Autophagic extracellular vesicles (AEVs) are distinct from exosomes and play crucial roles in viral infections

Domain: biology
Published: 2025-12-21
Editorial outcome: major_revision

Abstract
Emerging evidence indicates important interconnections between autophagy and the secretion of small extracellular vesicles. However, our understanding of these secretory vesicles remains incomplete. Here, we identify a subtype of small extracellular vesicles, termed autophagic extracellular vesicles (AEVs), which are distinct from exosomes. Extracellular AEVs characterized by a size less than 100 nm exhibit increased secretion when the autophagy response is induced. Amphisomes, the hybrid organelles, are essential for the secretion of these types of vesicles. Further exploration reveals that autophagic cargos, certain ESCRT III components and the Rab13 serve as distinctive markers for distinguishing AEVs from exosomes. Moreover, we find that the biogenesis of AEVs functionally requires components of the ESCRT III complex and the GTPase Rab27a. Finally, we confirm that the enteroviral particles or genomes can be encapsulated into AEVs and subsequently infect receptor-negative cells with high efficiency. This model represents a prominent pattern of EV-mediated virus transmission.

Introduction
Mammalian cells secrete extracellular vesicles (EVs), which can be distinguished on the basis of their origin, size, and other factors. Small extracellular vesicles (sEVs) are EVs smaller than 200 nm in diameter, and are the most widely studied types of EVs because of their many roles in different physiological and pathological conditions1,2. As an important subpopulation of sEVs, exosomes begin with inward budding of the plasma membrane to form early endosomes followed by maturation into multivesicular bodies (MVBs), which involves the formation of intraluminal vesicles (ILVs) via inward budding of the endosomal membrane. MVBs fuse with the plasma membrane and then release ILVs into the extracellular space as exosomes3. The mechanisms of exosome biogenesis are fairly well understood and importantly involve subunits of the endosomal sorting complex required for transport (ESCRT). The ESCRT machinery as a driver of membrane shape and scission, and is involved in the formation of MVBs and ILVs. As a result, some ESCRT components can act selectively on MVB and ILV subpopulations fated for secretion as exosomes, meanwhile, other components, such as ESCRT III, remain on the outside of ILVs until they are recycled4.

The canonical pathway of autophagy involves the coordinated degradation and recycling of intracellular components and pathogenic material. This occurs through degradation upon the fusion of autophagosomes with lysosomes. Unlike degradative autophagy, the autophagic machinery can, through a shared yet partially divergent pathway, result in the secretion of cytoplasmic components rather than their degradation. This process involves the fusion of autophagosomes and MVBs to create a secretory amphisome, which subsequently releases its cargo upon binding to plasma membrane in an event termed secretory autophagy5–7. The consequences of this secretory amphisome release may be the presence of sEVs positive for both exosomal markers and autophagy-associated proteins, which may contain poorly characterized cargos and functionalities that are distinctly different from those of conventional exosomes. The common step in exosome biogenesis and secretory amphisome formation is the formation of MVBs. Nevertheless, extracellular secretion of autophagy components and autophagy cargo receptors including p62/SQSTM1 and LC3B can be markedly enhanced in response to lysosomal inhibition or the accumulation of autophagosomes, but these autophagic dysfunction status has a negligible effect on the secretion of exosomal markers8,9. In addition, autophagy-related LC3 conjugation machinery regulates selective packaging and secretion of the secretome extracellularly10. These observations suggest that the secretion of autophagy-related proteins may occur in distinct extracellular vesicle populations. Importantly, many infectious pathogens such as viruses, specifically disrupt the lysosomal system, preventing clearance by autophagy-dependent degradation and impacting sEV or exosome secretion11–14. Therefore, exploring the relationship between secretory autophagy and exosomes not only offers a framework for a clearer understanding of extracellular vesicle heterogeneity, but also provides insights into disturbances in homeostasis such as during viral infection.

In this study, we identified a subtype of small extracellular vesicles, termed AEVs (autophagic extracellular vesicles) that were distinct from conventional exosomes. Extracellular AEVs characterized by a size less than 100 nm exhibited increased secretion when autophagy response was triggered in vitro and in vivo. Amphisomes, formed by the fusion of MVBs and autophagosomes, are essential for the secretion of these types of vesicles. Further analysis revealed that autophagic cargos, ESCRT III components and Rab13 serve as distinctive markers for distinguishing AEVs from exosomes. Moreover, we determined that the biogenesis of AEVs functionally requires certain ESCRT III components and the small GTPase Rab27a. Finally, using two enterovirus strains, we confirmed that the AEVs could carry and deliver viral particles or whole genomes into receptor-negative host cells with high infection efficiency, thereby broadening viral tropism and facilitating viral transmission.

Results
Starvation-induced autophagy affects the size distribution of small extracellular vesicles
Previous studies revealed that interconnections between the autophagy process and small extracellular vesicle (sEV)/exosome production is closely connected15. To investigate how the autophagic response affects the secretion yield and particle size of small extracellular vesicles, HEK-293T cells were cultured under serum-starved conditions to induce an autophagic response as determined by autophagic flux of cargos such as p62 and LC3B-Ⅱ (Fig. 1A). The vacuolar-type ATPase (v-ATPase) inhibitor bafilomycin A1 (BafA1), which disrupts lysosome acidification, was employed to facilitate the accumulation of autophagosomes (Fig. 1A). The sEV fractions were isolated from the culture supernatant using the traditional method that is the main one used in published studies (Fig. 1B). Nanoparticle tracking analysis (NTA) revealed a singular bell-shaped size distribution with peaks at 143 nm in the sEVs derived from 293T cells under normal conditions (Fig. 1C). Unexpectedly, a double-bell-shaped size distribution was detected in sEVs isolated from serum-starved 293T cells, with peaks at 92 nm and 138 nm. Similar double-peak contours were also observed in sEVs derived from serum-starved 293T cells after BafA1 treatment, which correlated with the accumulation of autophagosomes, with peaks at 99 nm and 136 nm (Fig. 1C). Further analysis revealed that the percentage or number of sEVs with a size smaller than 100 nm derived from serum-starved 293T cells, regardless of BafA1 treatment, was significantly greater than that from normally cultured cells (Fig. 1D–F). However, the secretion of sEVs with a size range of 100-200 nm varied minimally among normal cells, nutrient-deprived cells and cells treated with BafA1 (Fig. 1G). The double-bell-shaped size distributions of sEVs were also observed in other cell lines (Neuro-2a, THP-1 and SH-SY5Y cells) when autophagy was induced by serum deprivation, and the percentage of sEVs with a size less than 100 nm from serum-starved cells increased (Fig. S1). These results suggested that the induction of autophagy due to nutrient deprivation affects the size distribution of sEVs and that this phenomenon is a generalized response. Additionally, the increase in sEVs from starved 293T cells with a size smaller than 100 nm was predominantly within the range of 70-90 nm (Fig. 1H). To further characterize the increase in sEVs smaller than 100 nm induced by autophagy in vivo, we detected sEVs from mouse serum after the mice were maintained under normal feeding or starvation conditions. An induction of the autophagic response in the livers of starved mice was observed, as evidenced by a decrease in the expression of the autophagic cargo protein p62 (Fig. 1I). As shown in Fig. 1J, the percentage of sEVs less than 100 nm in size was notably greater in serum from starved mice than in serum from normal feeding mice. However, the quantity of sEVs in serum with a size range of 100-200 nm differed little between normal feeding mice and starved mice (Fig. 1K). Collectively, these findings suggest that the starvation-induced autophagic response influences the size distribution of small EVs, which might be attributed to autophagosome formation.

The stress-induced autophagy response led to increased secretion of sEVs less than 100 nm in size
To further investigate whether other inducers of the autophagy response affect the size distribution of sEVs, cells were treated with the autophagy inducer rapamycin to trigger an autophagy response, and the corresponding secreted sEVs exhibited a double-bell-shaped size distribution (Fig. 2A, B). Compared with the sEVs secreted from mock cells, the percentage of sEVs smaller than 100 nm increased (Fig. 2C, D). The quantity of sEVs with a size range of 100–200 nm differed little between mock cells and rapamycin-treated cells (Fig. 2D).
Virus infection induced host autophagy has been widely reviewed16. Our results confirmed that the Varicella-Zoster virus (VZV), a human herpesvirus responsible for chickenpox, could induce an autophagy response in human retinal pigment epithelium (RPE) cells, as determined by observing autophagic cargos such as NBR1, p62 and LC3B-Ⅱ (Fig. 2F). This autophagy process could be inhibited by treatment with acyclovir (ACV), an acceptable anti-herpesvirus drug (Fig. 2F). NTA revealed that the percentage of sEVs less than 100 nm in size from VZV-infected cells clearly increased compared with those from mock cells (Fig. 2G, H). The antiviral drug acyclovir, which inhibited VZV-induced autophagy, significantly decreased the secretion of sEVs smaller than 100 nm. Like in the starvation treatment group, the number of sEVs with a size less than 100 nm, which increased in the VZV-infected cells, was predominantly in the 70–100 nm range (Fig. 2I). In addition, VZV infection did not alter the number of secreted sEVs within the size range of 100–200 nm (Fig. 2J). Similarly, other human viral strains, such as coxsackievirus B4 (CVB4) and Herpes simplex virus type 1 (HSV-1), which have been confirmed to induce a cellular autophagy response (Fig. S2A, D), showed increased secretion of sEVs smaller than 100 nm (Fig. S2B, C, E, F).
Dysfunction of the autophagy-lysosome pathway is frequently associated with aging and age-related diseases17. Therefore, we detected the concentration of sEVs smaller than 100 nm in serum samples isolated from healthy older men aged over 65 years and observed a significantly greater percentage of sEVs less than 100 nm in size in the older population than in younger individuals aged 18–40 (Fig. 2K) (Supplementary Tables 1 and 2).
Although nanoparticle tracking analysis (NTA) is widely used to detect the size and concentration of extracellular vesicles, resistive pulse sensing (RPS) techniques that are calibrated with size-standards, and limit of detection can be reported in term of diameter, and are emerging as new methods for particle sizing and counting18. Therefore, the size distribution of sEVs from mock, starved, CVB4-infected and rapamycin-treated 293T cells was detected by RPS techniques through a nanopore chip with a measurement range of 50–240 nm. Compared with that from mock cells, the secreted sEVs in the size range of 50–100 nm was increased from starved, CVB4-infected and rapamycin-treated 293T cells compared to that from mock cells (Fig. S2G, K), which is consistent with the above NTA results. Moreover, when RPS was used, both Neuro-2a cells and SH-SY5Y neuronal cells presented higher percentages of sEVs ranging from 50–100 nm in size under starvation conditions (Fig. S2H, I, K). Compared with that from mock cells, the quantity of secreted sEVs smaller than 100 nm was significantly greater from VZV-infected RPE cells (Fig. S2J). Overall, our results indicated that induction of the autophagy response modulated the size distribution of small EVs, resulting in increased secretion of sEVs with sizes smaller than 100 nm.

Autophagic extracellular vesicles (AEVs) represent a subtype of small extracellular vesicles and are distinct from exosomes
Exosomes are typically described as having a diameter of 30-150 nm. The ESCRT pathway is involved in exosome biogenesis2. To investigate whether secreted exosomes contribute to the increase in the quantity of sEVs with size less than 100 nm induced by autophagy, we generated TSG101 and ATG7 gene knockout (TSG101-KO and ATG7-KO) HEK-293T cells, which were either subjected to starvation treatment or not. TSG101 plays a role in the process of exosome biogenesis and ATG7 is essential for autophagosome formation. Western blot analysis revealed that the vesicles secreted from starved wild-type cells (WT cells) or TSG101-KO cells contained high levels of autophagic cargos, including NBR1, p62 and lipidated LC3B (LC3B-II). In contrast, these autophagic cargos were barely detected in the vesicles secreted from either normal or starved ATG7-KO cells (Fig. 3A, B). CD63 and CD9, which are often considered markers associated with exosomes, were largely decreased in secreted vesicles from TSG101-KO cells compared with those from WT cells, whereas the expression of the marker CD81 decreased slightly (Fig. 3A, B). Importantly, compared with those in WT cells, the levels of both CD63 and CD9 in vesicles secreted from ATG7-KO cells were unaltered, whereas the level of CD81 was reduced. These results suggested that inhibition of exosome biogenesis did not affect the secretion of autophagic cargos via small extracellular vesicles. Moreover, the secretion of sEVs less than 100 nm in size from TSG101-KO cells was greater than that from WT cells under starvation conditions, but this secretion was markedly suppressed in ATG7-KO cells (Fig. 3C). These findings indicated that the inhibition of autophagosome formation, rather than exosome biogenesis, contributed to the release of sEVs less than 100 nm in size.
The Rab11 protein is required for the fusion of MVBs and autophagosomes to generate hybrid amphisomes, which are subsequently released into the extracellular space as vesicles19. To explore whether the fusion of MVBs and autophagosomes affect the sEVs smaller than 100 nm, we generated Rab11 gene knockout (Rab11-KO) cells. Deletion of Rab11 did not affect the fusion of autophagosomes with lysosomes under starvation conditions, as evidenced by decreased p62 levels and increased LC3B-II accumulation (Fig. 3D). We found that the secretion of vesicles less than 100 nm in size from Rab11-KO cells failed to increase despite the induction of autophagy by means of starvation, rapamycin treatment and CVB4 infection (Fig. 3E). Moreover, the autophagic cargos NBR1, p62 and LC3B-II were barely detected in the secreted vesicles of starved Rab11-KO cells (Fig. 3E). In contrast, the levels of the exosome markers CD63 and CD9 remained unchanged, with the exception of those of CD81 (Fig. 3F). Similarly, compared with those in starved WT cells, the levels of p62 and LC3B-II in the secreted vesicles of starved CD63 gene knockout (CD63-KO) cells were not affected (Fig. 3G). A previous study revealed that glutamine-depleted conditions could induce the secretion of Rab11a-exosomes that were unlike late endosomal exosomes20. To determine whether Rab11a-exosomes are involved in autophagy-induced vesicle formation, we detected the levels of Rab11a and caveolin-1 in vesicles secreted from WT and ATG7-KO cells, both of which were enriched in Rab11a-exosomes. These proteins were elevated in the secreted vesicles from starved ATG7-KO cells compared with those from normal culture conditions (Fig. 3H), suggesting that the subtypes of the Rab11a-exosomes were not the same as those of the autophagy-induced vesicles. Therefore, these results demonstrated that amphisomes, formed by the fusion of MVBs and autophagosomes, were essential for the secretion of vesicles less than 100 nm in size that carried autophagic cargos. Importantly, these amphisome-associated extracellular vesicles were distinct from exosomes derived from ILVs because of the inward budding of MVBs. Therefore, we termed these vesicles autophagic extracellular vesicles (AEVs), a subtype of small extracellular vesicles.
To further confirm that AEVs represent a subpopulation of small extracellular vesicles, we utilized a proximity barcoding (PBA) assay, a high-throughput method for single-sEV analysis, to simultaneously profile approximately 260 surface proteins on a single sEV. The proteomic characteristics of sEVs isolated from normal or starved 293T cells were determined via the FlowSOM algorithm, an unsupervised machine learning process, to generate a sEV cluster map. The clustering of sEVs from samples is shown in the t-distributed stochastic neighbor embedding (t-SNE) plot via FlowSOM in Fig. 3I. Next, we found a distinct subpopulation of vesicles (blue dots) present in sEVs derived from starved cells, accounting for 99.24% of the total, compared with only 0.76% of the sEVs from normally cultured cells. CD81 protein expression was relatively high in this subpopulation (Fig. 3J). These results may explain why the level of CD81, rather than that of CD63 and CD9, decreased in the sEV fraction from starved Rab11-KO cells, as shown in Fig. 3E. Taken together, our results suggest that AEVs, characterized by their transport of autophagic cargo proteins and sizes less than 100 nm, represent a subtype of small extracellular vesicles. They were distinguishable from both exosomes and the traditionally recognized small EVs.

AEVs exhibited a unique composition consisting of Rab GTPases and ESCRT components
To identify the characteristics of AEVs, we designed two separate strategies for AEV collection using differential centrifugation coupled with serial filtration (Method A and Method B in Fig. 4A). Using sequential filtration in conjunction with ultracentrifugation at 100,000g (Method A), approximately 60% of the total vesicles were less than 100 nm in size, suggesting that this method was insufficient for isolating high-purity AEVs (Fig. 4B). After many rounds of testing and improvement, we performed an additional ultracentrifugation at 40,000g to rule out large-sized vesicles from the supernatant after passage through a 0.1 μm filter, followed by a ultracentrifugation step at 100,000g, referred to as Method B. The proportion of AEVs with sizes less than 100 nm exceeded 80%, as determined by NTA (Fig. 4B). To further support these findings, we analyzed the autophagic markers associated with AEVs in collected vesicle fractions using either Method A or Method B and revealed that the levels of NBR1, p62 and LC3B-II were similarly enriched in these vesicle fractions (Fig. 4C). Importantly, the levels of CD63 and CD9 were markedly lower in the vesicle fractions isolated by Method B than in those obtained through Method A, whereas this effect was not observed for CD81 (Fig. 4C). In addition, the p62-positive AEVs with fluorochrome labeling mainly displayed sizes less than 100 nm, as determined by NTA (Fig. 4D). These results suggested that Method B for collecting purified AEVs was sufficiently valid.
To define the protein composition of the AEVs, we performed a quantitative proteomic analysis on the AEVs from starved HEK-293T cells and the vesicles from normally cultured cells, and Method B was used for isolation. Gene Ontology (GO) terms related to biological processes highlighted a profound enrichment in AEV proteins that localize to autophagy/macroautophagy, autophagosome maturation, the MVB assembly/sorting pathway and the ESCRT complex in control cells versus vesicles from normally cultured cells (Fig. 4E). This finding was consistent with our prior findings that revealed that the production of AEVs depended on the fusion of autophagosomes with MVBs. In addition, GO terms related to molecular function revealed a significant enrichment of AEV proteins associated with protein binding and RNA binding terms (Fig. 4F), suggesting that the loading and contents of proteins in AEVs were associated with specific proteins, such as protein and RNA binding proteins.
To further corroborate these results, we performed proteomics and compared vesicles purified from starved Rab11-KO cells with those from starved WT cells (Supplementary Data 1). We found that many proteins were dramatically decreased in vesicles from starved Rab11-KO cells and that the proteins whose expression was reduced were associated mainly with autophagy, the MVB functional pathway and ESCRT complex activity, as annotated by GO terms for biological processes (Fig. 4G), which is consistent with the role of Rab11 in controlling the fusion of autophagosomes with MVB to release their contents outside the cells as AEVs. The proteins whose expression was significantly reduced in vesicles from Rab11-KO cells exhibited a highly interconnected network enriched in protein binding, as annotated by GO molecular function terms (Fig. 4H). Notably, proteomic analyses of the AEV fraction from starved WT cells revealed that the expression of Rab13, a small GTPase, was greater in the AEV fraction than in the vesicle fraction from normally cultured cells. In addition, Rab11 knockout resulted in a significant reduction in Rab13 levels in AEVs (Fig. 4I), indicating that Rab13 acts as a cargo marker involved in the formation of AEVs. Moreover, we observed that several components of ESCRT, including VPS4A/B, CHMP2A/1A/2B, VAT1, TSG101, MVB12A and PDCD6IP/Alix, were also enriched in the AEV fraction derived from starved WT cells. In contrast, these ESCRT components were largely decreased in vesicle fractions from Rab11-KO cells (Fig. 4I). These observations highlight that specific ESCRT components, which are not typically present in defined exosomes, may be involved in AEV formation or secretion. Notably, the majority of these components were the ESCRT III complex and its associated accessory proteins. Taken together, our findings confirmed that the isolation of purified AEVs can be effectively achieved using a specific separation method. Moreover, this type of extracellular vesicle is enriched in Rab GTPases and ESCRT III components.

Autophagic cargos, ESCRT-III components, and Rab13 serve as distinct markers that differentiate AEVs from conventional exosomes
We next assessed whether the Rab13 and ESCRT components identified via proteomics analysis were released in association with AEVs in response to autophagy. AEV or vesicle fractions were isolated from starved WT, TSG101-KO, ATG7-KO or Rab11-KO cells using Method B, as described above. As shown in Fig. 5A, TSG101 knockout did not affect the secretion of Rab13, CHMP2A or VPS4A/B, or the release of AEVs under starvation conditions. In contrast, the knockout of ATG7 and Rab11 led to a decrease in the expression of these proteins in the vesicle fraction, suggesting that the expression of the Rab13 and ESCRT-III components was indeed coupled with AEV secretion.
We further attempted to separate the AEVs from total small EVs on the basis of density differences, and total small EVs prepared from starved 293T cells using differential centrifugation, as described in Fig. 1B, were sedimented in a density gradient (5%–40% iodixanol) for 18 h at 100,000g, leading to equilibration according to vesicle density. Western blot analysis revealed that the exosome/sEV markers CD63, CD9 and CD81 were present in fractions 7 and 8 (Fig. 5C). These fractions were also enriched in autophagic cargos, including the LC3B-II, Rab13, p62, and ESCRT III components VPS4A/B and CHMP2A, suggesting that the density of the AEVs is similar to that of the exosomes in iodixanol gradients, making it challenging to effectively separate the AEVs from the exosomes (Fig. 5C). Moreover, we identified lipidated LC3B-II in fraction 9, which was cofractionated with both Rab13 and Rab11 following continuous density gradient ultracentrifugation of the cell lysate. The early endosome marker EEA1 (fractions 4–5), endoplasmic reticulum marker calreticulin (fractions 2–5) and the cytoskeleton marker α-tubulin did not colocalize with the lipidation of LC3B-II (Fig. 5D). In addition, the late endosome/MVB marker CD63 was present mainly in fractions 6–8, as illustrated in Fig. 5D.
Protease protection assays were conducted in which starved 293T-derived AEVs were treated with either trypsin or trypsin plus detergent (Triton X-100). The autophagic cargos (p62 and NBR1), ESCRT-III components (VPS4A/B and CHMP2A) and Rab13 were dramatically digested by trypsin in the absence of detergent. However, LC3B-II was resistant to trypsin digestion in the absence of detergent but was degraded upon exposure to detergent. The protein TSG101, known for its presence in the luminal compartment of exosomes, maintained a stable abundance in AEVs regardless of trypsin digestion (Fig. 5E). Consistently, the immunoelectron microscopy showed that the AEVs isolated from starved 293T cells exhibited immunoreactivity with antibodies against p62 and Rab13 on the vesicle membranes, while antibodies against LC3B were present inside the vesicles (Fig. 5F).
Next, we determined whether these specific contents are present in AEVs from other cell types. The levels of autophagic cargos (NBR1, p62 and LC3-II), ESCRT-III components (VPS4A/B and CHMP2A) and Rab13 were markedly greater in the AEVs derived from leukemia monocytic THP-1 and neuroblastoma Neuro2a cells under starved conditions than in those derived from cells maintained under normal culture conditions (Fig. 5G, H). Overall, on the basis of these results, we proposed that the AEVs originating from intracellular hybrid amphisomes were rich in autophagic cargos, ESCRT III components and small GTPases. These elements serve as distinctive markers for distinguishing AEVs from conventional exosomes (Fig. 5I).

Rab13 contributes to the sorting of cargo proteins into AEVs without affecting their secretion
Given the significant enrichment of the small GTPase Rab13 in AEVs, we aimed to investigate its functional role in AEV biogenesis. Super-resolution (SR) microscopy revealed that endogenous Rab13 was localized around MVBs with CD63 labeling under normal conditions in 293T cells (Fig. 6A). Notably, upon autophagic activity induced by starvation, the structures of Rab13 closely bind to CD63-labeled MVBs (Fig. 6A), indicating that Rab13 may be involved in the amphisome generation process. Unexpectedly, Rab13 knockout did not affect the secretion of total sEVs regardless of whether the cells were subjected to starvation stimulation (Fig. 6B). Moreover, the secretion of AEVs differed little between WT and Rab13-KO cells under starvation conditions (Fig. 6C). These results suggest that Rab13 does not contribute to the formation or release of AEVs. Nevertheless, western blot analysis revealed that the levels of NBR1, p62 and LC3B-II in AEVs from starved Rab13-KO cells were largely lower than those in AEVs from starved WT cells, suggesting that the depletion of Rab13 disturbed the secretion of autophagic cargos via AEVs (Fig. 6D). The ESCRT components VPS4A/B, CHMP2A and TSG101 were also reduced in AEVs from starved Rab13-KO cells. In support of this observation, the colocalization of Rab13 with LC3B or p62 was observed in starved WT cells. In contrast, this colocalization was rarely observed in Rab11-KO cells (Fig. 6E). These results demonstrated that the interaction between Rab13 and autophagic cargos depended on amphisome formation, subsequently facilitating the sorting of these cargos into AEVs. Moreover, in starved WT cells, LC3B exhibited aggregate structures in close proximity to CD63-labeled MVBs without complete colocalization (Fig. 6F). However, those LC3B-positive aggregates were localized around CD63-labeled MVBs in starved Rab13-KO cells (Fig. 6F). Our results confirmed that LC3B was present inside AEVs.
On the basis of these results, we speculated that Rab13 may be involved in controlling the transfer of lipidated LC3B from the amphisome membrane to the interior of AEVs during the fusion of autophagosomes with MVBs. Depletion of Rab13 did not affect this fusion but redirected the localization of lipidated LC3B residing on the amphisome membrane.
Transmission electron microscopy (TEM) analysis of starved 293T cells revealed two distinct lumens in the amphisomes. One lumen consisted of a compact multivesicular structure (green arrow), possibly formed from autophagosomes after fusing with MVBs, while the other was filled with typical intraluminal vesicle (ILV) structures (yellow arrow), known as exosomes (Fig. 6G). These findings may explain why disruption of ILV formation through the deletion of ESCRT genes did not affect AEV secretion. To further corroborate these results, 293T cells were cotransfected with the LC3B-GFP fusion vector to label autophagosomes and the CD63-mCherry fusion vector to label MVBs. The presence of amphisomes could then be scored on the basis of both GFP and mCherry. After starvation, the transfected cells were disrupted using mechanical milling, and the GFP-mCherry-positive amphisomes were sorted by flow cytometry (Fig. 6H). As shown in Fig. 6I, the amphisomes, coexpressing CD63 and LC3B, exhibited two distinct groups, each labeled with either mCherry or GFP fluorescence. In support of this observation, the sEVs isolated from the cotransfected cells clearly presented two subtypes of vesicles, one labeled with GFP and the other with mCherry, as detected under super-resolution (SR) microscopy (60 nm SR) (Fig. 6J). These findings suggested that the amphisomes resulting from autophagosome-MVB fusion consisted of two distinct types: one type comprised the ILV-exosomes formed by the inward budding of MVBs, while the other constituted AEVs derived from autophagosomes. Both types could be released extracellularly as sEV-sized exosomes or sEV-sized AEVs when the amphisomes fused with the plasma membrane. In addition, amphisomes can also fuse with lysosomes to form autolysosomes, followed by degradation of the cargo. Therefore, we quantified intracellular amphisome numbers upon starvation or BafA1-mediated lysosome inhibition and observed an increase in the number of amphisomes when lysosomal function was compromised (Fig. 6K). Our data presented above in Fig. 1 show no significant alteration in AEV secretion in BafA1-treated cells, suggesting that the intracellular accumulation of amphisomes did not increase their fusion with the plasma membrane as an alternative route for extracellular release. These results suggest that the different fates of amphisomes, including lysosomal degradation and extracellular release, are independent.
Given that Rab13 affects the secretion of autophagic cargos and ESCRT components via AEV release, we subsequently investigated whether Rab13 plays a role in determining the composition of AEVs. Quantitative proteomics analysis of AEVs from starved Rab13-KO 293T cells compared with those from WT cells revealed that the levels of LC3B (MAP1LC3B) and ESCRT components were significantly decreased (Fig. 6K) (Supplementary Data 2), which is consistent with the results of the western blot analysis presented in Fig. 6D. GO terms of biological processes highlighted a significant decrease in AEV proteins from starved Rab13-KO cells that localize to autophagy-associated processes such as macroautophagy and autophagosome maturation, indicating that Rab13 depletion could disturb the packaging of autophagy-related proteins into AEVs (Fig. 6L). Importantly, this category also included a significant term related to protein localization to the plasma membrane, suggesting that Rab13 may be involved in regulating the localization of target proteins, such as autophagic cargo and ESCRT-III components, to the membrane. Taken together, our results suggest that Rab13 contributes to the sorting of autophagic cargos and ESCRT components into AEVs without significantly affecting the formation or secretion of these vesicles.

The biogenesis of AEVs depends on ESCRT-III/VPS4 and Rab27a
The ESCRT III complex is a conserved membrane remodeling machinery essential for the budding of endosomal vesicles and exosome formation21,22. Since the AEVs contained ESCRT III components, we aimed to investigate whether these components were involved in the membrane fission process that resulted in the formation of AEVs during amphisome formation. As shown in Fig. 7A, when the ESCRT-III components CHMP4A and CHMP4B were knocked down via transfection of small interfering RNAs (siRNAs), there was a marked decrease in AEV secretion relative to control or negative control siRNA (si-NC). The double knockdown of CHMP4A and CHMP4B resulted in an enhanced effect. Furthermore, when the ESCRT accessory proteins VPS4A and VPS4B were knocked down, the secretion of AEVs also decreased. Knockdown of other components, such as CHMP1B, CHMP2A and CHMP2B, did not influence AEV secretion (Fig. 7A). TEM analysis of CHMP4A and CHMP4B double-knockdown 293T cells under starvation conditions revealed the absence of a compacted multivesicular structure in amphisomes (green arrow, Fig. 7B). In addition, we also observed abnormal morphology for MVBs lacking ILVs (yellow arrow, Fig. 7B). In support of this observation, the compact multivesicular structure within the amphisomes reappeared when the interference effect of the siRNA was exhausted (Fig. 7C, red arrow). Furthermore, by using SR microscopy, the lipidated LC3B was found to not form aggregate structures, as shown in Fig. 7D, but instead colocalized with CD63-labeled MVBs in a punctate distribution, indicating that the lack of these ESCRT-III components led to the abnormal location of AEV cargos. These observations suggested that AEV biogenesis in amphisomes was required for CHMP4A-, CHMP4B- and VPS4A/B-mediated membrane fission.
Previous studies have demonstrated that Rab27a is involved in the release of exosomes originating from MVBs23. Thus, we tested whether Rab27a was required for AEV secretion. Compared with that from WT cells, the release of AEVs from stable Rab27a-knockout 293T cells (Rab27a-KO) was significantly decreased (Fig. 7E). Similarly, depletion of Rab27a suppressed the secretion of p62, LC3B-Ⅱ, p62, CHMP2A, VPS4A/B and TSG101 via AEVs (Fig. 7F). In further support of this finding, we detected no increase in AEV secretion from the serum of Rab27a-KO mice under starvation conditions compared with that under normal feeding conditions (Fig. 7G). These results revealed that Rab27a was functionally required for the extracellular secretion of autophagic cargos and ESCRT-III components via AEV populations.

AEVs that encapsulate enterovirus infectious elements exhibit infectivity in non-permissive cells, thereby expanding viral tropism
Viral infection not only induces a host autophagy response but also impacts the secretion of exosomes/sEVs24,25. Coxsackievirus B4 (CVB4), with one of the highest proportions of fatal outcomes among enterovirus serotypes, was confirmed in this study to induce an autophagy response and increase AEV secretion (Fig. S2A, C). Therefore, exploring the role of AEVs during CVB4 infection events is logical. Purified AEVs from CVB4-infected WT cells contained virus-like particles enclosed within membrane structures, as observed by TEM. Similar results were also obtained for AEVs from CVB4-infected TSG101-KO cells, with the presence of virus-like virions encapsulated within the vesicles (Fig. S4). In contrast, the AEVs from the CVB4-infected Rab11-KO cells were identified by TEM to not contain virus-like particles inside the vesicles (Fig. 8A). In further support of this observation, the CVB4 structural protein VP1, along with LC3B-II and p62, was detected in the AEV fraction derived from infected WT cells, suggesting that the virus-like particles enclosed within these AEVs were indeed authentic CVB4 virions (Fig. 8B). To determine whether the virions enclosed within the AEVs retain their infectivity, we utilized purified AEVs derived from CVB4-infected WT, TSG101-KO or Rab11-KO cells to treat susceptible HeLa cells. As shown in Fig. 8C, the level of viral RNA in HeLa cells did not significantly differ following treatment with various sources of AEVs at different time points. However, interestingly, we observed that the AEVs from infected WT or TSG101-KO cells, but not from Rab11-KO cells, efficiently entered and replicated over time in NIH3T3 cells, a non-permissive mouse cell lacking viral receptors (Fig. 8D), suggesting that AEVs containing the CVB4 virion exhibit high infectivity in non-permissive cells. Moreover, CVB4 RNA was significantly more enriched in both the brains and spleens of mice injected with AEVs from WT cells than in those of mice injected with AEVs from Rab11-KO cells (Fig. 8E). Compared with those from mice injected with WT cells, the survival rate from mice injected with AEVs from Rab11-KO cells was greater (~70%) (Fig. 8F). These findings indicated that CVB4 virions enclosed within AEVs have greater infection efficiency, thereby overcoming restrictions to viral tropism.
Our previous study demonstrated that the accumulation of enterovirus 71 (EV71)-induced autophagosomes facilitated their fusion with MVBs, leading to the packaging of the viral RNA genome into amphisomes for extracellular release26. Therefore, investigating whether AEVs from EV71-infected cells contain a viral genome is meaningful. EV71 infection triggered an autophagy response in 293T cells characterized by no alteration in p62 but an increase in LC3B-II, suggesting that incomplete autophagy was induced by EV71, whose accumulated autophagosomes did not fuse with lysosomes for degradation. In the following step, we isolated sEVs (EV71-sEVs, containing the AEVs) or AEVs (EV71-AEVs) from the culture medium derived from EV71-infected cells (Fig. 8H). Neither EV71-sEVs nor EV71-AEVs showed detectable levels of the viral structural protein VP1, indicating that no complete virus particles were present within these vesicles (Fig. 8I). However, we observed that both EV71-sEVs and EV71-AEVs contained the viral full-length genome (~7000 bp). Moreover, the levels of the EV71 RNA genome did not significantly differ between EV71-sEVs and EV71-AEVs from an equal number of infected cells (Fig. 8K). Our published study demonstrated that preventing autophagosome–MVB fusion via Rab11 knockdown could abolish the sorting of EV71 RNA into amphisomes, leading to a marked decrease in viral RNA for extracellular release26. These results collectively indicated that extracellular vesicle-associated EV71 genomes were primarily encapsulated within the AEVs. Importantly, the viral genomes encapsulated within AEVs exhibited high infectivity in a non-permissive L929 cells (a mouse fibroblast line), in contrast to EV71 free virions (Fig. 8L). Taken together, our data supported that AEVs could carry and deliver viral particles or whole genomes into receptor-negative host cells with high infection efficiency, overcoming restrictions to viral tropism.

Methods
Cell culture, virus strains and plasmids
HEK-293T, ARPE-19, HeLa, SH-SY5y, THP-1, Neuro2a, NIH3T3 and L929 cell lines were obtained from the American Type Culture Collection (ATCC, USA). The HEK-293T, ARPE-19, NIH3T3, HeLa and L929 cells were maintained in DMEM medium (HyClone, USA), the THP-1 cells were cultured in RPMI 1640 medium (HyClone, USA), the SH-SY5y and Neuro-2a cells were grown in DMEM/F12 medium (HyClone, USA). The cell culture supplemented with 10% fetal bovine serum (FBS, ExCell Bio, China) and incubated at 37°C in a 5% CO2 humidified incubator. FBS used in these cultures were depleted of EVs by ultracentrifugation at 120,000g for 3 h. All the cell lines used in this study tested negative for mycoplasma contamination using the reagent PlasmoTest (InvivoGen).
For serum starvation, cells were cultured in serum-free medium for 24 hours. If needed, bafilomycin A1 (20 nM, TargetMol, China) or rapamycin (100 nM, TargetMol, China) was added into the serum-free DMEM and incubated for 24 hours. To assess the extent of cell death induced by starvation, LDH release into the culture medium was measured using a commercially available LDH assay kit (DOJINDO, #CK12), to ensure that the cell mortality rate remained below 5%.
Human herpesvirus 1 strain (HSV-1, GenBank: GU734771.1), Coxsackievirus B4 strain (CVB4, GenBank: X05690.1) and Enterovirus 71 strain (EV71, GenBank: HQ882182.1) were kindly provided by Prof. Zhiwei Wu (Nanjing University, China) and passaged in Vero E6 cells. Varicella-zoster virus strain (VZV, GenBank: AB097933.1) was a kind gift from Prof. Tong Cheng (Xiamen University) and propagated in ARPE-19 cells. Both titers of viruses were determined by the 50% tissue culture infection dose (TCID50) assay and stored at -80°C until used.
The expression plasmid pCMV-GFP-LC3B (#D2815) and pCMV-CD63-mCherry (#D2841) were purchased from Beyotime Biotechnology (Shanghai, China). All the sequence of inserted genes in plasmids were verified by DNA Sequencing.

Generation of CRISPR-Cas9 KO cell lines
The guide RNAs (sgRNAs) targeting the human genes were designed using the CRISPick online software (Broad institute), and subcloned into the pSpCas9(BB)-2A-Puro plasmid (Addgene no. 48139). Forward and reverse oligos for sgRNAs were custom synthesized (Tsingke Biotech, Beijing, China) and annealed to generate sticky ends. pSpCas9 plasmid was cleaved with BbsI enzyme (#R3539; New England Biolabs) and ligated with annealed sgRNA using T4 ligase (Takara). HEK-293T cells were transfected with the corresponding sgRNA-ligated plasmids using Lipofectamine 3000 (Thermo Fisher Scientific) for 48 hours, followed by treating with 2 μg/mL puromycin for 3 days to select gene-knockout cells. Single-cell clones were selected for three weeks via limiting dilution assay. Knockout of corresponding genes were validated by western blot using specific antibodies. The sequences of sgRNAs used for CRISPR knockout are as follows: human TSG101: 5’-CTGTTCTGTTTTCAGGCCG-3’; human SDCBP: 5’-GACAGAATGTCATTGGATTGA-3’; human CD63: 5’-AACGAGAAGGCGATCCATAAGG-3’; human Rab11: 5’-GAGAGTGATCTACGTCATCTC-3’; human Rab13: 5’-GTATACTAGGATAATGCCCT-3’; human Rab27a: 5’-CCAAAGCTAAAAACTTGATG-3’.

Preparation of small extracellular vesicles (sEVs) and autophagic extracellular vesicles (AEVs)
The wild cell lines and knockout cell lines were seeded in 10 cm tissue culture dishes. Upon reaching approximately ~90% confluence, the medium containing 10% FBS was carefully removed. Cells were then washed with phosphate-buffered saline (PBS) slightly and subsequently cultured in 10% EV-depleted FBS medium or serum-free medium for 24 h. As for virus infection experiment, cells were infected with CVB4 at 0.05 TCID50 or EV71 at 0.03 TCID50 for 24 h under 10% EV-depleted FBS medium to avoid cell necrosis and apoptosis. The culture medium was centrifuged at 3000 × g for 15 min at 4°C and 10,000 × g for another 30 min at 4°C to remove cells, debris and large vesicles.
Small extracellular vesicles (sEVs) isolation according to standard protocols with some adaptations: the processed supernatants were filtered through 0.22 μm pore membrane filters (Millipore) and subjected to ultracentrifugation by 100,000 × g at 4°C for 90 minutes using Optima L-100 XP ultracentrifuge (Beckman Coulter) to pellet sEVs. Crude sEV pellets were then gently diluted in sterile PBS and ultracentrifuged for an additional 90 min at 100,000 × g. The final sEV pellet was resuspended in sterile PBS for further analysis.
For autophagic extracellular vesicles (AEVs) isolation, the processed supernatants were sequentially filtered through 0.22 μm and 0.1 μm pore filters. The filtered supernatants were centrifuged at 40,000 × g for 60 minutes at 4°C to remove large vesicles with size >100 nm, followed by ultracentrifuged by 100,000 × g for 90 minutes at 4°C. The resulting AEV pellets were resuspended in sterile PBS and centrifuged again at 100,000 × g for 90 minutes at 4°C to remove soluble factors for a series of subsequent experiments.
For serum isolation, the blood samples from mouse or donor were kept at 4°C overnight without anticoagulant to ensure serum separation. Equal volume of serum was centrifuged at 5000 × g for 10 min and then diluted with appropriate PBS. The diluted serum samples were filtered through 0.22 μm pore membrane filters and subjected to ultracentrifugation by 100,000 × g at 4°C for 90 minutes. The crude sEV pellets were cleaned by an additional 90 min at 100,000 × g to obtain purified sEVs for subsequent analysis.
Of note, for NTA assays, infectious activity, digestion protection and iodixanol gradient separation, the suspension of sEVs or AEVs were used within 12 h. For western blot, MS and PBA, they were stored at -80°C and used within three months. In addition, for all comparisons of sEVs or AEVs between experimental conditions, results from individual cohorts were corrected as indicated on the basis of total cell number to ensure that vesicle quantification was not confounded by seeding differences.

Nanoparticle tracking analysis (NTA) for particle size and concentration of EVs
The Nano NS300 instrument (Malvern Instruments, UK) equipped with laser-based light scattering system was used to track the size and number of EVs. Samples resuspended in PBS were diluted to achieve concentrations between 1×107/ml and 1×109/ml, and then manually pushed into the sample chamber, making sure there were no bubbles in the sample chamber and tubing. Laser light scattering was performed to visualize EVs in collected supernatants using a conventional optical microscope perpendicular to the beam axis. During this process, EVs were recorded and analyzed based on Brownian motion and the diffusion coefficient. Each sample was subjected to three 60-second video recordings under the following conditions: cell temperature: 25~26°C; Syringe speed: 40µl/s. The data were subsequently analyzed in triplicate to confirm reproducibility using NTA 2.3 software to calculate the number and size of EVs in each sample.
For detection of p62-positive fluorescent AEVs, the AEVs isolated from serum-free, 24-hour cultured HEK-293T cells and incubated with SQSTM1/p62 antibody conjugating Alexa Fluor 488 (#ab185015, Abcam, 1:500) at room temperature for 2 h. Then the AEVs were subjected to ultracentrifugation by 100,000 × g at 4°C for 90 minutes to remove excess antibody and were cleaned by PBS with an additional ultracentrifugation at 100,000 × g for 90 min. The particle size of p62-positive fluorescent AEVs was analyzed using NTA instrument in fluorescence mode (laser wavelength 488 nm). Sample was manually loaded into the sample chamber, and a fluorescence filter was inserted into the optical path. Camera levels and focus settings were adjusted to optimize particle tracking within the optimal threshold range. Subsequent analysis steps followed the standard NTA procedure.

Resistive pulse sensing (RPS) for particle size analysis
Particle size distribution was measured by Nanocoulter Counter (Resen Technology, Shenzhen, China) through a single-particle analysis approach, and the nanopore chip with the measuring range of 50-240 nm was employed. Samples underwent differential centrifugation and filtration as previously, followed by dilution in PBS to achieve an optimal concentration within the instrument’s detection range (1 × 10⁷/mL to 1 × 10⁹/mL), and then introduce into the sample port. The resistive pulse sensing technique relied on an electric induction zone generated by electrodes on either side of the nanopore in electrolyte solution. As individual particles traversed the nanopore, the particle displaced an equal volume of conductive solution, instantly increasing the resistance of the induction zone and generating a pulse signal. The signal was then analyzed by Nanocoulter software to obtain multi-dimensional particle information. The RPS assays were performed by Resen Technology (Shenzhen, China).

Iodixanol gradient ultracentrifugation
For subcellular fractionation, HEK-293T cells were serum starved for 24 h, harvested in tris-sucrose buffer (0.25 M sucrose, 10 mM Tris, pH 7.5) with Protease Inhibitor Cocktail (APExBIO, USA). Cells were mechanically lysed using the grinding tube and gently ground for 5 minutes to release cellular contents. The lysate was cleared of cellular debris and nuclei by centrifugation at 10,000g and the post-processing extract was loaded on a continuous iodixanol density gradient. To generate the 12 fractions continuous iodixanol density gradient, 5%, 10%, 20%, and 40% w/v iodixanol solutions (Sigma-Aldrich) were diluted in tris-sucrose buffer and incubated at 4°C overnight to generate a continuous gradient. The post-processing extract was loaded on the top of the gradient and centrifuged at 100,000 × g for 16 hours at 4°C in SW40 Ti rotor (Beckman Coulter). After centrifugation, fractions were collected and ultracentrifuged at 110,000 × g for 60 min at 4°C, and pellets were resuspended in 5× lysis buffer (Fudebiotech, China) for western blot analysis.
For small EV fractionation, sEV pellets were generated by differential centrifugation as described above, then resuspended in sterile PBS and centrifuged at 100,000g for 16 hours at 4°C. Fractions were carefully extracted from each gradient layer, diluted in PBS and further centrifuged at 100,000 × g for 90 min to obtain purified sEVs for subsequent analysis.

Trypsin protection assay
Autophagic extracellular vesicles (AEVs) were isolated from HEK-293T cell supernatants treated after 24 h of serum-free DMEM treatment. AEVs were purified as described and divided equally into three tubes (20 µL per tube). To each, 20 μL of PBS, 20 μL of 100 μg/ml trypsin (Gibco, USA) in PBS, or 20 μL of 100 μg/ml trypsin with 2% Triton X-100 (Sigma-Aldrich, USA) was added. After incubating at 4°C for 30 min with occasional flicking, the reactions were terminated by adding 5 μL 5× loading buffer (Fudebiotech, China) and heating at 95°C for 5 min. Each sample (25 µL) was then loaded for western blot analysis.

Surface proteins of single EV by proximity-dependent barcoding assay (PBA)
To profile single EV surface proteins, PBA was carried out using antibody-DNA conjugates and next-generation sequencing. PBA probes were prepared through chemical conjugation of antibodies with DNA oligonucleotides containing unique protein and molecular tags and a universal primer-binding sequence. For EV capture, biotinylated cholera toxin subunit B (CTB) was added to streptavidin-coated 96-well plates, incubated at room temperature for 20 minutes, and washed with PBST three times. Next, the sample-antibody mixture was diluted to 20 μL in PBA buffer and transferred to each CTB-coated well, allowing EVs to bind via ganglioside GM1 (which is enriched in membrane lipid rafts). Captured EVs were fixed with 4% paraformaldehyde.
For EV proteomic analysis, DNA polymerase extended the antibody-conjugated DNA, generating specific EV tags. The extended DNA fragments were compiled into a library and sequenced. Tag matching then produced EV counts and EV-protein data matrices, identifying individual EV surface protein profiles. For subpopulation analysis, FlowSOM was applied for dimensionality reduction and clustering of PBA data, with clusters visualized using tSNE and UMAP methods, enabling detailed analysis of EV subtypes at the single-vesicle level. The PBA assays were designed according to the protocols published by Vesicode (Solna, Sweden) and performed by Echobiotech (Beijing, China).

Mass spectrometry (MS) of extracellular vesicles
Small EVs or AEVs from wild-type, Rab11 knockout or Rab13 knockout HEK-293T cells were lysed using SDT buffer (4% SDS, 100 mM Tris-HCl, pH 7.6). Proteins were further purified by SDS-PAGE gels, and gel bands were excised and eluted. The proteins were then digested into peptide fragments by trypsin incubation at 37°C for 15-18 h, and the resulting peptides were collected as a filtrate. The peptides of each sample were desalted on C18 Cartridges (Thermo scientific). For mass spectrometry analysis, 15 μL of the prepared protein sample was loaded into the injection tube and subjected to liquid chromatography-mass spectrometry (LC-MS). Peptides were separated via liquid chromatography, ionized, and analyzed by Orbitrap Astral mass spectrometer (Thermo Scientific) connected to an Vanquish Neo system liquid chromatography (Thermo Scientific) in the data-independent acquisition (DIA) mode. DIA data was analyzed with DIA-NN 1.8.1. All reported data were based on 99% confidence for protein identification as determined by false discovery rate (FDR) ≤1%. For protein identification and quantification, the recorded spectra from three independent biological replicates were searched by UniProt human proteome database. GO enrichment analyses were performed using DAVID (Database for Annotation, Visualization and Integrated Discovery; https://david.ncifcrf.gov/).

Western blot
Samples of treated cell and sEV fractions were lysed in RIPA lysis buffer (phenylmethylsulfonyl fluoride, NaF, NaVO4 and protease inhibitor, Santa Cruz) for 15 minutes on ice and then subjected to a centrifugation at 12,000 × g for 10 minutes at 4°C. Lysates were quantified by BCA assay (#23225, Thermo Fisher Scientific), boiled in 5× sample loading buffer (Fudebiotech, China), separated by 12% SDS-PAGE gels (Bio-Rad, USA), and transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Membranes were blocked with 5% nonfat dry milk in TBST and then incubated with appropriate primary antibodies overnight at 4°C, followed by incubating with HRP-conjugated secondary antibody (Southern Biotech, 1:5000) for 1 hour at room temperature and exposed using an enhanced chemiluminescence system (NCM Biotech, Suzhou, China). Primary antibodies included the following: SQSTM1/p62 (#ab109012, Abcam, 1:2000), CD9 (#ab236630, Abcam, 1:1000), CD63 (#ab193349, Abcam, 1:1000), CHMP2A (#10477-1-AP, Proteintech, 1:500), VPS4A/B (#17673-1-AP, Proteintech, 1:1000), LC3B (#A19665, Abclonal, 1:1000), NBR1 (#16004-1-AP, Proteintech, 1:500), TSG101 (#14497-1-AP, Proteintech, 1:500) SDCBP/Syntenin-1 (#2239-1-AP, Proteintech, 1:500), CD81 (#sc-166029, Santa Cruz Biotechnology, 1:200). Rab13 (#A10571, Abclonal, 1:500), GAPDH (#AC033, Abclonal, 1:2000). CD81 (#MAB4615, R&D, 1:500), Calreticulin (#12238, Cell Signaling technology, 1:1000), Rab7 (#9367, Cell Signaling technology, 1:1000), α-tubulin (#5335, Cell Signaling technology, 1:2000), Rab11 (#5589, Cell Signaling technology, 1:1000), Rab5 (#3547, Cell Signaling technology, 1:1000), EEA1 (#sc-137130, Santa Cruz Biotechnology, 1:500). Enterovirus 71 VP1 (#GTX132339, GeneTex, 1:1000), Anti-Coxsackievirus B4 (This antibody developed in our laboratory through the immunization of mice with inactivated CVB4 particles, the serum obtained was purified and verified using Enzyme-linked immunosorbent assay (ELISA)).

Fluorescence and immunofluorescence microscopy
The wild-type, Rab13 knockout or CHMP4A/B knockdown HEK-293T cells were seeded to cover glass slides. After the designed treatment, the cells were washed for 3 times with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Subsequently, cells were permeabilized with 0.5% Triton X-100 for 15 minutes, followed by blocking with 5% BSA in PBS for 60 min. For endogenous protein detection, cells were incubated with diverse groups of primary antibodies as following: mouse anti-CD63 (#ab193349, Abcam, 1:500) and Rabbit anti-Rab13 (#A10572, 1:200); mouse anti-LC3B (#A17424, Abclonal, 1:400) and Rabbit anti-Rab13 (#A10572, Abclonal, 1:200); mouse anti-p62 (#56416, Abcam, 1:500) and Rabbit anti-Rab13 (#A10572, Abclonal, 1:200); Rabbit anti-CD63 (#ab252919, Abcam, 1:500) and mouse anti-LC3B (#A17424, Abclonal, 1:400). All primary antibodies were diluted in antibody dilution buffer (#003218, Life Technologies) and incubated overnight at 4°C. Coverslips were washed in PBS before being incubated with species-specific Alexa Fluor-conjugated (488 and 647) secondary antibodies (diluted 1:1000 in PBS) for 1 h at room temperature and washed again in PBS. DAPI dyes (Beyotime Biotechnology, Shanghai, China) was used for cell nucleic acid stains. Fluorescence microscopy was performed using a Nikon Eclipse Ti confocal microscope (Tokyo, Japan) equipped with s a Nikon A1R-HD25 confocal microscope (ran by NIS-Elements) equipped with an ApoTIRF 60x/1.49 NA oil immersion lens.

Transmission electron microscopy (TEM)
Wild-type cells and knockdown cells were fixed using 2% w/v PFA and 1.5% glutaraldehyde (#G5882, Sigma-Aldrich) in 0.1 M sodium cacodylate buffer (pH 7.4) at room temperature. Cells were washed in ice-cold distilled water and placed in 2% buffered osmium tetroxide at 4°C overnight. Then cells were incubated in graded alcohol concentrations (20%, 50%, 70%, 90%, 100%, 100%; 2 min in each), and embedded in epoxy resin (#45345, Sigma-Aldrich) according to standard protocols. Ultrathin sections of 70 nm thickness were prepared using a Leica Ultramicrotome UC7. The morphology of purified AEVs was measured by TEM as our previous description26. Images were collected on a Hitachi HT7700 transmission electron microscope (Tokyo, Japan).

Immunoelectron microscopy
A 10 μL drop of the sample solution containing AEVs was adsorbed to a glow-discharged carbon-coated copper grid (ZhongJingKeYi Ltd., Beijing, China), blocked with 0.5% BSA for 20 min at room temperature and incubated with an antibody against LC3B (#M152-3, MBL, 1:100), p62 ((#PM045, MBL, 1:100) and Rab13 (#A10571, Abclonal, 1:50) in 1% BSA for 2 hour at room temperature. The samples were washed 3 times with PBS for 5 min and incubated with a 10 nm anti-mouse (#G7777, Sigma-Aldrich) or anti-rabbit (#7402, Sigma-Aldrich) IgG-Gold antibody diluted 1:100 in 1% BSA for 1 hour at room temperature. After washing 3 times with PBS and deionized water for 5 min, samples were stained with freshly prepared 1% uranyl acetate. Samples were imaged on a Hitachi H-7700 transmission electron microscope and operated at an acceleration voltage of 80 kV.

Flow cytometric sorting of CD63 and LC3 positive amphisomes
HEK 293T cells were co-transfected with expression vectors CD63-mCherry and LC3-GFP using Lipofectamine 3000 (Life Technologies, USA) at 70% confluence. After 24 hours, the cells were cultured in serum-free DMEM for an additional 24 hours. The cells were collected and disrupted mechanically using a glass homogenizer in cold PBS on ice. The lysates were centrifuged at 10000 × g for 15 minutes, suspended and filtered through a 0.8 μm membrane filter (Millipore). The mCherry and GFP double-positive amphisomes were sorted using a flow cytometry (FACSAria Ⅲ Cell Sorter, BD). The sorting sample were further suspended in PBS and centrifugation at 40,000 × g for 30 minutes to precipitate the amphisomes. The mCherry-GFP-positive amphisome pellets were resuspended in PBS and were coated onto a cover slip intended for confocal imaging. Fluorescence microscopy was performed using a Nikon Eclipse Ti confocal microscope (Tokyo, Japan) equipped with s a Nikon A1R-HD25 confocal microscope equipped with an ApoTIRF 60x/1.49 NA oil immersion lens.

Analysis of vesicles-enclosed enterovirus infection
For detecting the vesicle-enclosed virion infection, equal number of wild-type or Rab11-knockout 293T cells infected with CVB4 at 0.05 TCID50 or EV71 at 0.03 TCID50 for 24 h under 10% EV-depleted FBS medium to avoid cell necrosis and apoptosis, and corresponding AEV or sEV pellets were isolated as described above. An immune-selection method was used to remove the free enteroviral virion that mixed in AEV or sEV pellets, the AEVs or sEV pellets were resuspended in 1 mL sterile PBS and incubated with excess CVB4- or EV71-specific antibodies at 4°C overnight. Then the suspension solutions mixed with Protein A/G magnetic beads (Millipore) for 30 minutes at room temperature. The tubes placed into the magnetic stand to capture the beads resulted in collecting free virions, then transferred supernatants to new centrifuge tubes (purified AEVs or purified vesicles) for TEM analysis or treating with various cell lines (NIH3T3 or L929 cells).
For determination of viral RNA copy number, the total RNA was isolated from purified AEV, sEV pellets or treated cells using TRIzol reagent (TaKaRa) according to the manufacturer’s specification and then reverse transcribed using random hexamers with the reverse-transcription kit (TaKaRa). The cDNA was subjected to quantitative PCR using SYBR-Green Master Mix (Life Technologies) on an ABI QuantStudio 6 Flex for 40 cycles. The primers were CVB4-Forward (5’-CGGGAGCGTCCTCTTACATC-3’) and CVB4-Reverse (5’-GACGTAAAAACTGCGTGCT-3’); EV71-Forward (5’-AGATAGGGTGGCAGATGT AATTGAAAG-3’) and EV71- Reverse (5’- TAGCATTTGATGATGCTC CAAT-3’). The fragment corresponding to nucleotides 1648-1780 of CVB4 and 2462-2635 of EV71 were adjusted to a concentration gradient (1×101 copies/μL to 1×108 copies/μL) and were used as the standard to calculate the copy number of CVB4 and EV71 RNA, respectively.
For determination of EV71 complete genome in sEVs and AEVs, the primers used were EV71-Forward (5’-AGAGCTTCGTTCAGCACTCC-3’) and EV71-Reverse (5’-GGAAGGTCGCATTACCCCAA-3’).

Clinical specimens
This study adhered to the ethical principles of the Declaration of Helsinki. Human specimens were obtained from voluntarily consenting patients at Affiliated Hospital of Medical School Nanjing University (Suzhou Hospital, Suzhou, China). To qualify as a healthy donor, individuals must fulfill health criteria, including the absence of chronic or communicable diseases and demonstration of normal findings in standard blood tests, urinalysis, liver function tests, and renal function evaluations. The serum samples from healthy individuals were selected and divided into two groups. The age distribution of the young group was 18-40 years old, and the age of the elderly group was over 65 years old. This study was approved by the Ethics Committee of the Suzhou Hospital of Nanjing University.

Animal experiment
Wild-type C57BL/6J mice were purchased form Jihui Laboratory Animal Care Co., Ltd (Shanghai, China). Rab27a-konckout (Rab27a-KO) mice (C57BL/6J background) were constructed by Cyagen Biosciences (Suzhou, China). Mice were maintained in a temperature-controlled environment at 25°C with a 12 h light and 12 h dark cycle. For starvation experiment, ten weeks-old of wild-type and Rab27a-KO mice were fasted for 48 hours but supplied with drinking water. At the end of the fasting period, the mice were euthanized, livers were collected for WB analysis and blood samples were collected for the assessment of the particle size distribution of sEV or AEVs. For vesicles-enclosed CVB4 infection experiment, each wild-type mouse (n=6) was injected with AEVs or vesicles isolated from supernatant of 5×107 CVB4-infected wild-type or Rab11-KO 293T cells through intra-peritoneally. The injected mice were continuously observed to record survival status. Mice were dissected at 24 h post-injection to collect brain, heart, spleen, lung, kidney, pancreas and small intestine tissues to screen virus replication. Total RNA was extracted from individual tissues using TRIzol (Life Technologies, USA) and the viral RNA copy number was determined as described above.
All animal experiments were performed in accordance with the recommendations of the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, PR China, 1998) and the guidelines of the Laboratory Animal Ethical Commission of Soochow University (#202309A0476).

Statistical analyses
All data were depicted using scatter plots where the mean ± SD were shown superimposed over the individual data points using GraphPad Prism 9.0 software (GraphPad Software Inc., San Diego CA). Two-tailed Student’s t-test was used to compare differences between two groups, while analysis of variance (ANOVA) followed by the Dunnett’s test was used to assess differences between more than two groups. P-value of less than 0.05 was considered statistically significant, indicated as *p<0.05, **p<0.01, ***p<0.001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Mao et al. reported a new subtype of small extracellular vesicles AEVs that distinct form conventional exosomes from serum-starved cells and starved mice. Methods are provided for the isolation of AEVs, and the importance of AEVs in the delivery of viral particles and viral genomes is emphasized. However, the mechanism and functional significance of AEVs are poorly defined. Also the novelty of this manuscript is limited, all involved molecules including Rab11, Rab13, Rab27 and ESCRT are already reported in exosome function previously. Mechanistic details how these proteins affect AEV secretion are missing.\n1. There are many mistakes in this manuscript, listed as following:\na. Line 456: The description “RAB13 cells” should to be correspond to the figure7A “RAB11 KO cells”.\nb. Line195: “Fig.3C” should be changed into “Fig.2C”.\nc. Line198: “Fig.3C” should be changed into “Fig.2C”.\nd. “NBRl” in Figure 1K, 4E and 5D should be changed into “NBR1”.\ne. The scar bar in ZOOM picture in Figure 5A,5E, 5F, 5G, 6B, 6C and 6D should be showed.\n2. The sampling in EM is of very small areas in small numbers of cells so is particularly prone to the pseudoreplication bias discussed above. Showing multiple examples in the supplementary information and including quantitative analysis are necessary.\n3. Multiple fields or cells should to be statistically quantified to better describe the colocalization of Rab13, CD63, LC3B and p62 under different treatment conditions. What’ more, CD63 and LC3B are well co-localized in Figure 5F, not as described in the line 365.\n4. In figure S1A and S1D, why p62 band was deeper in starved Neuro-2a cells and THP-1 cells than normal condition? It’s different from the trend in figure 1A.\n5. How cells promote AEVs secretion in response to starvation signals and how Rab11 and ESCRT III are involved in the formation and secretion of the AEVs? The authors need to provide more mechanistic details about the AEVs formation, sorting and release.\n6. If defined purely by diameter, it seems like AEVs are just a subtype of exosomes. What is the difference between the functions of AEVs and exosomes?\n7. The presence of autophagosome components in exosomes does not seem surprising，why are the amphisomes fused more with the plasma membrane to release AEVs than with lysosome for degradation in starvation condition? Lysosomal function in starvation condition should be measured to explore the cause of the increase release of AEVs.\n8. In Figure 1D and 1J, the framed areas in Normal or Mock conditions appears way more than 3% or 1.7%.\n9. The fonts in Figure 3E-H are too small to read.\n10. Figure 4E is not mentioned in the text.\n11. The EM images of AEVs are mostly descriptive. If the authors claim these vesicles are AEVs, the immunogold staining or CLEM should be performed.", "reviewer_2": "In this manuscript, the authors demonstrate that inducing autophagy through nutrient starvation or viral infection causes a proliferation of small extracellular vesicles with a diameter of 70-100 nm, which they term Autophagic Extracellular Vesicles (AEV), without impacting the overall production of conventional small extracellular vesicles (sEVs). These AEVs differ from sEVs in size, the type of cargos they envelop, and the composition of their secretome. Their secretion in contrast to sEVs, does not require TSG101 or SDCBP. Instead, their secretion and biogenesis depend on Rab11 and components of the ESCRT pathway, including VPS4A, CHMP4A, and CHMP4B. Interestingly, AEVs generated during enteroviral infection, they found, in contrast to naked virions, can infect nonpermissive cells.\nWhile these findings are of interest and might enrich our understanding of secretory autophagy, the manuscript is difficult to understand in places as written and the authors' intent may be obscured. While the data in this manuscript show that the AEVs are likely distinct from sEVs, many figures lack appropriate controls, and the figure legends miss important details. The nomenclature \"autophagic extracellular vesicles,\" is confusing, since the AEVs (70-100 nm) share a similar size range to small extracellular vesicles (30-200 nm). Furthermore, to confirm a role for autophagy in the induction of the AEVs, the authors should also knock out ATG7 or ATG5 and show that the increase of AEVs upon starvation in Figure 1 is attenuated in these knockout cells.\nIn Figure 1A, the authors demonstrate that starvation induces complete autophagy as indicated by the loss of p62 staining. Given that autophagy has been previously reported to redirect the MVBs to lysosomes for degradation and consequently reduce exosome release, could the authors discuss how inducing autophagy increases the secretion of sEVs >100 nm?\nIn Figure S2L, the authors demonstrated that the percentage of sEVs <100 nm in diameter is increased in older adults compared to younger adults. Since autophagy declines with age and the sEVs <100 nm are induced by autophagy, shouldn't younger adults have a higher amount of sEVs <100nm than older adults? The authors should comment on this connection.\nIn Figure 2B, the authors show that the knockout of TSG101 and SDCBP increased the percentage of sEVs >100 nm, indicating that these host proteins inhibit sEVs secretion during starvation. However, Figure 2B lacks the no-starvation controls for comparison for all three groups. Without these controls to show the baseline percentage of sEVs >100 nm, the interpretation of these results will be difficult. Similarly, appropriate controls should be included in Figures 2C, 2D, and 2E, and the figures should be properly labeled.\nThe authors show in Figures 2 and 3 that the AEVs have a distinct secretome from the sEVs. To further confirm these results, the authors should directly compare AEVs and sEVs with and without starvation by doing a western blot against proteins enriched in both sets of EVs.\nGiven that Rab27a KO reduced the number of AEVs/cell (Figure 6E) and attenuated the secretion of AEV-associated proteins (Figure 6F), the authors concluded (lines 339-341) that Rab27a is required for the fusion of secretory amphisomes with the plasma membrane. This is an overstatement because these assays did not directly examine the effects of Rab27a on exosome-plasma membrane fusion and should be revised accordingly.\nExosomes induced during enteroviral infection were previously shown to be around 300-900 nm in diameter and can contain many capsids. In Figure 7A, the authors show that the AEVs induced during CVB4 infection contain CVB4 virions. However, given the size of CVB4 capsids (around 30 nm), it is difficult to understand how AEVs with a diameter of 70-100nm can contain CVB4 capsids. Could the authors address this in the discussion?\nSince Rab11 KO blocks the generation of AEVs during starvation (Figure 2D), I'm surprised that treating HeLa cells with AEVs derived from RAB11 KO cells yielded comparable viral RNA copy numbers to those derived from WT cells (Figures 7C and D). Could the authors explain how these experiments were performed and how they obtained enough AEVs from Rab11 KO cells?", "reviewer_3": "The article by Mao et al describes a novel type of extracellular vesicles, induced by starvation, which the authors identify as formed by an autophagic pathway, and secreted from amphisomes. The authors also identify some molecules of the ESCRT-III family, Rab27 and Rab13 as required for their biogenesis, secretion and protein cargo sorting into them. These EVs are different from other small EVs whose secretion depends on other molecules. Finally, the authors show that these autophagic EVs are produced upon virus infection of the producing cells, contain components of the virus, and exhibit infectivity.\nThe results are interesting and novel, and well performed for the most part.\nHowever, it is not clearly explained in the results and throughout the text how much these autophagic EVs (AEVs) are identical or similar to the Rab11-enriched EVs induced by starvation described previously by the group of D. Goberdhan. The authors must clarify this upfront in the results section, and justify the use of a novel term (AEV) rather than the previous Rab11-enriched EV term, or change to the other terminology.\nI am also not very convinced by the immunofluorescence images (super resolution microscopy), which are very much over-exposed and impossible to interpret.\nSeveral grammatical mistakes are also present throughout the text, and the authors could improve by asking a native English speaker for corrections.\nFinally, the molecules and machineries the authors seem to consider as specific of exosomes (= MVB-derived EVs) are not really right, some of them are in fact abundant or required for production of plasma membrane-derived small EVs (eg CD9, TSG101), the authors should be more careful in their wording and message in that line.\nApart from that, I have a few specific comments listed below:\nFigure1: particles are analysed only for size by NTA. I am surprised of the results, since in our hands, NTA always gives a single peak around 100nm, and I never saw a smaller peak, but maybe the instruments of the settings used by the authors specifically allow this detection..However, a potential artefact should be excluded by performing the NTA analysis on non-conditioned medium containing EV-depleted serum (precultured in empty cell dishes at 37°C for the time used to condition medium in the presence of cells), to see how much of the remaining serum-derived particles may mask the 2nd peak observed in starvation (no serum) condition.\nIn panels K-P, the increase of small particles detected could be representing virus secretion, can the authors exclude this possibility?\nFig2A-B: please clarify if these quantifications are performed on cells exposed to starvation or not. And if one is without starvation (2A?) and one with (2B?) then please show number of particles released by the KO cells in starvation condition, not only the % of smaller than 100nm.\nAlso, in all these experiments where HEK293T cells are exposed to starvation, the authors should quantify the extent of cell death induced, because cell death could lead to release of some death-induced vesicles, and cells ko for one or the other machinery could possibly be more or less resistant to starvation-induced death.\nFig2D-E: I did not understand reading the text and looking at the results if the EVs the authors look at are the same as the Rab11-enriched EVs induced by starvation described by the group of D Goberdhan in several articles (Fan… Goberdhan, EMBO J 2020, Marie… Goberdhan, JEV 2023) or different: please quote the other group and describe more clearly how your vesicles and theirs are similar or different. In particular, is Rab11 present in the AEVs described here? If they are similar (likely, given the enrichment of Rab11 shown in WB of fig3C), why use now a new nomenclature (AEV) rather than the previous nomenclature of Rab11-enriched EV ?\nIn that line, in Fig2G: it would have been useful to compare starvation-induced EV from WT and Rab11-ko cells (although maybe not useful since the authors show a full proteomic analysis comparing wt and rab11 ko EV, in fig3G-I).\nIn the current Fig2G, 3 other clusters of EVs seem to be enriched in the starvation conditions: three blue and one deep red below the deep orange cluster 12. Can the authors comment also on these clusters and the proteins in them?\nFinally, from this analysis, CD151 is the major protein of cluster 12, CD151 is a tetraspanin, could the authors determine whether their EVs could be easily identified as CD151+ EVs?\nFig5A and 5E-F are highly overexposed and do not allow to visualize properly the distribution of the markers analysed.\nThere is also no quantification provided.\nSpecific wordings requiring corrections:\nResults paragraph 2, line 178: exosomes are not strictly described as smaller than 100nm in diameter, some articles propose rather a range of 50 to 150nm.\nLine 180: TSG101 is not only involved in exosome secretion, but also in plasma membrane-derived vesicles induced by ARRDC1. SDCBP (syntenin) is also not clearly specific of exosomes; The authors should rewrite with more caution this part, saying that they evaluate the dependence on various intracellular machineris previously proposed to be required for secretion of different subtypes of small EVs.\nLines 195 and 198: the authors probably refer to fig2C, rather than 3C.\nLines 206-7: “ the secretion of vesicles with size less than 100 nm were failed increase despite autophagy induction” what does it mean, did the % increase or not? Are the EVs the authors look at the same as the Rab11-EV induced by starvation described by the group of D Goberdhan in several articles (Fan… Goberdhan, EMBO J 2020, Marie… Goberdhan, JEV 2023): please quote the other group and describe more clearly how your vesicles and theirs are similar or different.\nLine 241 and 295: “the AEVs exhibit a different secretome” and “possesses a distinct secretome”, replace secretome by composition (the secretome is what cells release, not what EVs contain)\nLine 317: according to legend, Fig4C is EVs, whereas 4D is cells. LC3b is enriched in fraction 9 in 4D (hence cells), please clarify upfront in line 317 that the enrichment in fraction 9 is observed in cells and not EVs (this info arrives line 319, and mention “cell lysate supernatant”, why supernatant?)\nFigure 8: where would the authors place CD81 in the autophagosome, since their results show that it is more associated to AEVs than CD63 and CD9? It would have been interesting to see quantified immunofluorescence images for this molecule in the different cells and conditions.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: Regrettably, our manuscript did not receive a positive assessment from the reviewer.\nIn our study, we identified a novel subtype of small extracellular vesicles, termed AEVs\n(Autophagic Extracellular Vesicles) that were distinct from conventional exosomes. The AEVs\ncontained specific cargos such as autophagic proteins and ESCRT-III components which were\nnot present in classic exosomes. We also demonstrated that the biogenesis of AEVs functionally\nrequired ESCRT-Ⅲcomplex components, specifically CHMP4A and CHMP4B, alongside the\nGTPase Rab27a. To our knowledge, this type of vesicle has not been reported before and may\nbe confused with conventional exosomes. Our study not only highlighted the importance of\nrelationship between autophagy and extracellular vesicles, but also provided novel perspectives\non heterogeneity of extracellular vesicles, which facilitates the advancement of extracellular\nvesicles in clinical detection.\n1. There are many mistakes in this manuscript, listed as following:\na. Line 456: The description “RAB13 cells” should to be correspond to the figure7A “RAB11\nKO cells”.\nb. Line195: “Fig.3C” should be changed into “Fig.2C”.\nc. Line198: “Fig.3C” should be changed into “Fig.2C”.\nd. “NBRl” in Figure 1K, 4E and 5D should be changed into “NBR1”.\ne. The scar bar in ZOOM picture in Figure 5A,5E, 5F, 5G, 6B, 6C and 6D should be showed.\nResponse: We apologize for the errors in the paper and thanks for reviewer’s valuable\nsuggestions. We have corrected all errors and added the scar bar to all zoom figures.\n2. The sampling in EM is of very small areas in small numbers of cells so is particularly prone\nto the pseudo replication bias discussed above. Showing multiple examples in the\nsupplementary information and including quantitative analysis are necessary.\nResponse: Thanks for reviewer’s suggestion. In our opinion, the EM images presented in the\nFigure 6G, 7B and 7C clearly exhibited the structure adequately. We aimed to show that the\namphisome contained two distinct lumens in amphisomes. One lumen consisted of a compact\nmultivesicular structure, formed from autophagosome after fusing with MVBs, while the other\nwas filled with typical intraluminal vesicles (ILVs) structure, known as exosomes. We consider\nthat the quantitative analysis of this structure might lack significance.\n3. Multiple fields or cells should to be statistically quantified to better describe the\ncolocalization of Rab13, CD63, LC3B and p62 under different treatment conditions. What’\nmore, CD63 and LC3B are well co-localized in Figure 5F, not as described in the line 365.\nResponse: Thanks for reviewer’s suggestion. We have added the colocalization statistical chart\nin our manuscript (Figure 6E in revised manuscript). In addition, we revised the description of\nthe Figure 5F image co-localization to make it more rigorous.\n4. In figure S1A and S1D, why p62 band was deeper in starved Neuro-2a cells and THP-1 cells\nthan normal condition? It’s different from the trend in figure 1A.\nResponse: Thanks for reviewer’s suggestion. We carried out an in-depth examination of the\nproblem and found that the excessive loading amount on WB led to overly dark bands during\nexposure, masking the actual differences. Consequently, we reduced the loading amount and\nselected more prominent results.\n5. How cells promote AEVs secretion in response to starvation signals and how Rab11 and\nESCRT III are involved in the formation and secretion of the AEVs? The authors need to\nprovide more mechanistic details about the AEVs formation, sorting and release.\nResponse: We identified a novel subtype of small extracellular vesicles, termed AEVs\n(Autophagic Extracellular Vesicles). Amphisomes, a hybrid organelle formed by the fusion of\nmultivesicular bodies (MVBs) with autophagosomes, are essential for secretion of these type\nof vesicles. The Rab11 protein is required for fusion of MVBs and the autophagosomes to\ngenerate the hybrid amphisome, which is subsequently released into the extracellular space as\nAEVs. The ESCRT-III components CHMP4A and CHMP4B, along with VPS4A/B, were\nconfirmed to contribute to membrane fission in amphisomes, leading to intracellular AEV\nbiogenesis according our data. Evidences confirmed that the ESCRT-III/VPS4 complexes can\npromote both inside-out (positively curved membranes) and outside-in (negatively curved\nmembranes) membrane fission reactions, and emerging mechanistic models for ESCRT-\nmediated membrane fission. Therefore, we can speculate that the ESCRT-III components\nCHMP4A and CHMP4B, in conjunction with VPS4, could target to the side of autophagosome\nmembranes during amphisome formation and promote negatively curved membranes result in\ngenerating the luminal vesicles known as AEVs.\n6. If defined purely by diameter, it seems like AEVs are just a subtype of exosomes. What is\nthe difference between the functions of AEVs and exosomes?\nResponse: Exosomes are characterized as extracellular vesicles with a size range of 50 - 150\nnm, and are marked by proteins including CD63, CD9, CD81, and TSG101. In contrast, AEVs\npredominantly fall within the 70 -100 nm range. They are identified by markers such as Rab13,\nautophagy - related proteins, and ESCRT - III components. Notably, AEVs are released in large\nquantities during autophagic induction, whereas exosomes are secreted continuously.\n7. The presence of autophagosome components in exosomes does not seem surprising, why are\nthe amphisomes fused more with the plasma membrane to release AEVs than with lysosome\nfor degradation in starvation condition? Lysosomal function in starvation condition should be\nmeasured to explore the cause of the increase release of AEVs.\nResponse: We deem this to be a significant question. There are two pathways for\nautophagosomes: one is fusion with lysosomes and degradation of their contents, and the other\nis fusion with MVBs to form amphisomes, which are then released extracellularly. Although it\nhas been found that disrupting lysosomes leads to an increase in the fusion of autophagosomes\nwith MVBs, thereby promoting extracellular release, it remains unclear what mechanisms\nregulate the sorting between these two pathways, warranting further investigation. We are\ninvestigating the molecular mechanisms underlying this issue.\n8. In Figure 1D and 1J, the framed areas in Normal or Mock conditions appears way more than\n3% or 1.7%.\nResponse: We consider this to be a misunderstanding. Through several checks, we confirmed\nthat there were no mistakes in the outcomes.\n9. The fonts in Figure 3E-H are too small to read.\nResponse: Thanks for reviewer’s suggestion. We have magnified the text in the image to\nenhance its clarity.\n10. Figure 4E is not mentioned in the text.\nResponse: Thanks for reviewer’s suggestion. We have corrected this issue.\n11. The EM images of AEVs are mostly descriptive. If the authors claim these vesicles are\nAEVs, the immunogold staining or CLEM should be performed.\nResponse: Thanks for highlighting the need to strengthen the identification of AEVs in our EM\nimages. This problem has indeed been a persistent challenge for us. We have carried out no\nfewer than 11 experiments, yet the results have consistently failed to meet expectations. We\nanalyzed the reasons and consider that it might be due to the expression of AEV's marker\nproteins LC3B and p62 in multiple intracellular locations, such as autophagosomes, lysosomes,\nand amphisomes, etc., which leads to this situation. Nevertheless, we have supplemented the\nresults of super-resolution confocal microscopy on AEVs and exosomes, and essentially\nconfirmed the existence of AEVs and their distinct from exosomes(Figure 6J in revised\nmanuscript).\nReviewer #2 (Remarks to the Author):\nIn this manuscript, the authors demonstrate that inducing autophagy through nutrient starvation\nor viral infection causes a proliferation of small extracellular vesicles with a diameter of 70-\n100 nm, which they term Autophagic Extracellular Vesicles (AEV), without impacting the\noverall production of conventional small extracellular vesicles (sEVs). These AEVs differ from\nsEVs in size, the type of cargos they envelop, and the composition of their secretome. Their\nsecretion in contrast to sEVs, does not require TSG101 or SDCBP. Instead, their secretion and\nbiogenesis depend on Rab11 and components of the ESCRT pathway, including VPS4A,\nCHMP4A, and CHMP4B. Interestingly, AEVs generated during enteroviral infection, they\nfound, in contrast to naked virions, can infect nonpermissive cells.\nWhile these findings are of interest and might enrich our understanding of secretory autophagy,\nthe manuscript is difficult to understand in places as written and the authors' intent may be\nobscured. While the data in this manuscript show that the AEVs are likely distinct from sEVs,\nmany figures lack appropriate controls, and the figure legends miss important details.\nResponse: We gratefully thanks for reviewer’s professional review work. We genuinely\nappreciate reviewer’s constructive feedback regarding the grammar of our manuscript. We\nacknowledge the importance of clear and grammatically correct language in scientific\ncommunication. In response to your comments, we have undertaken a thorough revision of the\nentire manuscript, addressing grammatical errors and enhancing the overall clarity of sentences.\nIn addition, we have supplemented the missing control group experiments in this study, and\nmade comprehensive modifications to the figure legends.\nThe nomenclature \"autophagic extracellular vesicles,\" is confusing, since the AEVs (70-100\nnm) share a similar size range to small extracellular vesicles (30-200 nm). Furthermore, to\nconfirm a role for autophagy in the induction of the AEVs, the authors should also knock out\nATG7 or ATG5 and show that the increase of AEVs upon starvation in Figure 1 is attenuated in\nthese knockout cells.\nResponse: We sincerely appreciate the valuable comments. Although AEVs and exosomes\nexhibit different characteristics, these two types of vesicles belong to the category of small\nextracellular vesicles. Since the generation of AEVs is closely associated with autophagy, while\nthe biogenesis of exosomes has a weaker connection to autophagy, we select the term AEVs for\nthese vesicles. This nomenclature highlights their specific biogenesis traits and sets them apart\nfrom exosomes. Following the reviewer’s suggestions, we have supplemented the experimental\nresults of ATG7 knockout cells in our study (In revised manuscript Figure 3A-C).\nIn Figure 1A, the authors demonstrate that starvation induces complete autophagy as indicated\nby the loss of p62 staining. Given that autophagy has been previously reported to redirect the\nMVBs to lysosomes for degradation and consequently reduce exosome release, could the\nauthors discuss how inducing autophagy increases the secretion of sEVs >100 nm?\nResponse: The current research reports on how autophagy affects exosome secretion present\nseveral conflicts. We have conducted an analysis and consider the potential reason to be the\nnon-uniformity of the methods for extracting extracellular vesicles, which results in differences\nin experimental results. In addition, we think that many studies confuse AEVs with exosomes,\nleading to biased experimental results.\nIn Figure S2L, the authors demonstrated that the percentage of sEVs <100 nm in diameter is\nincreased in older adults compared to younger adults. Since autophagy declines with age and\nthe sEVs <100 nm are induced by autophagy, shouldn't younger adults have a higher amount\nof sEVs <100nm than older adults? The authors should comment on this connection.\nResponse: We thank the reviewer for the suggestions. This clinical sample testing experiment\ninvolved the detection of AEV in the serum of young healthy subjects and elderly healthy\npopulations. Previous researches had demonstrated that lysosomal degradation pathways\nfrequently malfunction in aging cells, leading to the accumulation of some proteins within the\ncell and accelerating the process of cellular aging (PMID: 34901876, 34563704; 37811693).\nConsequently, when the lysosomal pathway is impaired, autophagosomes accumulate inside\nthe cell, increasing the number of formed amphisome through fusion with MVBs, and thereby\nleading to a rise in AEV release. Remarkably, several studies demonstrated that the secretion of\nsmall extracellular vesicles was significantly increased in serum of elderly samples(PMID:\n34609061; 35665306; 39266768). Therefore, the conclusion that the increase of AEV secretion\nin the aging population is explainable.\nIn Figure 2B, the authors show that the knockout of TSG101 and SDCBP increased the\npercentage of sEVs >100 nm, indicating that these host proteins inhibit sEVs secretion during\nstarvation. However, Figure 2B lacks the no-starvation controls for comparison for all three\ngroups. Without these controls to show the baseline percentage of sEVs >100 nm, the\ninterpretation of these results will be difficult. Similarly, appropriate controls should be\nincluded in Figures 2C, 2D, and 2E, and the figures should be properly labeled.\nResponse: According to the reviewer’s suggestion. We have provided the no-starvation\ncontrols for comparison. (In revised manuscript Figure 3A-C).\nThe authors show in Figures 2 and 3 that the AEVs have a distinct secretome from the sEVs.\nTo further confirm these results, the authors should directly compare AEVs and sEVs with and\nwithout starvation by doing a western blot against proteins enriched in both sets of EVs.\nResponse: Thank for the reviewer’s suggestion. There might be a misunderstanding by the\nreviewer. In our study, we obtained extracellular vesicles, which are sEVs and AEVs, from\nnormally and starvation-cultured cells, and employed proteomics to contrast the protein\ncomponents of sEVs and AEVs (In revised manuscript Figure 4E,4F; Supplementary Table S3).\nIn addition, western blot analysis was carried out to confirm the marker proteins enriched within\nsEV and AEVs from sevral cell lines (In revised manuscript Figure 5A, 5E,5F and 5G).\nGiven that Rab27a KO reduced the number of AEVs/cell (Figure 6E) and attenuated the\nsecretion of AEV-associated proteins (Figure 6F), the authors concluded (lines 339-341) that\nRab27a is required for the fusion of secretory amphisomes with the plasma membrane. This is\nan overstatement because these assays did not directly examine the effects of Rab27a on\nexosome-plasma membrane fusion and should be revised accordingly.\nResponse: We sincerely appreciate the valuable suggestion. We have modified the relevant\ndescriptions in the revised manuscript as follows:\nThese results broached that Rab27a was functionally required for the extracellular secretion\nof autophagic cargos and ESCRT-III components via AEV populations.\nExosomes induced during enteroviral infection were previously shown to be around 300-900\nnm in diameter and can contain many capsids. In Figure 7A, the authors show that the AEVs\ninduced during CVB4 infection contain CVB4 virions. However, given the size of CVB4\ncapsids (around 30 nm), it is difficult to understand how AEVs with a diameter of 70-100nm\ncan contain CVB4 capsids. Could the authors address this in the discussion?\nResponse: It is not uncommon for extracellular vesicles/sEVs with a size of approximately 100\nnm to encapsulate whole enterovirus particles. Besides our study, reports from other research\nendeavors also exist. (PMID: 24722773, 33171580, 36056524, 32409751, 37665326). This\nphenomenon is frequently observed in enteroviral infections. We speculate this might be a\nshared characteristic of enteroviruses: they encapsulate themselves into extracellular vesicles\nto evade immune surveillance and expand their tropism.\nSince Rab11 KO blocks the generation of AEVs during starvation (Figure 2D), I'm surprised\nthat treating HeLa cells with AEVs derived from RAB11 KO cells yielded comparable viral\nRNA copy numbers to those derived from WT cells (Figures 7C and D). Could the authors\nexplain how these experiments were performed and how they obtained enough AEVs from\nRab11 KO cells?\nResponse: Initially, it should be pointed out that the knockout of Rab11 only inhibits the\nproduction of AEVs, but does not affect the release of other small extracellular vesicles such as\nexosomes. Therefore, after infecting equal numbers of wild-type(WT) and Rab11 knockout(KO)\ncells with CVB4 virus, we isolated sEVs and AEVs from WT and Rab11-KO cells, respectively.\nThen, equal numbers of HeLa and NIH 3T3 cells were treated with the isolated sEVs and AEVs\nrespectively. At various time points, total cellular RNA was extracted and viral loads was\ndetected via real-time PCR.\nReviewer #3 (Remarks to the Author):\nThe article by Mao et al describes a novel type of extracellular vesicles, induced by starvation,\nwhich the authors identify as formed by an autophagic pathway, and secreted from amphisomes.\nThe authors also identify some molecules of the ESCRT-III family, Rab27 and Rab13 as\nrequired for their biogenesis, secretion and protein cargo sorting into them. These EVs are\ndifferent from other small EVs whose secretion depends on other molecules. Finally, the authors\nshow that these autophagic EVs are produced upon virus infection of the producing cells,\ncontain components of the virus, and exhibit infectivity.\nThe results are interesting and novel, and well performed for the most part.\nHowever, it is not clearly explained in the results and throughout the text how much these\nautophagic EVs (AEVs) are identical or similar to the Rab11-enriched EVs induced by\nstarvation described previously by the group of D. Goberdhan. The authors must clarify this\nupfront in the results section, and justify the use of a novel term (AEV) rather than the previous\nRab11-enriched EV term, or change to the other terminology.\nResponse: We gratefully thanks for the reviewer’s professional review work. According to the\nreviewer’s suggestion, we explored whether Rab11a-exosomes involved in the autophagy-\ninduced vesicles, then we detected the levels of Rab11a and caveolin-1 in secreted vesicles\nfrom WT and ATG7-KO (Knockout) cells, both of which were enriched in Rab11a-exosomes.\nThen we found these proteins were elevated in secreted vesicles from starved ATG7-KO cells\nwhen compared to those from normally cultured condition (in revised manuscript Figure.3G),\nsuggesting that the Rab11a-exosomes were not of the same subtype as autophagy-induced\nvesicles. We also addressed this issue within the discussion section.\nI am also not very convinced by the immunofluorescence images (super resolution microscopy),\nwhich are very much over-exposed and impossible to interpret.\nResponse: Thanks for highlighting the issue with the immunofluorescence images. We have\nre- acquired the immunofluorescence images using optimized microscopy settings to prevent\nover -exposure. The new images can be found in the revised manuscript (Figures 6A, 6E, 6F,\n7D), and they are consistent with our hypothesis.\nSeveral grammatical mistakes are also present throughout the text, and the authors could\nimprove by asking a native English speaker for corrections.\nResponse: Thanks for pointing out the presence of grammatical mistakes in our manuscript.\nWe have thoroughly proofread the manuscript for common grammatical errors. After that, we\nhave asked two native English-speaking persons with experience in scientific writing to review\nand correct the language throughout the manuscript. All grammar-related changes have been\nmade to enhance the clarity and readability of the manuscript, ensuring that our scientific\nfindings are communicated as effectively as possible.\nFinally, the molecules and machineries the authors seem to consider as specific of exosomes\n(MVB-derived EVs) are not really right, some of them are in fact abundant or required for\nproduction of plasma membrane-derived small EVs (eg CD9, TSG101), the authors should be\nmore careful in their wording and message in that line.\nResponse: We sincerely appreciate the valuable suggestion. We conducted an in-depth\nliterature review to fully understand the roles of CD9, TSG101, and other relevant molecules\nin EV biogenesis. Based on this review, in the revised manuscript, we have adjusted the wording\nthroughout the relevant sections. We have also added appropriate citations in all the modified\nsections to support the new, more accurate statements. This ensures that our manuscript now\npresents a more precise understanding of the roles of these molecules in the context of different\nEV subtypes.\nApart from that, I have a few specific comments listed below:\nFigure1: particles are analysed only for size by NTA. I am surprised of the results, since in our\nhands, NTA always gives a single peak around 100nm, and I never saw a smaller peak, but\nmaybe the instruments of the settings used by the authors specifically allow this detection.\nHowever, a potential artefact should be excluded by performing the NTA analysis on non-\nconditioned medium containing EV-depleted serum (precultured in empty cell dishes at 37°C\nfor the time used to condition medium in the presence of cells), to see how much of the\nremaining serum-derived particles may mask the 2nd peak observed in starvation (no serum)\ncondition.\nResponse: Thank you for the reviewer’s insightful comments. Regarding the NTA setup,\nwe have added a detailed description in the Materials and Methods section. These settings were\noptimized to accurately detect particles in the size range relevant to our study (see line 755-767\nin revised manuscript).\nIn addition, we have performed the proposed control experiment. We cultured non-conditioned\nm\ne\nd\ni\nu\nm\nc\no\nn\nt\na\ni\nn\ni\nIn panels K-P, the increase of small particles detected could be representing virus secretion, can\nn\nthe authors exclude this possibility?\ng\nResponse: VZV is a highly cell-associated virus, and its spread mainly depends on cell-cell\nEtransmission. Therefore, infected cells generally do not release viral particles into the\nV\nsupernatant of the culture medium (PMID: 19193797, 28356523, 22674980).\n-\nd\neFig2A-B: please clarify if these quantifications are performed on cells exposed to starvation or\npnot. And if one is without starvation (2A?) and one with (2B?) then please show number of\nlparticles released by the KO cells in starvation condition, not only the % of smaller than 100nm.\neAlso, in all these experiments where HEK293T cells are exposed to starvation, the authors\ntshould quantify the extent of cell death induced, because cell death could lead to release of\nesome death-induced vesicles, and cells ko for one or the other machinery could possibly be\ndmore or less resistant to starvation-induced death.\nResponse: Thank you for the reviewer’s insightful comments. We have added clear statements\ns\nabout the culture conditions in figure legends. In addition, we had the data on the number of\ne\nparticles released by the cells under the normal or starvation condition (Figure 1F,1G,1H).\nr\nThanks for highlighting the importance of quantifying cell death in our experiments involving\nu\nstarved HEK293T cells. We have incorporated LDH release assays to quantify the extent of cell\nm\ni\nn\ne\ndeath in HEK293T cells exposed to starvation. In the revised manuscript, in the Materials and\nMethods section, we have added a detailed description of the LDH assay procedure: To assess\nthe extent of cell death induced by starvation, LDH release into the culture medium was\nmeasured using a commercially available LDH assay kit (DOJINDO, #CK12), to ensure that\nthe cell mortality rate remained below 5%.\nFig2D-E: I did not understand reading the text and looking at the results if the EVs the authors\nlook at are the same as the Rab11-enriched EVs induced by starvation described by the group\nof D Goberdhan in several articles (Fan… Goberdhan, EMBO J 2020, Marie… Goberdhan,\nJEV 2023) or different: please quote the other group and describe more clearly how your\nvesicles and theirs are similar or different. In particular, is Rab11 present in the AEVs described\nhere? If they are similar (likely, given the enrichment of Rab11 shown in WB of fig3C), why\nuse now a new nomenclature (AEV) rather than the previous nomenclature of Rab11-enriched\nEV ?\nResponse: We gratefully thanks for the reviewer’s professional review work. According to the\nreviewer’s suggestion, we explored whether Rab11a-exosomes involved in the autophagy-\ninduced vesicles, then we detected the levels of Rab11a and caveolin-1 in secreted vesicles\nfrom WT and ATG7-KO (Knockout) cells, both of which were enriched in Rab11a-exosomes.\nThen we found these proteins were elevated in secreted vesicles from starved ATG7-KO cells\nwhen compared to those from normally cultured condition (in revised manuscript Figure.3G),\nsuggesting that the Rab11a-exosomes were not of the same subtype as autophagy-induced\nvesicles. We also addressed this issue within the discussion section.\nIn that line, in Fig2G: it would have been useful to compare starvation-induced EV from WT\nand Rab11-ko cells (although maybe not useful since the authors show a full proteomic analysis\ncomparing wt and rab11 ko EV, in fig3G-I).\nResponse: Thanks for suggesting the comparison of starvation - induced EVs from WT and\nRab11 - ko cells in Fig2G. After careful consideration, we decided not to add this comparison.\nWhile it could potentially offer some insights, we believe that the full proteomic analysis\ncomparing WT and Rab11 ko EVs in Fig3G - I already provides comprehensive information\non the differences between these two cell types in terms of EV composition. Adding the\nproposed comparison in Fig2G might lead to some redundancy.\nIn the current Fig2G, 3 other clusters of EVs seem to be enriched in the starvation conditions:\nthree blue and one deep red below the deep orange cluster 12. Can the authors comment also\non these clusters and the proteins in them?\nResponse: Thanks for pointing out these additional clusters in Fig2G. It should be noted that\nthe cluster classification shown in the figure is a result of sorting and grouping the 260 proteins\ndetected. Upon careful consideration, we think that this mode of classification could potentially\nimpact the correctness of the PBA outcomes. Therefore, we modified the presentation form of\nthe results and displayed all 260 proteins. Through the comparison of the full spectra, it can be\nseen that the EV secreted by cells after starvation indeed exhibits a different subtype(in Figure\n3H of revised manuscript).\nFinally, from this analysis, CD151 is the major protein of cluster 12, CD151 is a tetraspanin,\ncould the authors determine whether their EVs could be easily identified as CD151+ EVs?\nFig5A and 5E-F are highly overexposed and do not allow to visualize properly the distribution\nof the markers analysed. There is also no quantification provided.\nResponse: Thanks for suggesting the determination of whether our EVs can be identified as\nCD151+ EVs. We previously analyzed the CD151 protein via Western blot. Unfortunately, we\nfound that this protein is not universally expressed as a marker in AEVs; instead, it is only\nexpressed in cells such as 293T.\nFor the image quality, we re-acquired the images with optimized exposure settings. In the\nrevised manuscript, the new images can be found on Figure 6A,6E-F. Regarding quantification,\nwe have added the colocalization statistical chart in our manuscript (Figure 6E in revised\nmanuscript).\nSpecific wordings requiring corrections:\nResults paragraph 2, line 178: exosomes are not strictly described as smaller than 100nm in\ndiameter, some articles propose rather a range of 50 to 150nm.\nResponse: The reviewer’s suggestion is well taken. The relevant correction is made in the\nrevised manuscript.\nLine 180: TSG101 is not only involved in exosome secretion, but also in plasma membrane-\nderived vesicles induced by ARRDC1. SDCBP (syntenin) is also not clearly specific of\nexosomes; The authors should rewrite with more caution this part, saying that they evaluate the\ndependence on various intracellular machineris previously proposed to be required for secretion\nof different subtypes of small EVs.\nResponse: Thanks for highlighting the inaccuracies in our description of the roles of TSG101\nand SDCBP. We first conducted a brief literature review to ensure we had the most current\nunderstanding of the functions of TSG101 and SDCBP. Based on this, we rewrote the relevant\nparts of the revised manuscript.\nLines 195 and 198: the authors probably refer to fig2C, rather than 3C.\nResponse: We apologize for the errors in the manuscript. The relevant correction is made in\nthe revised manuscript.\nLines 206-7: “the secretion of vesicles with size less than 100 nm were failed increase despite\nautophagy induction” what does it mean, did the % increase or not? Are the EVs the authors\nlook at the same as the Rab11-EV induced by starvation described by the group of D Goberdhan\nin several articles (Fan… Goberdhan, EMBO J 2020, Marie… Goberdhan, JEV 2023):\nplease quote the other group and describe more clearly how your vesicles and theirs are similar or\ndifferent.\nResponse: The issue has been exhaustively addressed previously.\nLine 241 and 295: “the AEVs exhibit a different secretome” and “possesses a distinct\nsecretome”, replace secretome by composition (the secretome is what cells release, not what\nEVs contain)\nResponse: The reviewer’s suggestion is well taken. The relevant correction is made in the\nrevised manuscript.\nLine 317: according to legend, Fig4C is EVs, whereas 4D is cells. LC3b is enriched in fraction\n9 in 4D (hence cells), please clarify upfront in line 317 that the enrichment in fraction 9 is\nobserved in cells and not EVs (this info arrives line 319, and mention “cell lysate supernatant”,\nwhy supernatant?)\nResponse: Thanks for pointing out the lack of clarity in our description of the data in Figure 4.\nThe relevant correction is made in the revised manuscript.\nFigure 8: where would the authors place CD81 in the autophagosome, since their results show\nthat it is more associated to AEVs than CD63 and CD9? It would have been interesting to see\nquantified immunofluorescence images for this molecule in the different cells and conditions.\nResponse: Thank you for these insightful comments. The reviewer raised an interesting\nquestion, which we have also considered. However, in the context of the focal issue of our\nmanuscript, the specific localization of CD81 may not be particularly important since CD81 is\nnot unique to AEVs.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: Thanks for sharing this information about the co-review process. We appreciate the\nefforts of both reviewers.\nResponse to Reviewers\nWe would like to thanks the reviewers and editors for the constructive comments. We hope\nthat we have satisfyingly addressed all the key points, and that the manuscript now reaches\nthe standards for publication.\nPlease find below our detailed response (blue ink).\nReviewer #1 (Remarks to the Author):\nWe appreciate that the authors have made partial revisions to the manuscript in response to our\ncomments, including the addition of statistical analysis for immunofluorescence colocalization\nand corrections to some experimental results and text. However, the authors did not provide\nfurther clarification on the functional differences between traditional exosomes and AEVs.\nAdditionally, no lysosomal function assays were conducted under starvation conditions, leaving\nunexplained why amphisomes tend to fuse more with the plasma membrane to release AEVs\nrather than with lysosomes for degradation in starvation conditions. More definitive\nexperiments, such as immunogold staining or correlative light-electron microscopy to confirm\nAEV presence, were also not performed. We believe the evidence defining AEVs remains\ninsufficient. The rigor and novelty of the article are not sufficient to meet the publication\nrequirements of Nature Communication.\nResponse: Thank you for your detailed feedback on the revised manuscript. We understand\nyour concerns regarding the areas that still need improvement, and we are committed to\naddressing them to enhance the quality of our work.\n1. Functional differences between traditional exosomes and AEVs.\nWe acknowledge the importance of the functional differences between traditional exosomes\nand AEVs. To address this, we explored the distinct roles of exosomes and AEVs in enterovirus\ninfection (as shown in Fig.8). Our results revealed that AEVs, rather than exosomes, could carry\nand deliver viral particles or whole genomes into receptor-negative host cells with high\ninfection efficiency, resulted in overcoming restrictions to viral tropism. Our data suggested\nthat the AEVs play the key role in enhancing virus transmission and provide potential antiviral\ntargets. In addition, according to your constructive comment, we added a new subsection to\ndiscuss the functional role differences between exosomes and AEVs in the Discussion section\n(page 18, line 596-603) (shown below).\n“Traditional exosomes are known primarily for their role in cell communication, and transport\nof various cargos including proteins, lipids and nucleic acids. They are derived from MVBs and\nreleased upon MVB-plasma membrane fusion. In contrast, AEVs are associated with autophagy\nprocesses. Our findings suggest that AEVs may play a unique role in maintaining cellular\nhomeostasis during stress conditions, such as starvation and virus infection. For example, while\nexosomes might transfer signaling molecules to neighboring cells under normal conditions,\nAEVs could be more involved in removing damaged cellular components or misfolded proteins\nduring stress. This difference in function could be related to their distinct biogenesis pathways.\nFuture studies, such as those focusing on the contents of exosomes and AEVs, could further\nelucidate these functional differences.”\n2. Lysosomal function assays under starvation conditions\nWe agree with the comment regarding the fate of amphisomes under lysosomal disorders. By\nusing the vacuolar-type ATPase inhibitor Bafilomycin A1 (BafA1), which disrupts lysosome\nacidification, we had confirmed that the BafA1 treatment did not affect the secretion of sEVs\nwith size less than 100 nm (AEVs) (see Fig.1C, D, E and F). To account for this result, we\nincorporated an experiment to detect intracellular amphisomes under starvation or BafA1-\nmediated lysosome inhibition (see Fig.6K, page 13, line 421-430). We added:\n“In addition, amphisomes can also fuse with lysosomes to form autolysosomes, followed by\ndegradation of the cargo. Therefore, we quantified intracellular amphisome numbers upon\nstarvation or BafA1-mediated lysosome inhibition and observed an increase in the number of\namphisomes when lysosomal function was compromised (Fig. 6K). Our data presented above\nin Fig. 1 show no significant alteration in AEV secretion in BafA1-treated cells, suggesting that\nthe intracellular accumulation of amphisomes did not increase their fusion with the plasma\nmembrane as an alternative route for extracellular release. These results suggest that the\ndifferent fates of amphisomes, including lysosomal degradation and extracellular release, are\nindependent”.\nFig.1C, D, E and F\nFig.6K\n3. Definitive experiments to confirm AEV presence\nWe understand that the lack of definitive experiments such as immunogold staining to confirm\nAEV presence undermines the strength of our claims. We have now performed immunogold\nstaining experiments. The immunogold-stained electron microscopy images are presented in\nFigure.5F with a description as follows (see Fig.5F, page 11, line 356-359):\n“Consistently, the immunoelectron microscopy showed that the AEVs isolated from starved\n293T cells exhibited immunoreactivity with antibodies against p62 and Rab13 on the vesicle\nmembranes, while antibodies against LC3B were present inside the vesicles (Fig.5F).”\nFig.5F\nIn the Materials and Methods section (page 30, line 962-973), we added the detail of operational\ninformation for the immunoelectron microscopy.\nWe believe these additional experiments and discussions significantly strengthen our\nmanuscript, addressing the concerns regarding the definition of AEVs. We are confident that\nthe enhanced rigor and novelty of the article now meet the high publication standards of Nature\nCommunications.\nReviewer #3 (Remarks to the Author):\nThe revised version of this article by Mao et al includes new panels in a figure to answer my\nand other reviewers’ comments (ko ATG7, conditions without starvation) and others.\nI am satisfied with the experimental and technical answers and find this article interesting\nand worth publishing.\nThe authors have not done an entirely satisfying job in answering the comments about wording\nand statements about exosomes : I still see incorrect mention that ESCRT (including TSG101)\nand CD9 are specific of exosomes, whereas they are both involved in plasma membrane-\nderived vesicles formation.\nE.g. line 193-194 : « exosomes have been described as having a diameter of 30-150 nm and are\ngenerated by ESCRT pathway mechanisms, » and line 198-199 : « Because TSG101 and ATG7\nhave essential functions in the process of exosome biogenesis and autophagosome formation,\nrespectively », line 204 : « The classical exosome markers CD63 and CD9 » : this is onlyartially\ntrue as other mechanisms than ESCRT have been described for ILV formation, PM-derived EVs\nare also generated via TSG101, and CD9 is now more recognized as a marker of Plasma\nmembrane-derived EVs.\nResponse: Thank you for pointing out these persistent inaccuracies in our wording regarding\nexosomes. We apologize for not fully addressing this issue in our previous revision. We have\nnow carefully reviewed and corrected all the relevant statements throughout the manuscript to\nprovide a more accurate representation.\nIn the revised manuscript:\nFor line 195 - 195: We have rewritten the sentence to read: “Exosomes are typically described\nas having a diameter of 30-150 nm. The ESCRT pathway is involved in exosome biogenesis”.\nFor line 199 - 200:\nThe revised text is: “TSG101 plays a role in the process of exosome biogenesis and ATG7 is\nessential for autophagosome formation”.\nFor line 204:\nWe changed it to: CD63 and CD9, which are often considered markers associated with\nexosomes, were largely decreased in secreted vesicles from TSG101-KO cells compared with\nthose from WT cells, whereas the expression of the marker CD81 decreased slightly.\nSome other minor mistakes here and there could be corrected, for example (not exhaustive) :\nLine 262 : « To identified the characteristic of AEVs » shoudleb « to identify »\nLine 337 : The lysosome marker EEA1, (fractions 4-5), (EEA1 is an early endosome marker,\nnot lysosome)\nResponse: Thank you for pointing out these minor mistakes. We apologize for these inaccurate\nsentences. The relevant corrections have been made in the revised manuscript.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThis is a much improved manuscript. I am mostly satisfied with the revised version of the\nmanuscript. However, I still have a couple of concerns listed below:\n1. The manuscript still requires extensive English editing\nResponse: We sincerely appreciate your feedback regarding the need for extensive English\nediting in our manuscript. We acknowledge that clear and precise language is crucial for\neffectively communicating our research findings. Our manuscript was edited for proper English\nlanguage use, grammar, punctuation, spelling, and overall style by one or more of the highly -\nqualified native English-speaking editors at Springer Nature Author Services (SNAS). The\nverification code is D455-4D72-815E-71F6-2AA0.\n2. The figure legends should be properly written. For instance, no information is provided on\nwhere the relative expression is from and how it is calculated in Figure 3B. I assume it's a\ndensitometry quantitation of the Western blot data in Figure 3A.\nResponse: Thank you for highlighting the issue with our figure legends and pointing out the\ndeficiency in the Figure 3B. We agree that they need to be properly written for better clarity\nand understanding. We applied a similar approach to all figure legends, ensuring that each one\ncontains detailed information about the experimental setup, methods, data analysis, and any\nrelevant controls. We also made sure to use clear and consistent language throughout the figure\nlegends. These changes can be found throughout the manuscript.\n3. Figure 3D shows that AEVs are not induced in starved Rab11-depleted cells. However, it\nappears that these cells are defective in autophagy, much like ATG7 KO cells. If Rab11 impairs\nAEV induction by modulating autophagy, then the conclusion that Rab11 is required for AEV\nbiogenesis should be revised. A western blot probing for p62 turnover upon starvation in these\ncells could help address this concern.\nResponse: Thank you for raising this important concern. We agree that the relationship between\nRab11, autophagy, and AEV induction needs further clarification. To address this, we have\nperformed the western blot analysis to detect the autophagy process in Rab11-depleted cells\nunder normal or starved conditions, using antibody against p62 and LC3B. In the Results\nsection (page7, line 220-223), we presented the new data. Figure 3D shows a decreased p62\nprotein and an increased LC3B-II in starved Rab11-KO cells compared to those in normally\ncultured Rab11-KO cells, indicating Rab11 deletion did not affect the fusion of\nautophagosomes with lysosomes.\nFig.3D"}, {"round": 2, "reviewer_comments": {"reviewer_1": "We appreciate that the authors have made partial revisions to the manuscript in response to our comments, including the addition of statistical analysis for immunofluorescence colocalization and corrections to some experimental results and text. However, the authors did not provide further clarification on the functional differences between traditional exosomes and AEVs. Additionally, no lysosomal function assays were conducted under starvation conditions, leaving unexplained why amphisomes tend to fuse more with the plasma membrane to release AEVs rather than with lysosomes for degradation in starvation conditions. More definitive experiments, such as immunogold staining or correlative light-electron microscopy to confirm AEV presence, were also not performed. We believe the evidence defining AEVs remains insufficient. The rigor and novelty of the article are not sufficient to meet the publication requirements of Nature Communication.", "reviewer_3": "The revised version of this article by Mao et al includes new panels in a figure to answer my and other reviewers’ comments (ko ATG7, conditions without starvation) and others.\nI am satisfied with the experiemntal and technical answers and find this article interesting and worth publishing.\nThe authors have not done an entirely satisfying job in answering the comments about wording and statements about exosomes : I still see incorrect mention that ESCRT (including TSG101) and CD9 are specific of exosomes, whereas they are both involved in plasma membrane-derived vesicles formation.\nE.g. line 193-194 : « exosomes have been described as having a diameter of 30-150 nm and are generated by ESCRT pathway mechanisms, » and line 198-199 : « Because TSG101 and ATG7 have essential functions in the process of exosome biogenesis and autophagosome formation, respectively », line 204 : « The classical exosome markers CD63 and CD9 » : this is onlyartially true as other mechanisms than ESCRT have been described for ILV formation, PM-derived EVs are also generated via TSG101, and CD9 is now more recognized as a marker of Plasma membrane-derived EVs.\nSome other minor mistakes here and there could be corrected, for example (not exhaustive) :\nLine 262 : « To identified the characteristic of AEVs » shoudl eb « to identify »\nLine 337 : The lysosome marker EEA1, (fractions 4-5), (EEA1 is an early endosome marker, not lysosome)", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThis is a much improved manuscript. I am mostly satisfied with the revised version of the manuscript. However, I still have a couple of concerns listed below:\n1. The manuscript still requires extensive English editing\n2. The figure legends should be properly written. For instance, no information is provided on where the relative expression is from and how it is calculated in Figure 3B. I assume it's a densitometry quantitation of the Western blot data in Figure 3A.\n3. Figure 3D shows that AEVs are not induced in starved Rab11-depleted cells. However, it appears that these cells are defective in autophagy, much like ATG7 KO cells. If Rab11 impairs AEV induction by modulating autophagy, then the conclusion that Rab11 is required for AEV biogenesis should be revised. A western blot probing for p62 turnover upon starvation in these cells could help address this concern."}, "author_response": "Response to Reviewers\nWe would like to thanks the reviewers and editors for the constructive comments. We hope\nthat we have satisfyingly addressed all the key points, and that the manuscript now reaches\nthe standards for publication.\nPlease find below our detailed response (blue ink).\nReviewer #1 (Remarks to the Author):\nWe appreciate that the authors have made partial revisions to the manuscript in response to our\ncomments, including the addition of statistical analysis for immunofluorescence colocalization\nand corrections to some experimental results and text. However, the authors did not provide\nfurther clarification on the functional differences between traditional exosomes and AEVs.\nAdditionally, no lysosomal function assays were conducted under starvation conditions, leaving\nunexplained why amphisomes tend to fuse more with the plasma membrane to release AEVs\nrather than with lysosomes for degradation in starvation conditions. More definitive\nexperiments, such as immunogold staining or correlative light-electron microscopy to confirm\nAEV presence, were also not performed. We believe the evidence defining AEVs remains\ninsufficient. The rigor and novelty of the article are not sufficient to meet the publication\nrequirements of Nature Communication.\nResponse: Thank you for your detailed feedback on the revised manuscript. We understand\nyour concerns regarding the areas that still need improvement, and we are committed to\naddressing them to enhance the quality of our work.\n1. Functional differences between traditional exosomes and AEVs.\nWe acknowledge the importance of the functional differences between traditional exosomes\nand AEVs. To address this, we explored the distinct roles of exosomes and AEVs in enterovirus\ninfection (as shown in Fig.8). Our results revealed that AEVs, rather than exosomes, could carry\nand deliver viral particles or whole genomes into receptor-negative host cells with high\ninfection efficiency, resulted in overcoming restrictions to viral tropism. Our data suggested\nthat the AEVs play the key role in enhancing virus transmission and provide potential antiviral\ntargets. In addition, according to your constructive comment, we added a new subsection to\ndiscuss the functional role differences between exosomes and AEVs in the Discussion section\n(page 18, line 596-603) (shown below).\n“Traditional exosomes are known primarily for their role in cell communication, and transport\nof various cargos including proteins, lipids and nucleic acids. They are derived from MVBs and\nreleased upon MVB-plasma membrane fusion. In contrast, AEVs are associated with autophagy\nprocesses. Our findings suggest that AEVs may play a unique role in maintaining cellular\nhomeostasis during stress conditions, such as starvation and virus infection. For example, while\nexosomes might transfer signaling molecules to neighboring cells under normal conditions,\nAEVs could be more involved in removing damaged cellular components or misfolded proteins\nduring stress. This difference in function could be related to their distinct biogenesis pathways.\nFuture studies, such as those focusing on the contents of exosomes and AEVs, could further\nelucidate these functional differences.”\n2. Lysosomal function assays under starvation conditions\nWe agree with the comment regarding the fate of amphisomes under lysosomal disorders. By\nusing the vacuolar-type ATPase inhibitor Bafilomycin A1 (BafA1), which disrupts lysosome\nacidification, we had confirmed that the BafA1 treatment did not affect the secretion of sEVs\nwith size less than 100 nm (AEVs) (see Fig.1C, D, E and F). To account for this result, we\nincorporated an experiment to detect intracellular amphisomes under starvation or BafA1-\nmediated lysosome inhibition (see Fig.6K, page 13, line 421-430). We added:\n“In addition, amphisomes can also fuse with lysosomes to form autolysosomes, followed by\ndegradation of the cargo. Therefore, we quantified intracellular amphisome numbers upon\nstarvation or BafA1-mediated lysosome inhibition and observed an increase in the number of\namphisomes when lysosomal function was compromised (Fig. 6K). Our data presented above\nin Fig. 1 show no significant alteration in AEV secretion in BafA1-treated cells, suggesting that\nthe intracellular accumulation of amphisomes did not increase their fusion with the plasma\nmembrane as an alternative route for extracellular release. These results suggest that the\ndifferent fates of amphisomes, including lysosomal degradation and extracellular release, are\nindependent”.\nFig.1C, D, E and F\nFig.6K\n3. Definitive experiments to confirm AEV presence\nWe understand that the lack of definitive experiments such as immunogold staining to confirm\nAEV presence undermines the strength of our claims. We have now performed immunogold\nstaining experiments. The immunogold-stained electron microscopy images are presented in\nFigure.5F with a description as follows (see Fig.5F, page 11, line 356-359):\n“Consistently, the immunoelectron microscopy showed that the AEVs isolated from starved\n293T cells exhibited immunoreactivity with antibodies against p62 and Rab13 on the vesicle\nmembranes, while antibodies against LC3B were present inside the vesicles (Fig.5F).”\nFig.5F\nIn the Materials and Methods section (page 30, line 962-973), we added the detail of operational\ninformation for the immunoelectron microscopy.\nWe believe these additional experiments and discussions significantly strengthen our\nmanuscript, addressing the concerns regarding the definition of AEVs. We are confident that\nthe enhanced rigor and novelty of the article now meet the high publication standards of Nature\nCommunications.\nReviewer #3 (Remarks to the Author):\nThe revised version of this article by Mao et al includes new panels in a figure to answer my\nand other reviewers’ comments (ko ATG7, conditions without starvation) and others.\nI am satisfied with the experimental and technical answers and find this article interesting\nand worth publishing.\nThe authors have not done an entirely satisfying job in answering the comments about wording\nand statements about exosomes : I still see incorrect mention that ESCRT (including TSG101)\nand CD9 are specific of exosomes, whereas they are both involved in plasma membrane-\nderived vesicles formation.\nE.g. line 193-194 : « exosomes have been described as having a diameter of 30-150 nm and are\ngenerated by ESCRT pathway mechanisms, » and line 198-199 : « Because TSG101 and ATG7\nhave essential functions in the process of exosome biogenesis and autophagosome formation,\nrespectively », line 204 : « The classical exosome markers CD63 and CD9 » : this is onlyartially\ntrue as other mechanisms than ESCRT have been described for ILV formation, PM-derived EVs\nare also generated via TSG101, and CD9 is now more recognized as a marker of Plasma\nmembrane-derived EVs.\nResponse: Thank you for pointing out these persistent inaccuracies in our wording regarding\nexosomes. We apologize for not fully addressing this issue in our previous revision. We have\nnow carefully reviewed and corrected all the relevant statements throughout the manuscript to\nprovide a more accurate representation.\nIn the revised manuscript:\nFor line 195 - 195: We have rewritten the sentence to read: “Exosomes are typically described\nas having a diameter of 30-150 nm. The ESCRT pathway is involved in exosome biogenesis”.\nFor line 199 - 200:\nThe revised text is: “TSG101 plays a role in the process of exosome biogenesis and ATG7 is\nessential for autophagosome formation”.\nFor line 204:\nWe changed it to: CD63 and CD9, which are often considered markers associated with\nexosomes, were largely decreased in secreted vesicles from TSG101-KO cells compared with\nthose from WT cells, whereas the expression of the marker CD81 decreased slightly.\nSome other minor mistakes here and there could be corrected, for example (not exhaustive) :\nLine 262 : « To identified the characteristic of AEVs » shoudleb « to identify »\nLine 337 : The lysosome marker EEA1, (fractions 4-5), (EEA1 is an early endosome marker,\nnot lysosome)\nResponse: Thank you for pointing out these minor mistakes. We apologize for these inaccurate\nsentences. The relevant corrections have been made in the revised manuscript.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThis is a much improved manuscript. I am mostly satisfied with the revised version of the\nmanuscript. However, I still have a couple of concerns listed below:\n1. The manuscript still requires extensive English editing\nResponse: We sincerely appreciate your feedback regarding the need for extensive English\nediting in our manuscript. We acknowledge that clear and precise language is crucial for\neffectively communicating our research findings. Our manuscript was edited for proper English\nlanguage use, grammar, punctuation, spelling, and overall style by one or more of the highly -\nqualified native English-speaking editors at Springer Nature Author Services (SNAS). The\nverification code is D455-4D72-815E-71F6-2AA0.\n2. The figure legends should be properly written. For instance, no information is provided on\nwhere the relative expression is from and how it is calculated in Figure 3B. I assume it's a\ndensitometry quantitation of the Western blot data in Figure 3A.\nResponse: Thank you for highlighting the issue with our figure legends and pointing out the\ndeficiency in the Figure 3B. We agree that they need to be properly written for better clarity\nand understanding. We applied a similar approach to all figure legends, ensuring that each one\ncontains detailed information about the experimental setup, methods, data analysis, and any\nrelevant controls. We also made sure to use clear and consistent language throughout the figure\nlegends. These changes can be found throughout the manuscript.\n3. Figure 3D shows that AEVs are not induced in starved Rab11-depleted cells. However, it\nappears that these cells are defective in autophagy, much like ATG7 KO cells. If Rab11 impairs\nAEV induction by modulating autophagy, then the conclusion that Rab11 is required for AEV\nbiogenesis should be revised. A western blot probing for p62 turnover upon starvation in these\ncells could help address this concern.\nResponse: Thank you for raising this important concern. We agree that the relationship between\nRab11, autophagy, and AEV induction needs further clarification. To address this, we have\nperformed the western blot analysis to detect the autophagy process in Rab11-depleted cells\nunder normal or starved conditions, using antibody against p62 and LC3B. In the Results\nsection (page7, line 220-223), we presented the new data. Figure 3D shows a decreased p62\nprotein and an increased LC3B-II in starved Rab11-KO cells compared to those in normally\ncultured Rab11-KO cells, indicating Rab11 deletion did not affect the fusion of\nautophagosomes with lysosomes.\nFig.3D"}, {"round": 3, "reviewer_comments": {"reviewer_1": "I have no further questions.", "reviewer_3": "I am satisfied with the current verison and do not require any new modifications."}, "author_response": ""}]

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