGT KD cells and found that wild-type OGT, but not the catalytic dead mutant OGTK852M, restored the formation of migrasomes. Thus, O-GlcNAcylation facilitates migrasome formation.

We then investigated whether O-GlcNAcylation regulate the stability of TSPAN4. TMG slightly enhanced, while OSMI-1 treatment decreased the protein level of TSPAN4, suggesting that O-GlcNAcylation may inhibit the degradation of TSPAN4. Overexpression of wild-type OGT, but not the OGTK852M mutant, attenuated TSPAN4 degradation in the CHX assay. Additionally, knockdown of OGT promoted TSPAN4 degradation, which could be blocked by treatment with MG132. Rescue experiments in OGT KD cells demonstrated that expression of wild-type OGT, but not OGTK852M mutant, reversed the degradation of TSPAN4. Thus, OGT stabilizes TSPAN4 dependent on its transferase activity.

We then tested whether O-GlcNAcylation regulate the stability of TSPAN4 through its ubiquitination. Treatment with TMG significantly reduced TSPAN4 ubiquitination, while treatment with OSMI-1 enhanced it. Consistently, we observed that the ubiquitination of TSPAN4 increased upon OTG knockdown, whereas it decreased under OGT overexpression conditions, but not with OGTK852M overexpression. Together, these data indicate that O-GlcNAcylation inhibits the ubiquitination of TSPAN4, thereby enhancing its stability to facilitate migrasome formation.

O-GlcNAcylation of AMFR at T643
Next, we sought to determine the mechanism(s) by which O-GlcNAcylation regulate migrasome formation. Using an anti-O-GlcNAc antibody, we unexpectedly found that AMFR, but not TSPAN4, was O-GlcNAcylated. The level of AMFR O-GlcNAcylation was increased following TMG treatment, while it decreased under OSMI-1 treatment. Through in vivo co-IP and in vitro GST pull-down assays, we confirmed that AMFR directly interacts with the O-GlcNAcylation enzyme OGT. In vitro, Escherichia coli expressed His-tagged AMFR was incubated with recombinant wild-type glutathione S-transferase (GST)-tagged OGT or the catalytic dead mutant GST-OGTK852M, along with UDP-GlcNAc, and subjected to an O-GlcNAcylation assay. We found that wild-type OGT, but not the OGTK852M mutant, catalyzed the O-GlcNAcylation of AMFR. Noticeably, AMFR O-GlcNAcylation was abolished by co-incubation with OGA. In cellular experiments, the amount of O-GlcNAc-modified AMFR was lower in OGT KD cells compared to controls, whereas overexpression of wild-type OGT, but not OGTK852M, promoted AMFR O-GlcNAcylation. Consistently, co-expression of OGA impaired OGT-mediated AMFR O-GlcNAcylation in cells.

We then sought to unambiguously identify the O-GlcNAcylation site(s) on AMFR. Based on predictions for O-GlcNAcylation attachment sites in Yin O Yang, six potential O-GlcNAcylation sites were identified. To comprehensively map these possible sites, we generated point mutations at the indicated residues. Notably, T643A mutation significantly reduced AMFR O-GlcNAcylation, while other mutations had minimal impact. Through mass spectrometry analysis of the synthesized AMFR peptide, we further confirmed that AMFR T643 can be O-GlcNAcylated. Therefore, both mass spectrometry and extensive mutation studies suggested that T643 is the primary O-GlcNAcylation site on AMFR.

We were surprised found that T643 is not evolutionarily conserved. Except mouse and rat, in other species, such as Homo sapiens the last amino acid is Ser. We then verified whether the last amino acid Ser of human AMFR (abbreviated as hAMFR) can also be O-GlcNAcylated. In vivo and in vitro assays suggested that hAMFR S643A showed reduced O-GlcNAcylation, suggesting that S643 of human AMFR is the primary site for O-GlcNAcylation.

O-GlcNAcylation of AMFR at T643 stabilizes TSPAN4 for migrasome formation
Next, we sought to determine whether O-GlcNAcylation of AMFR regulate TSPAN4 degradation and migrasome formation. We first treated cells with the OGT inhibitor OSMI-1 or the OGA inhibitor TMG and examined TSPAN4 degradation and migrasome formation. OSMI-1 treatment resulted in increased AMFR-mediated TSPAN4 degradation and reduced migrasome formation, whereas TMG treatment partially impaired AMFR-mediated TSPAN4 degradation and the defect of migrasome formation. Neither OSMI-1 nor TMG treatment had any effect on AMFR T643A-induced TSPAN4 degradation or on AMFR T643A-inhibited migrasome formation.

Furthermore, we noticed that co-expression of OGT reversed AMFR (but not AMFR T643A)-abolished migrasome formation. Consistently, co-expression of OGT restored TSPAN4 degradation induced by AMFR, but did not affect TSPAN4 degradation induced by AMFR T643A.

Together, these observations demonstrate that O-GlcNAcylation of AMFR at T643 suppresses the degradation of TSPAN4 and facilitates migrasome formation.

O-GlcNAcylation of AMFR disrupts AMFR-TSPAN4 interactions
Next, we sought to determine the mechanism(s) by which O-GlcNAcylation of AMFR inhibit TSPAN4 degradation. We first assayed whether the TSPAN4-AMFR interactions were affected by O-GlcNAcylation. Indeed, TSPAN4-AMFR interactions were suppressed upon TMG treatment. Conversely, OSMI-1 treatment enhanced TSPAN4-AMFR interactions. We further found that knockdown of OGT enhanced AMFR-TSPAN4 interactions, whereas overexpression of wild-type OGT, but not OGTK852M mutant, disrupted AMFR-TSPAN4 interactions. OGT overexpression failed to affect the interactions between AMFR T643A and TSPAN4. Consistently, human TSPAN4-AMFR interactions were also affected by O-GlcNAcylation in cells. Furthermore, His-AMFR recombinantly expressed and purified from Escherichia coli was incubated with OGT and UDP-GlcNAc to elevate the level of O-GlcNAcylation. In vitro pull-down of the differentially O-GlcNAcylated AMFR proteins with recombinant TSPAN4 showed that upregulated O-GlcNAcylation blocked AMFR-TSPAN4 interactions. To further investigate the molecular mechanism by which O-GlcNAcylation disrupts the AMFR-TSPAN4 interaction, we employed AlphaFold to predict the structure of the AMFR-TSPAN4 complex and generated an AMFR mutant (W345A/E634A/H420A/R484A/R428A/F424A/F436A) lacking the putative TSPAN4-binding domain. This AMFR mutant exhibited significantly reduced binding affinity toward TSPAN4, supporting the reliability of the structural prediction. Given that the potential O-GlcNAcylation site is located in proximity to the AMFR-TSPAN4 interaction interface, we hypothesize that O-GlcNAcylation at residue T643 may induce steric hindrance, thereby disrupting the AMFR-TSPAN4 association.

Put together, these results support a mechanism in which OGT-mediated O-GlcNAcylation of AMFR attenuates AMFR-TSPAN4 interactions, and this inhibition subsequently prevents AMFR-mediated ubiquitination and degradation of TSPAN4, thereby facilitating migrasome formation.

AMFR-TSPAN4 axis regulates viral release
Given that AMFR O-GlcNAcylation and TSPAN4 ubiquitination regulate migrasome formation and migrasomes facilitate viral release and spread, we aimed to determine whether the AMFR-TSPAN4 axis modulate viral release via migrasomes. AMFR has been reported to catalyze K27-linked polyubiquitination of STING upon microbial DNA challenge. To exclude the effect of AMFR on viral release through the regulation of STING, we assayed the viral release in Sting KO cells, and found that overexpression of wild-type AMFR, but not AMFR3CS, still inhibited viral release in Sting KO cells. To confirm that AMFR regulates viral release dependent on TSPAN4, we gradually increased TSPAN4 expression and observed that the AMFR expression-induced reduction in viral release was reversed. In Tspan4 KO cells, AMFR overexpression had a minor effect on viral release. To excluded the possibility that AMFR could theoretically suppress viral infection by degrading viral proteins, we co-expressed AMFR with VSV or HSV-1 proteins, and found that AMFR expression cased no effect on the protein level of viral proteins. Additionally, overexpression of TSPAN4 still enhanced the viral release in Amfr KO cells, suggesting that the pro-viral function of TSPAN4 is not dependent on the antiviral effects of AMFR. Thus, AMFR expression inhibits viral release through degrading TSPAN4.

Next, we sought to determine whether viral release is affected by the O-GlcNAcylation of AMFR. The reduction in viral release induced by AMFR expression was partially reversed by TMG treatment or overexpression of wild-type OGT (but not OGTK852M), indicating that the O-GlcNAcylation of AMFR promotes viral release. We further conducted rescue experiments in OGT KD cells and found that expression of wild-type OGT, but not the OGTK852M mutant, reversed the reduction of viral release. Together, these results indicate that OGT-mediated O-GlcNAcylation of AMFR enhances viral release by stabilizing TSPAN4.

Virus infection dynamically regulates AMFR O-GlcNAcylation and TSPAN4 ubiquitination
Given that both the migrasome formation and TSPAN4 stability were dynamically regulated at increasing time intervals following viral infection, we then investigated whether AMFR O-GlcNAcylation, TSPAN4 ubiquitination, and AMFR-TSPAN4 interactions are dynamically affected by viral infection. Based on the protein levels of TSPAN4, we set up the early stage of infection using 6 h post-infection (hpi) for VSV and 12 hpi for HSV-1 when TSPAN4 was upregulated. The late stage of infection using 12 hpi for VSV and 24 hpi for HSV-1, when TSPAN4 was downregulated. We first evaluated the K48-linked polyubiquitination levels of TSPAN4 in both mock and virus-infected cells. Consistent with the protein levels of TSPAN4 following virus infection, we observed that both VSV and HSV-1 infections decreased TSPAN4 ubiquitination at early stage of infection but increased TSPAN4 ubiquitination at late stage of infection. Indeed, virus infections enhanced AMFR-OGT interactions and AMFR O-GlcNAcylation at the early stage, followed by a decrease at the late stage. Additionally, AMFR-TSPAN4 interactions were also downregulated at the early stage of infection and slightly upregulated at the late stage of infection. These results suggest that virus infection dynamically regulates AMFR O-GlcNAcylation and subsequently AMFR-mediated TSPAN4 ubiquitination.

Next, we sought to determine why AMFR O-GlcNAcylation is reduced during the late stages of viral infection. High-glucose conditions are known to promote O-GlcNAcylation. Intracellular glucose levels were measured and showed a significant decrease during the late stages of viral infection, indicating that viral infection reduces intracellular glucose levels, thereby contributing to the reduction in AMFR O-GlcNAcylation. Interestingly, compare to mock cells, there was no significant increase of glucose level during the early stages of infection, indicating that the upregulated AMFR O-GlcNAcylation during the early stages of infection is not caused by increased glucose level, and AMFR may undergo additional post-translational modifications that contribute to its enhanced O-GlcNAcylation.

We then investigated whether glucose regulate migrasome formation and subsequent viral release. TSPAN4-mCherry-NRK cells were cultured in DMEM medium containing varying glucose concentrations. Compared to the 25 mM glucose condition, low glucose (5.5 mM) decreased migrasome formation and viral release, whereas high glucose (50 mM) slightly enhanced these processes. These findings suggest that during the early stages of infection, elevated glucose levels promote AMFR O-GlcNAcylation and subsequent migrasome formation to facilitate viral release, while during the late stages, viral infection lead to diminished intracellular glucose levels, resulting in reduced AMFR O-GlcNAcylation and impaired migrasome formation.

Virus infection regulates migrasome formation via AMFR-TSPAN4 axis
We then determine whether virus infection dynamically regulate migrasome formation by modulating AMFR O-GlcNAcylation and TSPAN4 ubiquitination. Our findings indicate that at the early stage of infection, VSV or HSV-1 failed to enhance the protein levels of TSPAN4 or promote migrasome formation in OGT knockdown cells, suggesting that during the early stage of infection viral infection enhances migrasome formation by upregulating AMFR O-GlcNAcylation.

At the late stage of infection, TSPAN4 exhibited greater stability in Amfr KO cells compared to wild-type cells, and virus infection failed to impair migrasome formation in Amfr KO cells. Wild-type AMFR (but not AMFR3CS) effectively rescued viral infection-induced TSPAN4 degradation and the inhibition of migrasome formation in Amfr KO cells. TSPAN46KR mutant demonstrated increased stability compared to the wild-type during viral infection, and viral infection did not hinder TSPAN46KR-mediated migrasome formation. Notably, overexpression of OGT, but not OGTK852M, counteracted the degradation of TSPAN4 and the inhibition of migrasome formation induced by viral infection.

Together, these data indicated that during the early stage of infection, viruses enhanced AMFR O-GlcNAcylation, which resulted in the reduction of TSPAN4 ubiquitination and the increased migrasome formation to facilitate viral release. Conversely, at the late stage of infection, AMFR O-GlcNAcylation was downregulated, leading to an increase in TSPAN4 ubiquitination. Consequently, the degradation of TSPAN4 in infected cells inhibited migrasome formation.

Methods
Cell culture
HEK293T cells (CRL-1573) and HeLa cells (CCL-2) were obtained from ATCC. BHK21 cells were obtained from Mingzhou Chen (Wuhan University). Vero-E6 cells were obtained from Kun Cai (Hubei Provincial Center for Disease Control and Prevention). L929 TSPAN4-mCherry cells, NRK cells, and L929 ITGA5-GFP cells were obtained from Li Yu (Tsinghua University). TSPAN4 WT and KO HeLa, AMFR WT and KO HeLa, Tspan4 WT and KO NRK, Amfr WT and KO NRK, TSPAN4-mCherry NRK, TSPAN4-GFP NRK, TSPAN46KR-mCherry NRK, ITGA5-mCherry NRK, TSPAN4-Flag NRK, and TSPAN4-Flag L929 cells were constructed in our lab. All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, 11995065) supplemented with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, 12303C) and 1% penicillin-streptomycin (Gibco, 15140163) at 37°C in 5% CO2. All cells were tested for mycoplasma negative.

Antibodies and reagents
Mouse anti-Flag (AE005, western blot (WB) 1:5000), mouse anti-HA (AE008, WB 1:5000), rabbit anti-Myc (AE070, WB 1:10000), mouse anti-GFP (AE012, WB 1:5000), mouse anti-GST (AE001, WB 1:5000), and mouse anti-His (AE003, WB 1:5000) were obtained from ABclonal. Rabbit anti-HA (3724, WB 1:1000), rabbit anti-Rab11 (5589, WB 1:2000), and rabbit anti-Integrin α5 (4705, WB 1:1000) were obtained from Cell Signaling Technology. Mouse anti-Tubulin (E7, WB 1:10000) was obtained from Developmental Studies Hybridoma Bank. Mouse anti-GAPDH (sc-365062, WB 1:5000), mouse anti-HSV-1/2 ICP27 (SC-69807, WB 1:500), mouse anti-VSV-G (SC-365019, WB 1:500, IF 1:200) and mouse anti-TOM20 (SC-17764, WB 1:500) were obtained from Santa Cruz Biotechnology. Mouse anti-VSV-M (EB0011, WB 1:1000) was obtained from Kerafast. Rabbit anti-HSV-1/2 (PA1-7214, IF 1:2000) was obtained from ThermoFisher Scientific. Mouse anti-O-Linked N-Acetylglucosamine (RL2) antibody (ab2739, WB 1:1000), Rabbit anti-PIGK antibody (ab201693, WB 1:2000), Rabbit anti-Calnexin antibody (ab22595, WB 1:800), and Rabbit anti-EOGT antibody (ab190693, WB 1:5000) were obtained from Abcam. Rabbit anti-AMFR (16675-1-AP, WB 1:5000) and Mouse anti-HSP90 (60318-1-Ig, WB 1:10000) were obtained from Proteintech. HRP-conjugated goat anti-mouse IgG (H+L) (AS003, WB 1:10000), HRP-conjugated goat anti-rabbit IgG (H+L) (AS014, WB 1:10000), and HRP-conjugated goat anti-mouse IgG heavy chain (AS064, WB 1:10000) were obtained from ABclonal. Peroxidase-AffiniPure goat anti-mouse IgG light-chain-specific (115-035-174, WB 1:10000) was obtained from Jackson Immunoresearch. Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) (A-11031, IF 1:500) and goat anti-rabbit IgG (H+L) (A-11036, IF 1:500) were purchased from ThermoFisher Scientific.

Plasmid construction
shCert, shCers5, shSgms2, sgTSPAN4, sgRab35, sgPIP5K1A, GFP tagged TSPAN4, and ITGA5 were provided by Li Yu (Tsinghua University). mCherry-TSPAN1, mCherry-TSPAN2, pmCherry-TSPAN8, and mCherry-TSPAN9 were provided by Yuwei Huang (Xian Jiaotong University). OGA-Myc was provided by Lidong Sun (Huazhong University of Science and Technology). GST tagged full length OGT was a gift from Hong Zhang (Chinese Academy of Sciences). VSV-N-Myc and VSV-G-Myc were provided by Mingzhou Chen (Hubei University). HSV-1-UL17-Flag, HSV-1-UL5-Flag, and HSV-1-ICP4-Flag were provided by Yujie Ren (Wuhan Institute of Virology) and Junjie Zhang (Wuhan University). pCDNA4-ICP27-strep was cloned into pCDNA4-strep vector. pCDNA4-VSV-M-HA was cloned into pCDNA4-HA vector. cDNA encoding human and mouse-AMFR and its point mutants were respectively cloned into pCDNA4 vector with a HA, Flag, or Myc tag, or pCOLD-6×His-sumo vector. pLenti-mCherry-Ramp4 was cloned into the mammalian expression vector pLenti-N1-mCherry. pLenti-mTSPAN4-Flag and its mutants were cloned into pLenti-Flag vector. pLenti-hTSPAN4-Flag was cloned into pLenti-Flag vector. pLenti-mAMFR-Flag and its mutants were cloned into pLenti-Flag vector. pLenti-TSPAN4-GFP was cloned into pLenti-GFP vector. pLenti-ITGA5-mCherry was cloned into pLenti-mCherry vector. pLenti-AMFR-GFP and its point mutants were cloned into pLenti-GFP vector. pLenti-OGT-GFP and its point mutant was cloned into pLenti-GFP vector. pLenti-AMFR-Halo and its point mutants were cloned into pLenti-Halo vector. pLenti-OGT-Halo and its point mutants were cloned into pLenti-Halo vector. pCDNA3.0-HA-UB, pCDNA3.0-HA-UBK48, and pCDNA3.0-HA-UBK63 were cloned into the mammalian expression vector pCDNA3.0-HA. GST-OGA (31-624), GST-TSPAN4, GST-OGT (323-1041), and its point mutant were cloned into pGEX-2T vector. Full length OGT and its point mutant were cloned into pCDNA4 vector with a HA, Flag, or Myc tag. All mutant constructs in this study were introduced by using a PCR-mediated site-directed mutagenesis on the basis of their respective wild-type constructs. All constructs were confirmed by sequencing.

DNA transfection and RNA interference
Plasmid DNAs were transfected into HEK293T cells using Polyethylenimine Linear (Yeasen, 40815ES03). siRNA oligonucleotides were purchased from GenePharma. siRNAs were transfected into cells with iboFECT CP Transfection Kit (RiboBio, C10511-05). Cells were harvested 48 h after transfection, the efficiency of protein knockdown was determined by Western blot analysis. siRNA targeting sequences for OGT: GAUUAAGCCUGUUGAAGUCTT, siRNA targeting sequences for TRIM21: AGAUUCUUGAGCAGAAAGCGC. siRNA targeting sequences for UBA1: UGAAAAGGAGACAAAGUCCCC. siRNA targeting sequences for AMFR: AAAAUUUGUCUUUGAGAUGCU. siRNA targeting sequences for UBQLN4: UCGGCCCCGCUCGGCUCCGCC. siRNA targeting sequences for UBQLN2: UUUUUCCGGCAAAAAUCAGCA. siRNA targeting sequences for LTN1: UUGAAUAGCAGGAACAUAGCC.

CRISPR/Cas9 knockout
Single guide RNAs (sgRNAs) were produced using Synthego (https://www.synthego.com/products/bioinformatics/crispr-design-tool), and annealed and ligated into BsmBI-cut pLenti-CRISPR-V2 vectors. The plasmid was transfected together with pMD2.G and psPAX2 into HEK293T cells to produce recombinant lentivirus. The supernatant from transfected HEK293T cells were filtered through a syringe with 0.22 μm filters. Cells were infected with Lentivirus for 24 h, and then selected with puromycin. sgRNA targeting for mouse-AMFR #1: AAGCGCTCGAGGAAGAGCAG. mouse-AMFR #2: TGAGGCCCGTGTAGGTGCGG. sgRNA targeting sequences for mouse-STING #1: GCCGTGGGATGGCTGGATGC, mouse-STING #2: GTGACCTCTGGGCCGTGGGA, mouse-STING #3: AGCGGTGACCTCTGGGCCGT. sgRab35 and sgPIP5K1A were obtained from Li Yu (Tsinghua University).

In vivo O-GlcNAcylation assay
HEK293T cells were co-transfected with AMFR-Flag, OGT-Myc, and other plasmids, where indicated for 48 h. Cells were collected into TAP lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, Protease cocktail) and lysed for 30 min on ice, and cleared by centrifugation at 13,000 rpm (16,060 × g) for 20 min at 4°C. Cell lysate was incubated with anti-Flag magnetic beads (Bimake, B26102) by rotation at 4°C for 6 h. Beads were then washed with TAP wash buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.5% NP-40, 1 mM EDTA) for three times. Samples were then added 1× SDS protein loading buffer and analyzed by western blot with O-GlcNAc antibody (Abcam, ab2739).

In vitro O-GlcNAcylation assay
The purified wild-type His-AMFR or its mutants were incubated with recombinant and purified GST-OGT protein (323-1041) in 50 μL reactions buffer (50 mM Tris-HCl, 12.5 mM MgCl2, 1 mM DTT, 2 mM UDP-GlcNAc, pH 7.5) at 37°C for 4 h. The samples were then added with 5× SDS protein loading buffer and detected by western blot with anti-O-GlcNAc (RL2) antibody (Abcam, ab2739).

OGA treatment assay
For O-GlcNAc cleavage, O-GlcNAcylated His-AMFR (OGT assay product) were treated with 5 μg purified OGA (31-624) at 37°C for 2 h in a volume of 100 μl. Samples were then added 5× SDS protein loading buffer and analyzed by western blot with O-GlcNAc antibody (Abcam, ab2739).

Western blot
Cells were lysed by SDS lysis buffer (Beyotime Biotechnology, P0015L) and heated at 100°C for 10 min. Equal amounts of protein were separated on 10% or 12% SDS-PAGE gels according to the molecular weight of target proteins, followed by electrophoretic transfer onto nitrocellulose membranes (Millipore, HATF00010). After blocking with 5% skim milk in PBST (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, 0.1% Tween-20) for 1 h at 37°C, membranes were incubated with the relevant primary antibodies in the primary antibody dilution buffer (Absin, ABS954-1L) overnight at 4°C. After washing 3 times with PBST for 5 min each, the membranes were incubated with HRP-conjugated secondary antibodies in 1% skim milk for 1 h at 37°C. Membranes were washed and signals were developed using ECL Plus western blot substrate (Biosharp, BL520B). Western blotting images were obtained using Tanon 5200 and analyzed using Bio-Rad Image Lab Software for PC Version 6.1.

Co-immunoprecipitation
HEK293T cells were transfected with indicated plasmids for 36 h, then cells were lysed in TAP lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, Protease cocktail) for 30 min on ice. The lysates were centrifuged at 13,000 rpm (16,060 × g) for 20 min at 4°C. For HA, Flag and GFP tag immunoprecipitation, tag magnetic beads were added in supernatants and incubated C together with rotation at 4°. Precipitated immunocomplexes were washed three times with TAP lysis buffer, five minutes each time. Finally, beads-bound proteins were added in 1× SDS loading buffer and subjected to western blot. The immunoprecipitated fractions were analyzed via mass spectrometry using 30 min LC-MS MS (Wuhan Genecreate Inc).

In vitro binding assay
His-tagged AMFR (WT, T643A, and S643A) was purified from Escherichia coli. The purified AMFR was subjected to in vitro O-GlcNAcylation assay for 4 h at 37°C with different concentrations of UDP-GlcNAc to obtain different levels of O-GlcNAcylation. GST-tagged TSPAN4 proteins were obtained by immunoprecipitation with anti-GST affinity beads from Escherichia coli cells. The O-GlcNAcylated proteins were then incubated with GST-TSPAN4, in cytosolic buffer (40 mM HEPES pH 7.4, 0.1% Triton ×-100, 10 mM NaCl, 150 mM KCl, and 2.5 mM MgCl2) by rotating at 4°C for 2 h. The beads were then washed three times with Cytosolic Buffer and denatured by heating at 100°C in 1× loading buffer for 10 min.

GST pull-down assay
His-fusion proteins or GST-fusion proteins were expressed in Escherichia coli strain BL21 (DE3). Then bacteria were grown at 37°C until the OD value reached 0.6–0.8, the proteins expression were induced with 0.4 mM IPTG for 18 h at 16°C. Bacteria were collected and lysed in lysis buffer (20 mM Tris, pH 7.4, 300 mM NaCl, 0.1% NP-40, 1 mM PMSF) by sonication on ice. Lysates were centrifuged at 12,000 × g for 20 min at 4°C, and the supernatants mixed the indicated protein samples were incubated with corresponding agarose beads overnight at 4°C. Then the beads were washed with lysis buffer and resuspended in 1× loading buffer, bound proteins were analyzed by western blot.

Cell imaging and image analysis
NRK cells were cultured in 35-mm confocal dishes (Biosharp, BS-20-GJM) pre-coated with 10 μg/ml fibronectin (Solarbio, F8180) in PBS for 3 h at 37°C and grew for 12–15 h. Cells were stained with 5 nM Janilia Fluo 646 HaloTag Ligand for 30 min or 1 μg/ml WGA-Alexa 647 for 5 min at 37°C. Images were acquired using Zeiss LSM900 confocal microscopes.

To determine the cell migration speed, time-lapse images were acquired by Zeiss LSM900, the interval was 2 min, and the duration was at least 4 h. For Time-lapse imaging of VSV-GFP-infected NRK cells stable expressing TSPAN4-mCherry. Cells were infected with VSV-GFP at an MOI of 0.2. Images were acquired using an Olympus FV-3000 (60×/1.30 silicone oil objective). Images were collected at 1024 × 1024 pixels resolution. The samples were maintained at 37°C in an atmosphere containing 5% CO2. The recording was set as every 6 min for 12 h, and one focal plane was recorded for all live-cell videos. Images were processed with ImageJ (RRID: SCR_003070), and statistical analyses were conducted using GraphPad Prism v9.5.1.

Immunostaining
Cells grown in 35 mm glass-bottom dishes were fixed with methanol for 5 min at −20°C, washed 3 times with PBS and permeabilized with Saponin (Solarbio, P1070) in PBS for 5 min. Cells were then blocked with 5% BSA for 30 min at room temperature, and further incubated with the specific primary and secondary antibodies at room temperature in 5% BSA. After 3 washes with PBS. Images were acquired by Zeiss LSM900 confocal microscopes.

Virus titration and viral plaque assays
Vero and BHK21 cells were seeded into 24-well plates, respectively one day prior. Next day, the cells were infected with ten fold diluted series of VSV-GFP and HSV-1 aliquots. Cells were incubated at 37°C for 2 h. After infection, cells were replenished with a maintenance cover layer (DMEM with 2% FBS and 0.9% carboxy-methylcellulose). After 72 h incubation at 37°C the plates were fixed with 4% paraformaldehyde overnight at 4°C. Then the cells were stained with crystal violet for 15 min at room temperature and the viral plaque numbers were visualized. The viral titer in plaque-forming units (PFU) was then calculated according to the plaque number and dilution ratio.

The migrasomes produced by the VSV-GFP-infected cells from forty 150 mm dishes were isolated according to the method described in this study and suspended with 200 μl PBS. 15 μL of suspension-containing medium was serially diluted and then added to confluent Vero-E6 cells for viral plaque assays.

Attachment assay
NRK monolayers grown in 6-well plates were pre-chilled at 4°C for 0.5 h and infected with VSV-GFP and HSV-1 at a MOI of 5 PFU/cell. Cells were incubated for 2 h at 4°C and followed by three washes with cold PBS. The RNA was extracted and analyzed by real-time PCR to detect viral genomes.

Entry assay
For entry assays, NRK monolayers grown in 12-well plates were infected with VSV-GFP and HSV-1 at a MOI of 5 PFU/cell. Cells were incubated for 2 h at 4°C and the unbound viruses were removed by washing with cold PBS. The cells were cultured with fresh medium supplemented with 10% FBS was added for 2 h at 37°C. After a single wash with acidic glycine to remove virus particles from the cell surface, the RNA was extracted and analyzed by real-time PCR to detect viral genomes.

Detection of glucose concentrations
Glucose concentrations using a glucose detection kit (BestBio, BB-4731) according to the manufacturer’s instructions. Briefly, cells were collected in cold PBS and centrifuged at 1000 × g for 5 min at 4°C. The supernatant was removed then resuspended the cells in 200 μl lysis buffer and incubated for 30 min on ice. Lysates were centrifuged at 12,000 × g for 15 min at 4°C and collected the supernatant. Then 10 μL of supernatant was added into the wells followed by the addition of 220 μL of the work solution and standard. After incubation at 37°C for 15 min, the absorbance at 510 nm was measured.

Quantitative RT-PCR
Total RNA was extracted using TRIzol (Biosharp, BS259A). 1 μg of RNA was used for reverse-transcribed using Reverse Transcription Kit (Biosharp, BL699A) according to the manufacturer’s protocol. The quantifications of cDNAs were performed by real-time PCR using qPCR Mix (Bio-Rad, 1725120). The PCR primers used as follows: hGAPDH forward 5′- GACAAGCTTCCCGTTCTCAG-3′, reverse 5′- GACAAGCTTCCCGTTCTCAG-3′; for Rat β-ACTIN forward 5′- GCGCAAGTACTCTGTGTGGA-3′, reverse 5′- AAGGGTGTAAAACGCAGCTC-3′; for VSV forward 5′- ACGGCGTACTTCCAGATGG-3′, reverse 5′- CTCGGTTCAAGATCCAGGT-3′; for HSV-1 forward 5′- CATCACCGACCCGGAGAGGGAC-3′, reverse 5′- GGGCCAGGCGCTTGTTGGTGTA-3′. For hIFN-β forward 5′- ATGACCAACAAGTGTCTCCTCC-3′, reverse 5′- GGAATCCAAGCAAGTTGTAGCTC-3′.

Generation of stable cell line
For generation of stable TSPAN4-Flag overexpressed HeLa cell line. The TSPAN4 coding sequence were cloned into pLenti-puro-Flag vector. Lentivirus were produced by co-transfecting pLenti-TSPAN4-Flag with packaging plasmids pMD2.G and psPAX2 into HEK293T cells. Forty-eight hours later, the supernatant from transfected HEK293T cells were collected and followed by filtering through a syringe with 0.22 μm filters. For HeLa cells were infected with Lentivirus for 48 h, puromycin (5 μg/ml) was added to select positive cells for two days. For the NRK cell lines with overexpression of GFP-tagged, mCherry-tagged, or Halo-tagged targets, L929 and NRK cell lines with overexpression of Flag-tagged targets were also constructed using the above method. For NRK and L929 cells were infected with Lentivirus for 48 h, puromycin (10 μg/ml) was added to select positive cells for two days. The cell lines with stable overexpression were used for subsequent experiments.

Mass spectrometry for protein O-GlcNAcylation
AMFR peptide MLAAAAERRLQRQRTT (628-643) was synthesized (Zhuantai Biotech Inc., Anhui). In a protein O-GlcNAcylation experiment, 1.0 mg of peptide was dissolved in 100 μL of ddH2O and prepared stock solution. Peptide stock solution was incubated with recombinant and purified GST-OGT protein (323-1041) in 50 μL reactions. Reaction buffer containing 50 mM Tris-HCl, 12.5 mM MgCl2, 1 mM DTT, 2 mM UDP-GlcNAc, pH 7.5) were incubated at 37°C for 4 h and samples were conserved in liquid nitrogen. Samples analyzed via mass spectrometry using 60 min timsTOF Pro 2 (Wuhan Baizhen Bio-technologies Inc.) to detect O-GlcNAcylated residues in peptide of AMFR.

Transmission electron microscopy
NRK cells were grown in 35 mm dishes precoated with fibronectin (10 μg/ml), and infected with VSV-GFP at an MOI of 0.2. At 8 hpi. The cells were pre-fixed at room temperature for 5 min using a 1:1 ratio of growth medium to 2.5% glutaraldehyde. Cells were further fixed with 2.5% glutaraldehyde in PB buffer for 2 h at room temperature, washed three times with PBS and dehydrated with a graded series of ethanol (50%, 70%, 90%, 95%, and 100%) for 8 min each. Samples were infiltrated with and embedded in SPON12 resin. After polymerizing for 48 h at 60°C, 70-nm-thick ultrathin sections were cut using a diamond knife, and then picked up with Formvar-coated copper grids (100 mesh). The sections were double-stained with uranyl acetate and lead citrate. After air drying, samples were examined with a Hitachi H-7000FA transmission electron microscopy at 80 kV.

ER fractionation
Cells were collected in cold PBS and centrifuged at 600 × g for 5 min at 4°C, removed the supernatant and resuspended the cells in 1 ml buffer E (20 mM HEPES, 250 mM sucrose, 1 mM EDTA, pH 7.4, 1 mM PMSF) and homogenized on ice by ultrasonic. The homogenates were centrifuged at 1000 × g for 10 min at 4°C, then collected the supernatant as cytosol. Two-thirds of the cytosol fraction was further centrifuged at 8000 × g for 20 min at 4°C, then the supernatant was transferred to ultracentrifuge tube and ultracentrifuged at 100,000 × g for 1 h at 4°C. The obtained pellet was collected as the ER fraction.

Isolation of migrasomes
Migrasome isolation was performed by iodixanol-sucrose density gradient centrifugation using a Lysosome Isolation Kit (Sigma-Aldrich, 233-140-8). Cells were seeded in 150 mm dishes precoated with fibronectin (10 μg/ml) at 50% confluence, followed by VSV-GFP infection (MOI=0.2). At 8 hpi, growth medium was disposed of and cells were washed to remove other extracellular vesicles in the medium. Cells were digested with trypsin and collected in 50 ml tubes. All subsequent manipulations were performed at 4°C. Cells and large debris were removed and supernatants were collected by centrifugation at 1000 × g for 10 min followed by 4000 × g for 20 min. Crude migrasomes were then collected as the pellet by centrifugation at 18,000 × g for 30 min. Migrasomes fractionation was performed by density gradient centrifugation, using 5–50% Optiprep as the density medium (Sigma-Aldrich, LYSISO1). First, a step gradient was built starting with 50% (500 μl), followed by 40% (500 μl), 35% (500 μl), 30% (500 μl), 25% (500 μl), 20% (500 μl), 15% (500 μl), 10% (500 μl), 5% (500 μl) and crude migrasomes (5%, 800 μl). The crude migrasome sample was prepared by first resuspending the pellet with 400 μl 1× extraction buffer mixed with 400 μl 10% Optiprep. Second, the prepared gradient was centrifuged at 150,000 × g for 4 h at 4°C in an MLS-50 rotor (Beckman). Finally, samples were collected from top to bottom (500 μl per fraction). Each fraction was mixed with the same volume of PBS (500 μl) and centrifuged at 18,000 × g for 30 min to collect the pellet. The pellets were washed with PBS and centrifuged again at 18,000 × g for 30 min. Fractions were prepared for TEM, plaque assay, infecting cells and western blot analysis.

Proteinase K protection assay
The isolated migrasomes pellet from VSV-GFP-infected cells were resuspended in PBS buffer and incubated on ice with 10 μg/ml proteinase K in the presence or absence of 0.1% Triton ×-100 for 30 min on ice. The reaction was terminated by addition of 1 mM PMSF. Samples were then subjected to western blot and viral plaque.

Statistics and reproducibility
Data are expressed as mean ± standard deviation (SD). The significance of the variability between different groups was determined by two-way analyses of variance using GraphPad Prism software (v9.5.1). Error bars, mean ± SD of two or three independent experiments. Two-tailed Unpaired Student’s t-test. A p value of < 0.05 was considered statistically significant and a p value of > 0.05 was considered statistically non-significant (ns). P value as exact values were given.

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

Data availability
All data generated or analyzed during this study are included in this article and its supplemental materials. Source data are provided with this paper.

Code availability
Code applied to the analyses in this study can be found on GlycoPOST at: https://glycopost.glycosmos.org (ID: GPST000657).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript investigates a novel mechanism by which viruses, specifically VSV and HSV-1, utilize migrasomes to facilitate their release and spread. The study focuses on the critical roles of TSPAN4 and the E3 ubiquitin ligase AMFR. The authors provide detailed evidence describing a pathway involving O-GlcNAcylation and ubiquitination processes that regulate TSPAN4 stability in the context of viral infection. This research highlights the significance of migrasomes in viral release and identifies potential strategies to inhibit viral dissemination. However, while the biochemistry experiments are well-designed and the data are clearly presented, the evidence falls short of convincingly demonstrating the role of TSPAN4 or migrasomes in viral infection.\n\nMajor Concerns:\n1, The authors should specify which step of the viral life cycle TSPAN4 targets. Is TSPAN4 critical for the entry of HSV-1 and VSV? Members of the tetraspanin family are known to play important roles in viral entry. For example, TSPAN9 has been implicated in alphavirus entry, TSPAN8 is essential for coronavirus entry, TSPAN27 is involved in HTLV-1 entry, and CD81 serves as the receptor for HCV. The manuscript must address whether TSPAN4 functions in a similar manner for HSV-1 or VSV entry.\n2, The authors used qPCR to conclude that migrasomes are critical for viral infection. However, these conclusions are based on experiments with a Multiplicity of Infection (MOI) of 1. To investigate viral replication effectively, researchers typically use MOIs of 3 or higher. On the other hand, studies focused on viral release and spread are usually conducted with lower MOIs, such as 0.01. The authors should revisit their experimental design and provide evidence supporting the model outlined in Fig. 7.\n3, AMFR could theoretically suppress viral infection by degrading viral proteins. In this model, TSPAN4 would simply antagonize AMFR’s antiviral effects by consuming its functional capacity, without any direct relevance to migrasome formation. To clarify the proviral function of TSPAN4, the authors should investigate its role in AMFR knockout (KO) cells. Additionally, it is important to determine whether AMFR directly degrades viral protein(s) from VSV or HSV-1.\n4, Why is AMFR O-GlcNAcylation reduced during the late stages of viral infection? High-glucose conditions typically promote O-GlcNAcylation. The authors should explore whether viral infection lowers intracellular glucose levels and whether high-glucose conditions enhance migrasome formation and subsequent viral release.\n5, AMFR-mediated degradation is unlikely to be the sole mechanism regulating TSPAN4 stability. In Fig. 2I, 6KR-Flag is still degraded, and in Fig. 3F, TSPAN4 degradation persists even when AMFR is knocked out. Alternative pathways must be considered and discussed.\n6, Most experiments in the study were conducted using HSV-1 and VSV; however, the last figure of the study inexplicably shifts focus to SARS-CoV-2. The manuscript must clarify whether migrasomes or TSPAN4 are relevant to SARS-CoV-2 infection, or explain the rationale for including this virus.\n7, Western blot (WB) results need to be repeated and quantitatively analyzed for statistical support. Key figures require quantification, including Fig. 5I, 5O, 5P, 5Q, 5R, 6G-6L, S1F, S2F, S3A, S3B, S3C, S3K-S3N, and S5.\n\nMinor Concerns:\n1, In the abstract, the authors overstate their findings by claiming that “During the early stages of infection, viruses enhance migrasome formation and exploit these structures to spread among neighboring cells, whereas abolish migrasome formation during the late stages of infection.”. This hypothesis was validated in a limited number of viruses, specifically HSV-1 and VSV. Further studies are needed to generalize this claim. The authors should revise the abstract to specify \"in HSV-1 and VSV models\" rather than generalizing across all viruses.\n2, Some Figures lack scale bars, such as Fig. 1A. Scale bars should be added where necessary.\n3, The labeling of protein markers is inconsistent across the manuscript. Within a single panel, if one blot is labeled, all other blots in the panel should also be labeled. For example, this inconsistency is observed in Fig. 2E, 2F, 2H, 3C, 4H-4K, 6E, and 6F.\n4, It remains unclear why the authors chose EV-A71 and RSV for Fig. S1H and S1I. Do EV-A71 and RSV induce migrasome formation? If not, the rationale for using these viruses should be clarified.\n5, The author contributions section lists R.C. and W.Z., who do not appear to be authors of this manuscript. This should be corrected.\n6, In lines 790 and 973, the \"2\" in \"CO2\" should be written as a subscript.\n7, Throughout the text, \"EV71\" should be corrected to \"EV-A71\" to reflect the standard nomenclature.\n8, The authors should discuss whether TSPAN4 undergoes other post-translational modifications that could be relevant to its function in viral infection.", "reviewer_2": "The authors present a compelling investigation into the dynamic interplay between viral infection and migrasome biogenesis. The study demonstrates that viruses regulate migrasome formation in infected cells, which in turn enhances viral replication. Key components of this regulatory axis, including OGT, AMFR, and Tspan4, were identified, alongside critical post-translational modifications such as O-GlcNAcylation and ubiquitination. These findings provide important mechanistic insights into migrasome biogenesis and its functional link to viral infection.\nOverall, this is a well-executed study that contributes novel insights into the molecular mechanisms governing migrasome formation in the context of viral infection. However, several key issues need to be addressed to strengthen the conclusions and improve clarity.\n\nMajor Points\n1. Limited Contribution of AMFR-K48 Ubiquitination to Tspan4 Degradation\nFigures 2B–D suggest that proteasome-mediated degradation is the primary pathway, as MG132 treatment nearly completely blocks Tspan4 degradation. However, Figure 2I shows that the 6KR mutant still undergoes substantial degradation, indicating a limited role for ubiquitination. The authors should clarify whether alternative pathways contribute to Tspan4 turnover.\nThe effect of AMFR knockout in Figure 3F appears minor. A statistical analysis is needed to determine whether the observed differences are significant.\nLines 178–185: The proportion of Tspan4 localized in the ER relative to its total cellular pool should be quantified. Given that most Tspan4 is found in the plasma membrane and endolysosomal pathway, the contribution of ER-resident AMFR may be limited.\n2. Mechanism of O-GlcNAcylation-Mediated Disruption of AMFR-Tspan4 Interaction\nThe study shows that OGT-mediated O-GlcNAcylation of AMFR disrupts its interaction with Tspan4 (Figures 5O, 5P), reducing Tspan4 ubiquitination and promoting migrasome formation. However, the molecular basis of this disruption remains unclear. The authors should provide mechanistic insights into how O-GlcNAcylation disrupts AMFR-Tspan4 binding or, at least, discuss a hypothesis to explain this effect.\n3. Viral Regulation of OGT-Mediated AMFR Glycosylation\nThe study establishes that viral infection regulates AMFR O-GlcNAcylation in an OGT-dependent manner (Figures 6G–J, 7J–L), yet the underlying mechanism is not addressed. Notably, Figures 6G and 6H show that OGT and AMFR protein levels remain relatively stable during infection, suggesting the involvement of post-translational regulation. The authors should provide mechanistic insights into how viral infection modulates OGT-mediated AMFR glycosylation or, at the very least, discuss plausible hypotheses that account for this regulation.\n\nMinor Points\nCorrection (Lines 240–241): The statement that “TSPAN4 directly interacts with OGT” should be revised, as Figures 5B and 5C show that AMFR, not TSPAN4, interacts with OGT.\nTspan4-6KR Stability vs. Migrasome Formation: The authors claim that Tspan4-6KR is less susceptible to degradation than Tspan4-WT, yet Figure 3L and 3M show similar migrasome formation between the two. If degradation is reduced, a higher migrasome output would be expected. The authors should resolve this inconsistency by quantifying Tspan4 protein levels, using fluorescence intensity measurements or Western blot analysis.\nSample Size for Imaging Experiments: In experiments such as Figure 5M, only 20 cells per group were analyzed. To improve statistical robustness, increasing the sample size to at least 50 cells per group per biological replicate is recommended.\nClarity and Readability: The manuscript presents a complex signaling network but could be more clearly structured. The authors should minimize long sentences, avoid redundant descriptions, and enhance logical coherence to improve readability.\nBy addressing these points, the study’s conclusions will be significantly strengthened."}, "author_response": "Point-by-point response:\nReviewer #1 (Remarks to the Author):\nThis manuscript investigates a novel mechanism by which viruses, specifically VSV and\nHSV-1, utilize migrasomes to facilitate their release and spread. The study focuses on the\ncritical roles of TSPAN4 and the E3 ubiquitin ligase AMFR. The authors provide detailed\nevidence describing a pathway involving O-GlcNAcylation and ubiquitination processes that\nregulate TSPAN4 stability in the context of viral infection. This research highlights the\nsignificance of migrasomes in viral release and identifies potential strategies to inhibit viral\ndissemination. However, while the biochemistry experiments are well-designed and the data\nare clearly presented, the evidence falls short of convincingly demonstrating the role of\nTSPAN4 or migrasomes in viral infection.\nResponse: Many thanks for your valuable and positive comments and conclusion of our work.\nIn response to your Major Concerns 6 and Minor Concerns 1: “The authors should revise the\nabstract to specify \"in HSV-1 and VSV models\" rather than generalizing across all viruses”.\nWe have deleted the data related to EV-A71, RSV and SARS-CoV-2, and revised the manuscript\nto specify “in HSV-1 and VSV models”.\nMajor Concerns:\n1, The authors should specify which step of the viral life cycle TSPAN4 targets. Is TSPAN4\ncritical for the entry of HSV-1 and VSV? Members of the tetraspanin family are known to\nplay important roles in viral entry. For example, TSPAN9 has been implicated in alphavirus\nentry, TSPAN8 is essential for coronavirus entry, TSPAN27 is involved in HTLV-1 entry, and\nCD81 serves as the receptor for HCV. The manuscript must address whether TSPAN4\nfunctions in a similar manner for HSV-1 or VSV entry.\nResponse: We are grateful for the Reviewer’s instructive suggestions. Through attachment and\ninternalization assays, we demonstrated that neither TSPAN4 overexpression nor Tspan4\nknockout affected the entry of VSV or HSV-1 (Supplementary Fig.1c, d) (Revised\nmanuscript line 108-110).\nWe also add the information about tetraspanin family and viral entry in the revised manuscript:\n“Members of the tetraspanin family are known to play important roles in viral entry, as TSPAN9\nhas been implicated in alphavirus entry, TSPAN8 is essential for coronavirus entry, TSPAN27\nis involved in HTLV-1 entry, and CD81 serves as the receptor for HCV.” (Revised manuscript\nline 64-67).\n2, The authors used qPCR to conclude that migrasomes are critical for viral infection.\nHowever, these conclusions are based on experiments with a Multiplicity of Infection (MOI)\nof 1. To investigate viral replication effectively, researchers typically use MOIs of 3 or higher.\nOn the other hand, studies focused on viral release and spread are usually conducted with\nlower MOIs, such as 0.01. The authors should revisit their experimental design and provide\nevidence supporting the model outlined in Fig. 7.\nResponse: We appreciate this critical advice and absolutely agree that low MOI (0.01) should\nbe used to study viral release. In the revised manuscript, to study the roles of TSPAN4-mediated\nmigrasome in viral release and spread, cells were infected with VSV or HSV-1 at MOI of 0.01,\nand viral titers were measured via plaque assays (Fig. 1a-d and 6a-c; Supplementary Fig. 4b-\nd, 4f and 4i).\n3, AMFR could theoretically suppress viral infection by degrading viral proteins. In this\nmodel, TSPAN4 would simply antagonize AMFR’s antiviral effects by consuming its\nfunctional capacity, without any direct relevance to migrasome formation. To clarify the\nproviral function of TSPAN4, the authors should investigate its role in AMFR knockout (KO)\ncells. Additionally, it is important to determine whether AMFR directly degrades viral\nprotein(s) from VSV or HSV-1.\nResponse: We appreciate this important comment. We analyzed the viral release of TSPAN4\noverexpression in Amfr KO cells, and found that overexpression of TSPAN4 still enhanced the\nviral release in Amfr KO cells, suggesting that the pro-viral function of TSPAN4 is not\ndependent on the antiviral effects of AMFR (Supplementary Fig. 4f) (Revised manuscript\nline 353-355).\nTo excluded the possibility that AMFR could theoretically suppress viral infection by degrading\nviral proteins, we co-expressed AMFR with VSV proteins (N, M, or G) or HSV-1 proteins\n(UL17, UL5, UL38, ICP4, or ICP27), and found that AMFR cased no effect on the protein level\nof these viral proteins (Supplementary Fig. 4e) (Revised manuscript line 350-353).\n4, Why is AMFR O-GlcNAcylation reduced during the late stages of viral infection? High-\nglucose conditions typically promote O-GlcNAcylation. The authors should explore whether\nviral infection lowers intracellular glucose levels and whether high-glucose conditions\nenhance migrasome formation and subsequent viral release.\nResponse: Thank you for your insightful comments. Using a glucose assay kit, intracellular\nglucose levels were measured and the results showed the significant decreased in glucose levels\nduring the late stages of viral infection (Supplementary Fig. 4g), indicate that viral infection\nlowers intracellular glucose levels, leading to the reduction of AMFR O-GlcNAcylation.\nWe then investigated whether glucose regulate migrasome formation and subsequent viral\nrelease. TSPAN4-mCherry-NRK cells were cultured in DMEM medium with different glucose\nconcentrations. We found that compared to 25 mM glucose group, low (5.5 mM) glucose\ncondition decreased while high (50 mM) glucose condition slightly enhanced migrasome\nformation and viral release (Supplementary Fig. 4h, i).\nThus, these data indicate that during the early stages of viral infection, high glucose conditions\npromote AMFR O-GlcNAcylation and subsequent migrasome formation for viral release,\nwhereas during the late stages viral infection lowers intracellular glucose levels, leading to the\nreduction of AMFR O-GlcNAcylation and migrasome formation (Revised manuscript line\n387-400).\n5, AMFR-mediated degradation is unlikely to be the sole mechanism regulating TSPAN4\nstability. In Fig. 2I, 6KR-Flag is still degraded, and in Fig. 3F, TSPAN4 degradation persists\neven when AMFR is knocked out. Alternative pathways must be considered and discussed.\nResponse: Thank you for your instructive suggestions. TSPAN4-Flag was remarkably\ndegraded following CHX treatment, even after just 30 minutes (Fig. 2a), indicating that\nTSPAN4 is susceptible to degradation, and several pathways may be involved in its degradation.\nWe did notice that TSPAN4 degradation induced by AMFR expression or virus infection can\nbe significantly blocked by MG132 treatment, and also partly blocked by CQ treatment (Fig.\n2c, d and Supplementary Fig. 2j), indicating that beside ubiquitin-proteasome,\nautophagosome-lysosome may function as alternative pathways to AMFR-mediated\ndegradation contribute to TSPAN4 turnover (Revised manuscript line 202-206).\nBesides, we co-expressed TSPAN4 with UbK6, UbK11, UbK27, UbK29, UbK33, UbK48, and UbK63\nproteins in HEK293T cells. Co-IP assays revealed that TSPAN4 undergoes K48 and K63\nubiquitination (Response letter Fig. 2), indicating that other E3 ligases may be involved in\nTSPAN4 degradation.\nResponse letter Fig.2 HEK293T cells were transfected with the indicated plasmid for 36 h.\nInteractions were analyzed by co-immunoprecipitation.\nWe discussed alternative degradation pathways of TSPAN4 in the revised manuscript: “Besides,\nwe noticed that TSPAN46KR is still degraded, TSPAN4 degradation persists even when AMFR is\nknocked out, and more importantly virus infection or AMFR expression-induced TSPAN4\ndegradation can also be partly inhibited upon CQ treatment. These results indicate that\nautophagy-lysosome pathway may also be involved in TSPAN4 degradation.” (Revised\nmanuscript line 491-499).\n6, Most experiments in the study were conducted using HSV-1 and VSV; however, the last\nfigure of the study inexplicably shifts focus to SARS-CoV-2. The manuscript must clarify\nwhether migrasomes or TSPAN4 are relevant to SARS-CoV-2 infection, or explain the\nrationale for including this virus.\nResponse: Thank you for your insightful comments. We have deleted these data and revised\nthe manuscript to specify “in HSV-1 and VSV models”.\nTo answer your concerns, we overexpressed or knocked out TSPAN4 in Vero-E6 cells, followed\nby infection with SARS-CoV-2 (MOI = 0.05) for 24 hours. The effect of TSPAN4 on SARS-\nCoV-2 replication was assessed using RT-qPCR. The results showed that, compared to the\ncontrol group, overexpression of TSPAN4 obviously promoted SARS-CoV-2 replication. In\ncontrast, knockout of TSPAN4 inhibited SARS-CoV-2 replication compared to the wild-type\n(WT) group (Response letter Fig.1).\nResponse letter Fig.1 Vero-E6 cells with overexpressed TSPAN4 or TSPAN4 knocked out Vero-E6\ncells were infected with SARS-CoV-2 (MOI = 0.05) for 24 hours. SARS-CoV-2 replication was\nassessed using RT-qPCR.\n7, Western blot (WB) results need to be repeated and quantitatively analyzed for statistical\nsupport. Key figures require quantification, including Fig. 5I, 5O, 5P, 5Q, 5R, 6G-6L, S1F,\nS2F, S3A, S3B, S3C, S3K-S3N, and S5.\nResponse: Thank you for your instructive suggestions. These results have been repeated three\ntimes and the statistical analysis have been added in the revised manuscript.\nMinor Concerns:\n1, In the abstract, the authors overstate their findings by claiming that “During the early\nstages of infection, viruses enhance migrasome formation and exploit these structures to\nspread among neighboring cells, whereas abolish migrasome formation during the late stages\nof infection.”. This hypothesis was validated in a limited number of viruses, specifically HSV-\n1 and VSV. Further studies are needed to generalize this claim. The authors should revise the\nabstract to specify \"in HSV-1 and VSV models\" rather than generalizing across all viruses.\nResponse: Thank you for your instructive suggestions. We apologize for overstating\nelaboration. We have revised the claim to specify \"in HSV-1 and VSV models\".\n2, Some Figures lack scale bars, such as Fig. 1A. Scale bars should be added where necessary.\nResponse: Thank you for your helpful suggestions. We have deleted the original Fig1A and 1B\ndue to the inappropriate MOI. Scale bars have been added for all image figures.\n3, The labeling of protein markers is inconsistent across the manuscript. Within a single panel,\nif one blot is labeled, all other blots in the panel should also be labeled. For example, this\ninconsistency is observed in Fig. 2E, 2F, 2H, 3C, 4H-4K, 6E, and 6F.\nResponse: Thank you for your instructive suggestions. We have labeled the protein markers\nof all panels.\n4, It remains unclear why the authors chose EV-A71 and RSV for Fig. S1H and S1I. Do EV-\nA71 and RSV induce migrasome formation? If not, the rationale for using these viruses\nshould be clarified.\nResponse: Thank you for your instructive suggestions. We have deleted these data and revised\nthe manuscript to specify “in HSV-1 and VSV models”.\n5, The author contributions section lists R.C. and W.Z., who do not appear to be authors of\nthis manuscript. This should be corrected.\nResponse: Thank you for your instructive suggestions. We have updated the author list and\ncontributions.\n6, In lines 790 and 973, the \"2\" in \"CO2\" should be written as a subscript.\nResponse: We apologize for this error. This has now been corrected.\n7, Throughout the text, \"EV71\" should be corrected to \"EV-A71\" to reflect the standard\nnomenclature.\nResponse: Thank you for your instructive suggestions. We have deleted these data and revised\nthe manuscript to specify “in HSV-1 and VSV models”.\n8, The authors should discuss whether TSPAN4 undergoes other post-translational\nmodifications that could be relevant to its function in viral infection.\nResponse: Thank you for your instructive suggestions. We have discussed this possibility in\nthe revised manuscript: “Additionally, TSPAN4 may undergo other post-translational\nmodifications that could be relevant to its function in viral infection, and more evidences are\nrequired to investigate the potential mechanism(s) by which TSPAN4 regulate migrasome\nformation and viral release.” (Revised manuscript line 496-499).\nReviewer #2 (Remarks to the Author):\nThe authors present a compelling investigation into the dynamic interplay between viral\ninfection and migrasome biogenesis. The study demonstrates that viruses regulate migrasome\nformation in infected cells, which in turn enhances viral replication. Key components of this\nregulatory axis, including OGT, AMFR, and Tspan4, were identified, alongside critical post-\ntranslational modifications such as O-GlcNAcylation and ubiquitination. These findings\nprovide important mechanistic insights into migrasome biogenesis and its functional link to\nviral infection.Overall, this is a well-executed study that contributes novel insights into the\nmolecular mechanisms governing migrasome formation in the context of viral infection.\nHowever, several key issues need to be addressed to strengthen the conclusions and improve\nclarity.\nResponse: Many thanks for your conclusion of our work. Your constructive suggestions help\nus significantly improve the quality of this manuscript.\nMajor Points\n1. Limited Contribution of AMFR-K48 Ubiquitination to Tspan4 Degradation\nFigures 2B–D suggest that proteasome-mediated degradation is the primary pathway, as\nMG132 treatment nearly completely blocks Tspan4 degradation. However, Figure 2I shows\nthat the 6KR mutant still undergoes substantial degradation, indicating a limited role for\nubiquitination. The authors should clarify whether alternative pathways contribute to Tspan4\nturnover\nResponse: Thank you for your instructive suggestions. TSPAN4-Flag was remarkably\ndegraded following CHX treatment, even after just 30 minutes (Fig. 2a), indicating that\nTSPAN4 is susceptible to degradation, and several pathways may be involved in its degradation.\nWe did notice that TSPAN4 degradation induced by AMFR expression or virus infection can\nbe significantly blocked by MG132 treatment, and also partly blocked by CQ treatment (Fig.\n2c, d and Supplementary Fig. 2j), indicating that beside ubiquitin-proteasome,\nautophagosome-lysosome may function as alternative pathways to AMFR-mediated\ndegradation contribute to TSPAN4 turnover (Revised manuscript line 202-206).\nBesides, we co-expressed TSPAN4 with UbK6, UbK11, UbK27, UbK29, UbK33, UbK48, and UbK63\nproteins in HEK293T cells. Co-IP assays revealed that TSPAN4 undergoes K48 and K63\nubiquitination (Response letter Fig. 2), indicating that other E3 ligases may be involved in\nTSPAN4 degradation.\nResponse letter Fig.2 HEK293T cells were transfected with the indicated plasmid for 36 h.\nInteractions were analyzed by co-immunoprecipitation.\nWe discussed alternative degradation pathways of TSPAN4 in the revised manuscript: “Besides,\nwe noticed that TSPAN46KR is still degraded, TSPAN4 degradation persists even when AMFR is\nknocked out, and more importantly virus infection or AMFR expression-induced TSPAN4\ndegradation can also be partly inhibited upon CQ treatment. These results indicate that\nautophagy-lysosome pathway may also be involved in TSPAN4 degradation.” (Revised\nmanuscript line 492-497).\nThe effect of AMFR knockout in Figure 3F appears minor. A statistical analysis is needed to\ndetermine whether the observed differences are significant.\nResponse: Thank you for your instructive suggestions. We have replaced with repeated data\nand statistical analysis has been added (Fig. 3f).\nLines 178–185: The proportion of Tspan4 localized in the ER relative to its total cellular pool\nshould be quantified. Given that most Tspan4 is found in the plasma membrane and\nendolysosomal pathway, the contribution of ER-resident AMFR may be limited.\nResponse: We are grateful for the Reviewer’s critical comments. We have discussed the\npossibility of ER-resident TSPAN4 degradation contribute to migrasome formation in the\noriginal manuscript: “According to reports, TSPAN4 can be found on the cytoplasmic membrane,\nendosomes, and migrasomes. We found that TSPAN4 also partially colocalizes with ER and\nonly ER localized TSPAN4 is unstable and can be degraded upon AMFR overexpression,\nbut not non-ER TSPAN4 (Fig. 3h). The degradation of ER-localized TSPAN4 induced by AMFR\noverexpression impaired migrasome formation. Our model predicted that ER-localized TSPAN4\nmay move to the cytoplasmic membrane by endosomes, where TSPAN4 was dynamically\nrecruited to migrasomes via retraction fibres. Since ER-localized TSPAN4 might be the source\nof migrasome-localized TSPAN4, this could help to explain why the degradation of ER-\nlocalized TSPAN4 caused the defect of migrasome formation.” (Revised manuscript line 482-\n491).\nAs your suggested, we quantified the proportion of TSPAN4 localized in the ER relative to its\ntotal cellular pool, and found that 32.12 ± 2.31% of total TSPAN4 was enrich in the ER\n(Supplementary Fig. 2h) (Revised manuscript line 191-193).\n2. Mechanism of O-GlcNAcylation-Mediated Disruption of AMFR-Tspan4 Interaction.\nThe study shows that OGT-mediated O-GlcNAcylation of AMFR disrupts its interaction with\nTspan4 (Figures 5O, 5P), reducing Tspan4 ubiquitination and promoting migrasome\nformation. However, the molecular basis of this disruption remains unclear. The authors\nshould provide mechanistic insights into how O-GlcNAcylation disrupts AMFR-Tspan4\nbinding or, at least, discuss a hypothesis to explain this effect.\nResponse: Thank you for your instructive suggestions. To further investigate the molecular\nmechanism by which O-GlcNAcylation disrupts the AMFR-TSPAN4 interaction, we employed\nAlphaFold to predict the structure of the AMFR-TSPAN4 complex and generated an AMFR\nmutant (W345A/E634A/H420A/R484A/R428A/F424A/F436A) lacking the putative TSPAN4-\nbinding domain (Supplementary Fig. 3o). This AMFR mutant exhibited significantly reduced\nbinding affinity toward TSPAN4 (Supplementary Fig. 3p), supporting the reliability of the\nstructural prediction. Given that the potential O-GlcNAcylation site is located in proximity to\nthe AMFR-TSPAN4 interaction interface, we hypothesize that O-GlcNAcylation at residue\nT643 may induce steric hindrance, thereby disrupting the AMFR-TSPAN4 association\n(Revised manuscript line 322-331).\n3. Viral Regulation of OGT-Mediated AMFR Glycosylation\nThe study establishes that viral infection regulates AMFR O-GlcNAcylation in an OGT-\ndependent manner (Figures 6G–J, 7J–L), yet the underlying mechanism is not addressed.\nNotably, Figures 6G and 6H show that OGT and AMFR protein levels remain relatively stable\nduring infection, suggesting the involvement of post-translational regulation. The authors\nshould provide mechanistic insights into how viral infection modulates OGT-mediated AMFR\nglycosylation or, at the very least, discuss plausible hypotheses that account for this\nregulation.\nResponse: Thank you for your insightful comments. High-glucose conditions are known to\npromote O-GlcNAcylation. To determine why AMFR O-GlcNAcylation is reduced during the\nlate stages of viral infection, intracellular glucose levels were measured and showed a\nsignificant decrease during the late stages of viral infection (Supplementary Fig. 4g),\nindicating that viral infection reduces intracellular glucose levels, thereby contributing to the\nreduction in AMFR O-GlcNAcylation.\nWe then investigated whether glucose regulates migrasome formation and subsequent viral\nrelease. TSPAN4-mCherry-NRK cells were cultured in DMEM medium containing varying\nglucose concentrations. Compared to the 25 mM glucose condition, low glucose (5.5 mM)\ndecreased migrasome formation and viral release, whereas high glucose (50 mM) slightly\nenhanced these processes (Supplementary Fig. 4h, i).\nThese findings suggest that during the early stages of infection, elevated glucose levels promote\nAMFR O-GlcNAcylation and subsequent migrasome formation to facilitate viral release, while\nduring the late stages, viral infection leads to diminished intracellular glucose levels, resulting\nin reduced AMFR O-GlcNAcylation and impaired migrasome formation (Revised manuscript\nline 387-400).\nMinor Points\nCorrection (Lines 240–241): The statement that “TSPAN4 directly interacts with OGT”\nshould be revised, as Figures 5B and 5C show that AMFR, not TSPAN4, interacts with OGT.\nResponse: We apologize for this error. This has now been corrected.\nTspan4-6KR Stability vs. Migrasome Formation: The authors claim that Tspan4-6KR is less\nsusceptible to degradation than Tspan4-WT, yet Figure 3L and 3M show similar migrasome\nformation between the two. If degradation is reduced, a higher migrasome output would be\nexpected. The authors should resolve this inconsistency by quantifying Tspan4 protein levels,\nusing fluorescence intensity measurements or Western blot analysis\nResponse: Thank you for your instructive suggestions. We have shown that when co-expressed\nwith AMFR or under CHX treatment, Tspan4-6KR is less susceptible to degradation and form\nhigher migrasome output than Tspan4-WT, whereas 6KR and WT showed similar protein level\nand migrasome output under mock condition (Original manuscript Fig. 3d, 3l, 3m).\nWe have quantified the protein levels of Tspan4 via Western blot analysis for Fig. 3l (Revised\nManuscript Fig. 3n) (Revised manuscript line 215-220).\nSample Size for Imaging Experiments: In experiments such as Figure 5M, only 20 cells per\ngroup were analyzed. To improve statistical robustness, increasing the sample size to at least\n50 cells per group per biological replicate is recommended.\nResponse: Thank you for your instructive suggestions. We have added the quantifications of\nmigrasome numbers by increasing the sample size to at least 50 cells per group per biological\nreplicate, three independent experiments (Revised Manuscript Fig. 1j, 3k, 3m, 4b, 4d, 5m,\n7c, 7f, 7i and 7l).\nClarity and Readability: The manuscript presents a complex signaling network but could be\nmore clearly structured. The authors should minimize long sentences, avoid redundant\ndescriptions, and enhance logical coherence to improve readability.\nResponse: Thank you for your instructive suggestions. We have revised the manuscript\naccordingly.\nBy addressing these points, the study’s conclusions will be significantly strengthened."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The data and text in the revised manuscript have significantly improved. However, there\nare still some issues that need to be addressed.\n1, The current data do not convincingly demonstrate the presence of VSV virions in\nmigrasomes. The TEM results shown in Fig. 1g are not persuasive. In Case 1, the overall\nstructure does not resemble a migrasome, which typically appears rounded. The authors\nshould provide more examples of virus-induced migrasomes, rather than just partial\nstructures like Case 1. Furthermore, the structures marked by the arrows do not appear\nto be typical VSV virions; instead, they resemble tubules. It is known that VSV-GP alone\ncan form bullet-shaped particles. Therefore, Fig. 1g does not provide strong evidence for\nthe presence of VSV virions within migrasomes. I suggest that the authors investigate\nwhether VSV virions are located inside the migrasomes that they have already purified. A\nrecent study (PMID: 40914694) reported that VSV-GP is found on the surface of\nmigrasomes, while viral RNA is present within the migrasomes. This has led to the\nproposal of the concept of migrasome-mediated VSV transmission. In this context, it was\nnoted that VSV produces migrasomes, but virions were not identified inside them, as\nultrasonication eliminated the infectivity of the migrasomes without affecting free virions.\nThus, it appears that VSV could not generate migrasomes that contain virions.\n2, The glucose level decreases during the later stages of infection (Supplementary Figure\n4g), which is consistent with the protein levels of TSPAN4 (Figure 1k and 1l). However,\nTSPAN4 is upregulated during the early stages of infection (Figure 1k and 1l). Does\nglucose level increase during the early stages of infection? If so, how does this occur?\n3, Some representative images appear to be inconsistent with the quantified data, such\nas the data presented in lane 4 of Figure 2d, lane 5 of Figure 7j.\n4, The source and the information about VSV-GFP are missing. Is the GFP fused to the\nviral protein, or is it used alone?\n5, In line 1149, “R” should not be capitalized.", "reviewer_2": "The authors have addressed my comments satisfactorily. I recommend acceptance of the revised manuscript."}, "author_response": "Response:\nReviewer #1 (Remarks to the Author):\nThe data and text in the revised manuscript have significantly improved. However, there\nare still some issues that need to be addressed.\n1, The current data do not convincingly demonstrate the presence of VSV virions in\nmigrasomes. The TEM results shown in Fig. 1g are not persuasive. In Case 1, the overall\nstructure does not resemble a migrasome, which typically appears rounded. The authors\nshould provide more examples of virus-induced migrasomes, rather than just partial\nstructures like Case 1. Furthermore, the structures marked by the arrows do not appear\nto be typical VSV virions; instead, they resemble tubules. It is known that VSV-GP alone\ncan form bullet-shaped particles. Therefore, Fig. 1g does not provide strong evidence for\nthe presence of VSV virions within migrasomes. I suggest that the authors investigate\nwhether VSV virions are located inside the migrasomes that they have already purified. A\nrecent study (PMID: 40914694) reported that VSV-GP is found on the surface of\nmigrasomes, while viral RNA is present within the migrasomes. This has led to the\nproposal of the concept of migrasome-mediated VSV transmission. In this context, it was\nnoted that VSV produces migrasomes, but virions were not identified inside them, as\nultrasonication eliminated the infectivity of the migrasomes without affecting free virions.\nThus, it appears that VSV could not generate migrasomes that contain virions.\nResponse: We appreciate this critical advice. Using proteinase K protection assay and\nconfocal microscopy, we have confirmed that VSV-G protein was partly enriched on\nthe out membrane of migrasomes (Supplementary Fig. 1f, g).\nTo further confirm that VSV virions are located inside the migrasomes, we treated the\npurified migrasomes isolated from VSV-infected cells with proteinase K to degrade the\nmigrasome outer membrane localized VSV-G. These treated migrasomes were then\nincubated with control cells. Compared to the PBS-treated control, migrasomes treated\nwith proteinase K alone exhibited a slightly reduction in infectivity, whereas co-\ntreatment with proteinase K and Triton X-100 abolished migrasome-mediated\ninfectivity (Fig. 1i), indicating that the primary mechanism of migrasome-mediated\nviral transmission is mediated by VSV virions contained inside the migrasomes,\nwhereas the VSV-G protein localized on the outer membrane of migrasomes\ncontributes only minimally to this process (Revised manuscript line 135-144).\nFurthermore, we provided additional transmission electron microscopy (TEM) data,\nthrough which hexagonal virus particles were directly visualized within migrasomes\nfrom VSV-infected cells (Response letter Fig 1).\nResponse letter Fig 1. Representative transmission electron micrograph (TEM) images of\nVSV-GFP (at an MOI of 0.2) infected NRK cells. Red arrows indicated virions in migrasomes.\nMito, mitochondria.\n2, The glucose level decreases during the later stages of infection (Supplementary Figure\n4g), which is consistent with the protein levels of TSPAN4 (Figure 1k and 1l). However,\nTSPAN4 is upregulated during the early stages of infection (Figure 1k and 1l). Does\nglucose level increase during the early stages of infection? If so, how does this occur?\nResponse: Thank you for your insightful comments. Compare to mock cells, there was\nno significant increase of glucose level during the early stages of infection, indicate that\nthe upregulated AMFR O-GlcNAcylation during the early stages of infection is not\ncaused by increased glucose level (Supplementary Fig. 4g).\nOur data indicate that the upregulated O-GlcNAcylation of AMFR suppresses AMFR-\nmediated degradation of TSPAN4, resulting in the accumulation of TSPAN4 during the\nearly stages of infection. However, the mechanism underlying the increased O-\nGlcNAcylation despite unchanged glucose levels remains unclear. As suggested by\nreviewer#1 from the previous comments, we have discussed in the revised manuscript\nthe possibility that AMFR may undergo additional post-translational modifications that\ncontribute to its enhanced O-GlcNAcylation: “Interestingly, compare to mock cells,\nthere was no significant increase of glucose level during the early stages of infection\n(Supplementary Fig. 4g), indicating that the upregulated AMFR O-GlcNAcylation\nduring the early stages of infection is not caused by increased glucose level, and AMFR\nmay undergo additional post-translational modifications that contribute to its\nenhanced O-GlcNAcylation” (Revised manuscript line 401-405).\n3, Some representative images appear to be inconsistent with the quantified data, such\nas the data presented in lane 4 of Figure 2d, lane 5 of Figure 7j.\nResponse: Many thanks for this important comment. We meticulously verified the\noriginal and quantified data, and re-performed multiple statistical analyses on images\nacquired under different exposure times (2c, 2d, 4f, 7a, 7d, 7j, S2c, S2f, S5c, S5g). All\nraw blot data used for subsequent quantification were provided in the Source data file.\n4, The source and the information about VSV-GFP are missing. Is the GFP fused to the\nviral protein, or is it used alone?\nResponse: We appreciate this valuable comment. VSV-GFP was kindly provided by\nMingzhou Chen (Hubei University). GFP gene is inserted into the G gene and L gene\njunction and the method was described in previous study (PMID: 27373596).\n5, In line 1149, “R” should not be capitalized.\nResponse: We apologize for this error. This has now been corrected.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed my comments satisfactorily. I recommend acceptance of\nthe revised manuscript.\nResponse: Many thanks for your valuable and positive comments of our work."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed all my concerns."}, "author_response": ""}]

Decision letter