Enterovirus A71 co-opts the IQGAP1 for a non-lytic release of viral particles

Enterovirus A71 co-opts the IQGAP1 for a non-lytic release of viral particles | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Journal of Medical Virology This is a preprint and has not been peer reviewed. Data may be preliminary. 16 July 2025 V1 Latest version Share on Enterovirus A71 co-opts the IQGAP1 for a non-lytic release of viral particles Authors : Han-Hsiang Chen , Guang-Huar Young , Chin-Jung Lin , Heng-I Su , Tzu-Chieh Ho , Yih-Cherng Liou , Chih-Ching Huang , Robert Y.-L. Wang 0000-0002-1322-3890 [email protected] , and Chin-Kuo Chen Authors Info & Affiliations https://doi.org/10.22541/au.175263847.75476434/v1 Published Journal of Medical Virology Version of record Peer review timeline 394 views 171 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The human enterovirus A71 (EV-A71) is known to infect host cells, replicate, and assemble viral particles, which are released by cell lysis. However, studies have demonstrated that EV-A71 can also exit cells via a non-lytic pathway. Nevertheless, research on this phenomenon remains limited. The present study aims to elucidate the mechanisms by which EV-A71 employs non-lytic pathways to release viral particles. A combination of mass spectrometry and gene ontology analysis was utilized to identify the host protein IQGAP1 as interacting with the EV-A71 3CD protein. The extracellular vesicles released from EV-A71-infected cells with IQGAP1 knockdown exhibited diminished expression of both IQGAP1 and EV-A71 VP1 proteins, accompanied by a substantial decrease in Tsg101 level. In IQGAP1 knockout cells or after dihydroartemisinin-mediated IQGAP1 inhibition, unclosed phagosomes were observed, impairing viral particle release. Furthermore, IQGAP1 has been found to facilitate the closure of phagosomes, thus forming virus-containing autophagosomes and promoting the non-lytic release of EV-A71 particles. The mechanism of action of the EV-A71 virus involves the exploitation of a host cell’s IQGAP1 to regulate autophagy, leading to the formation of virus-containing autophagosomes. This process ultimately results in the promotion of non-lytic viral particle release. Enterovirus A71 co-opts the IQGAP1 for a non-lytic release of viral particles Han-Hsiang Chen 1,a , Guang-Huar Young 1,2,a , Chin-Jung Lin 3 , Heng-I Su 1 , Tzu-Chieh Ho 4 , Yih-Cherng Liou 5 , Chih-Ching Huang 3,6 , Robert Y.L. Wang 1,7,* , Chin-Kuo Chen 7,8,** 1 Department of Biomedical Sciences, College of Medicine, Chang Gung University, TaoYuan 333323, Taiwan 2 Department of Nephrology, Chang Gung Memorial Hospital, Linkou Medical Center, 333323, Taiwan 3 Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, 202301, Taiwan 4 Department of Pediatrics, School of Medicine, Indiana University, Indianapolis, IN 46202, USA 5 Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore 6 Center of Excellence for the Oceans, National Taiwan Ocean University, 202301, Keelung, Taiwan 7 Department of Otolaryngology-Head and Neck Surgery, Chang Gung Memorial Hospital, Keelung 204201, Taiwan 8 School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, TaoYuan 333323, Taiwan a These authors contributed equally to this work * Corresponding author: [email protected] (Robert Y.L. Wang) ** Corresponding author: [email protected] (Chin-Kuo Chen) ABSTRACT The human enterovirus A71 (EV-A71) is known to infect host cells, replicate, and assemble viral particles, which are released by cell lysis. However, studies have demonstrated that EV-A71 can also exit cells via a non-lytic pathway. Nevertheless, research on this phenomenon remains limited. The present study aims to elucidate the mechanisms by which EV-A71 employs non-lytic pathways to release viral particles. A combination of mass spectrometry and gene ontology analysis was utilized to identify the host protein IQGAP1 as interacting with the EV-A71 3CD protein. The extracellular vesicles released from EV-A71-infected cells with IQGAP1 knockdown exhibited diminished expression of both IQGAP1 and EV-A71 VP1 proteins, accompanied by a substantial decrease in Tsg101 level. In IQGAP1 knockout cells or after dihydroartemisinin-mediated IQGAP1 inhibition, unclosed phagosomes were observed, impairing viral particle release. Furthermore, IQGAP1 has been found to facilitate the closure of phagosomes, thus forming virus-containing autophagosomes and promoting the non-lytic release of EV-A71 particles. The mechanism of action of the EV-A71 virus involves the exploitation of a host cell’s IQGAP1 to regulate autophagy, leading to the formation of virus-containing autophagosomes. This process ultimately results in the promotion of non-lytic viral particle release. Keywords: Enterovirus A71, IQGAP1, Autophagosomes, Extracellular vesicles INTRODUCTION Enterovirus A71 (EV-A71) is classified as a member of the Enterovirus genus within the Picornaviridae family 1 . In 1970, these viruses were classified and designated as enteroviruses 2,3 . Subsequent to this, enteroviruses were reclassified into four distinct types through the application of virological gene sequence analysis. Enterovirus A71 is classified as enterovirus A. The transmission of enterovirus A71 occurs primarily through the respiratory and gastrointestinal systems 4-6 . The majority of patients who become infected do so asymptomatically; however, mild symptoms may resemble those of a cold, including fever, sore throat, and headache. A minute proportion of infected individuals may manifest severe symptoms, primarily herpangina and hand-foot-mouth disease (HFMD) 7-12 . The extant literature suggests that enterovirus A71 has the capacity to infect the nervous system and trigger inflammatory responses in the brainstem and spinal cord, resulting in the development of neurological symptoms in affected patients 13 . Current research on the viral release of enteroviruses primarily indicates that these viruses release viral particles through lysis. However, numerous studies have demonstrated that enteroviruses also employ a secretory autophagy pathway to facilitate viral release in a non-lytic manner. For instance, following the infection of neural progenitor stem cells (NPSCs) by Coxsackievirus B3, the autophagy pathway is activated, resulting in the generation of extracellular microvesicles containing Coxsackievirus B3. It is important to note that neutralizing antibodies are ineffective in preventing the infection of normal cells by these infectious extracellular microvesicles. Similarly, following the infection of HeLa cells by poliovirus, the assembled viral particles interact with the viral 3CD protein. During this process, the cells undergo autophagy, leading to the formation of omegasomes through the action of the LC3 II protein, which subsequently develop into phagophores. The viral particles and the viral 3CD protein complex bind to the LC3 II protein and are subsequently transported to the phagophore. Subsequent to this, the phagophore undergoes a closure process, resulting in its transformation into an autophagosome. This newly formed autophagosome is then released from the cell in a non-lytic manner, as previously described 14 . IQGAP1, also known as Ras GTPase-activating-like protein IQGAP1, is a scaffold protein that is widely expressed in mammals. It’s typical location is in the nucleus, plasma membrane, and cytoplasm. Its primary functions include the regulation of cell adhesion, the actin cytoskeleton, and cell cycle-related signaling pathways 15-17 . A plethora of studies have demonstrated that IQGAP1 is overexpressed in various types of cancers, including aggressive forms of colorectal and ovarian cancer 18 . Additionally, approximately 10% of genes demonstrate augmented expression in cancer cells, and these genes interact with IQGAP1. For instance, IQGAP1 has been shown to regulate the activation of intracellular MAPK signaling pathways, thereby promoting cell proliferation, and contributing to the development of pancreatic cancer 19-21 . Furthermore, IQGAP1 has been shown to interact with β-catenin, thereby enhancing the activation of the Wnt signaling pathway, which has been implicated in the progression of colon cancer 22,23 . Numerous studies have demonstrated that IQGAP1 plays a crucial role in the release of various viruses. For instance, the Epstein-Barr virus (EBV) utilizes IQGAP1 to regulate its maturation process and enhance its release 24 . The WW domain of IQGAP1 interacts with the L domain of the VP40 protein from the Ebola virus (EBOV), initiating the recruitment of Cdc42 and facilitating viral budding and release 25 . In the aftermath of infection by the Marburg virus (MARV), IQGAP1 facilitates its interaction with the Tsg101 protein and is recruited to viral inclusions by the nucleoprotein (NP). Thereafter, IQGAP1 binds to the nucleocapsid, thereby supporting viral release 26 . Furthermore, IQGAP1 has been observed to bind to the core protein of the Classical swine fever virus (CSFV), aiding in its release. The binding of IQGAP1 to the core protein is dependent on the specific amino acids (207-209; 213-215) within the protein. Mutations in these amino acids result in a reduction in CSFV release 27 . However, the mechanism by which EV-A71 employs IQGAP1 to regulate the viral infection cycle remains poorly understood. The objective of this study was to investigate the host proteins that interact with the EV-A71 3CD protein to facilitate the formation of replication organelles. The results obtained from mass spectrometry and Gene Ontology (GO) analysis identified IQGAP1 as a protein that interacts with 3CD and exhibits the highest binding affinity for lipids. In pull-down assays, we demonstrated that EV-A71 3CD binds to the IQ domain of IQGAP1. During IQGAP1 knockdown experiments, it was observed that a reduction in IQGAP1 expression in cells resulted in an increase in EV-A71 RNA levels, while the extracellular viral titer decreased. This finding suggests that the host protein IQGAP1 plays a crucial role in the release of EV-A71. In addition, experimental findings from extracellular vesicle analysis suggest that EV-A71 assembles extracellular vesicles containing viral particles through IQGAP1, thereby facilitating the release of EV-A71. This study signifies the inaugural discovery that EV-A71, in addition to utilizing lytic release, also forms extracellular vesicles containing viral particles through host proteins to enhance viral release. It is evident that these findings will play a substantial role in future research endeavors concerning other viruses and their interactions with extracellular vesicles. MATERIALS AND METHODS Cells and Virus The Hela cells and RD cells (Rhabdomyosarcoma cells) were obtained from BCRC Strain Collection Catalog & Shopping Cart. The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) supplemented with 10% FBS in a humidified incubator at 37℃ under 5% CO 2 . EV-A71/2231/TW strain was propagated in RD cells and determined the viral titers by plaque assay. Site-Directed Mutagenesis and Transformation The polymerase chain reaction (PCR) was conducted using the Agilent QuikChange Lightning Site-Directed Mutagenesis Kit for site-directed mutagenesis. The mutated oligonucleotide primer sequences employed were pCMV8-3CD, pCMV8-3Tag-8-EV71-3D, and pCMV8-3Tag-8-EV71-3CD. The plasmids pCMV8-3Tag-8-EV71-3CD (C147G), pCMV8-3Tag-8-EV71-3C (C147G), and pCMV8-3Tag-8-EV71-3D were transformed into E. coli DH5α (Medical Supply Company Ltd) and screened using ampicillin. The purification of the plasmids was conducted using a plasmid mini/prep kit (GeneDireX), and their sequences were subsequently confirmed to ensure integrity. mRNA Preparation and Purification The pCMV8-3Tag-8-EV-A71-3CD (C147G), pCMV8-3Tag-8-EV-A71-3C (C147G), and pCMV8-3Tag-8-EV-A71-3D plasmids are transcribed into RNA using the T7 mScript™ Standard mRNA Production System (CELLSCRIPT™). The resulting EV-A71 3C, 3D, and 3CD mRNA solution is mixed with an equal volume of phenol–chloroform (24:25) and thoroughly mixed, followed by centrifugation at 12,000 g for 5 minutes at 4°C. This step is repeated twice. Subsequently, the supernatant is transferred to a new Eppendorf tube, and an equal volume of 5 M ammonium acetate is added, followed by incubation at -20°C for 15 minutes. Next, 2.5 volumes of 100% ethanol are added, and the mixture is centrifuged at 12,000 g for 5 minutes at 4°C. The supernatant is discarded, and 1 mL of 75% ethanol is added, followed by another centrifugation at 12,000 g for 5 minutes at 4°C. The supernatant is again removed, and 50 μL of DEPC-treated water is added to resuspend the RNA. EV-A71 3C, 3D, and 3CD mRNA Transfection and Protein Extraction The EV-A71 3C, 3D, and 3CD mRNA were transfected into HeLa cells (totaling 6 × 10^6 cells) using Lipofectamine RNAiMAX, following the established protocol for a duration of 8 hours. Subsequently, cell lysis was performed using NP-40 lysis buffer (TBS, 50 mM Tris-HCl pH 7.4 + 150 mM NaCl + 1 mM EDTA + 1% Triton X-100 or NP-40 + 1x Protease Inhibitor Cocktail) to extract total protein and assess protein concentration. Subsequently, western blotting and the ChemiDoc™ MP Imaging System was utilized to analyze the expression of EV-A71 3C, 3D, and 3CD proteins. In this study, the primary antibody used was rabbit anti-Flag (1:10,000, Sigma), and the secondary antibody was goat anti-mouse (1:10,000, GeneTex). EV-A71 3C, 3D, and 3CD Recombinant Protein Purification The transfected EV-A71 3C, 3D, and 3CD mRNA of HeLa cells was lysed using NP-40 lysis buffer to extract total protein. Subsequently, 20 mg of protein was mixed with 40 μL of ANTI-FLAG® M2 Affinity Gel (Flag-resin, Sigma-Aldrich®) and incubated for 2 hours. Following incubation, the mixture was centrifuged at 1,000 g for 5 minutes, and the pellet was washed with 400 μL of TBS five times. Next, 300 ng/μL of Flag peptide (25 mg/mL, Sigma-Aldrich®) was added to the bound protein on the Flag-resin and incubated for an additional 2 hours, after which the protein eluate was collected. Silver Staining After purifying the protein sample using SDS-PAGE, immerse the SDS gel in Solution A (50% methanol, 25% acetic acid, and 25% distilled deionized water) for overnight treatment. Next, add Solution B (30% methanol) for fixation for 15 minutes, followed by washing with distilled deionized water three times, each for 5 minutes. Then, add Solution C (25.3 mg of sodium thiosulfate (Na2S2O3) per 200 mL) for 2 minutes, and wash with distilled deionized water three times, each for 30 seconds. Following this, add Solution D (0.2% silver nitrate (AgNO3)) and incubate in the dark for 25 minutes, then wash with distilled deionized water three times, each for 5 minutes. Subsequently, prepare Solution E (5.93 g of sodium carbonate (Na2CO3) per 200 mL, combined with 4 mL of Solution C and 200 μL of 40% formaldehyde) for rapid coloration. After washing with distilled deionized water once, immerse the gel in Solution F (3.13 g of disodium ethylenediaminetetraacetic acid dihydrate (Na2EDTA·2H2O) per 200 mL) to halt the coloration reaction and analyze protein expression. Immunofluorescence Staining First, seed 2x10 4 HeLa cells in an 8-well slide (Millicell® EZ SLIDES) and culture them for 24 hours. Next, transfect the cells with EV-A71 3CD mRNA using Lipofectamine RNAiMAX, following the protocol for an 8-hour incubation. After transfection, remove the supernatant and wash the cells three times with 1X PBS, allowing 5 minutes for each wash. Subsequently, fix the cells with 4% paraformaldehyde, followed by three additional washes with 1X PBS. Add 0.1% Triton X-100 and incubate for 30 minutes, then wash three times with 1X PBS, again allowing 5 minutes for each wash. Next, add 2% BSA and incubate for 1 hour, followed by three washes with PBS, each lasting 5 minutes. Proceed with fluorescence staining and fluorescence microscopy analysis. In this study, the primary antibodies used were mouse anti-Flag (1:200; Sigma), mouse anti-IQGAP1 (1:200; Invitrogen), and rabbit anti-Clanexin (1:200; GeneTex). The secondary antibodies included goat anti-mouse antibody (1:125; GeneTex) and goat anti-rabbit antibody (1:125; GeneTex). Cytosolic Fractionation After collecting HeLa cells transfected with EV-A71 3CD mRNA and EV-A71-infected HeLa cells, Tris-buffered saline (TBS) without NP-40 was added, mixed thoroughly, and incubated at 4°C for 30 minutes. The mixture was then subjected to three cycles of freezing and thawing in liquid nitrogen, followed by centrifugation at 12,000 g for 10 minutes at 4°C to collect the supernatant, which contained non-membrane proteins. Next, TBS containing NP-40 was added to the pellet in an Eppendorf tube, and the mixture underwent three additional cycles of freezing and thawing in liquid nitrogen, followed by centrifugation at 12,000 g for 10 minutes at 4°C to collect the supernatant, which contained membrane proteins. Subsequently, Western blotting was performed to analyze the expression of viral and host proteins, and Coomassie blue staining was conducted. The protein bands were then excised for mass spectrometry analysis. In this study, the primary antibodies used were mouse anti-Flag (1: 10,000; Sigma), mouse anti-IQGAP1 (1: 1,000; Invitrogen), rabbit anti-3D (1: 5,000), and rabbit anti-Clanexin (1: 10,000; GeneTex). The secondary antibodies included goat anti-mouse antibody (1:10,000; GeneTex) and goat anti-rabbit antibody (1: 10,000; GeneTex). Gene Ontology The utilization of the Gene Ontology (Gene Ontology Consortium) approach facilitated the analysis of the proteins. This analysis was conducted subsequent to a comparison of the results of mass spectrometry. The selected species is Homo sapiens. The comparison is conducted on the basis of biological processes, molecular functions, and cellular components. The focus is on proteins related to lipid response and metabolism, as well as phospholipid response and metabolism. The significance level is set at p < 0.05. IQGAP1 Knockdown Assay The IQGAP1 shRNA plasmids utilized in this study were TRCN0000047483 [#83], TRCN0000047485 [#85], and TRCN0000047486 [#86] (RNAiCore). These plasmid strains were selected by streaking to isolate single colonies, followed by plasmid amplification and purification. Subsequently, TransIT-X2® (Mirus Bio) was employed to transfect IQGAP1 shRNA-83, 85, and 86 (10 μg each) into HeLa cells for a duration of 72 hours. After transfection, the cells were collected, and proteins were extracted for the analysis of host protein expression using Western blotting. Additionally, EV-A71 (MOI=1) was used to infect the IQGAP1 knockdown cells for 48 hours. Collect cells and extract RNA, utilizing reverse transcription quantitative polymerase chain reaction (RT-qPCR) to analyze viral RNA expression. Additionally, collect the supernatant and perform a plaque assay to determine viral titers. Furthermore, collect cells and extract proteins, employing Western blotting to assess the expression of both viral and host proteins. In this study, the primary antibodies used included mouse anti-IQGAP1 (1:1000; Invitrogen), rabbit anti-3D (1:5000), and mouse anti-β-actin (1:10000; GeneTex). The secondary antibodies employed were goat anti-mouse antibody (1:10000; GeneTex) and goat anti-rabbit antibody (1:10000; GeneTex). IQGAP1 inhibitor treatment The IQGAP1 inhibitor utilized in this study, dihydroartemisinin (TCI Materials Science), was employed to investigate the inhibition of IQGAP1 function. Initially, a cell viability assay was performed on RD cells treated with dihydroartemisinin at concentrations of 1, 5, 10, 20, 50, and 100 μg/mL. Subsequently, RD cells were infected with EV-A71 at a multiplicity of infection (MOI) of 1 for 1 hour. After this incubation, the supernatant was removed and replaced with DMEM medium lacking fetal bovine serum (FBS) but containing 5 μg/mL of dihydroartemisinin for an additional hour. FBS was then added to achieve a final concentration of 10%, and the cells were cultured for 24 hours. Following this period, the cells were collected for RNA extraction, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) was employed to analyze viral RNA expression. The supernatant was also collected for a plaque assay to determine viral titers. Construction of Transgenic Vectors and Preparation and Purification of IQGAP1 Recombinant Protein The gene synthesis of the IQGAP1-CH, IQ, and GRD/RGCT domains, along with the pET-30(a)+ vector, was digested using the restriction enzymes BamH I and Xba I. The enzyme-digested fragments were then purified using a gel extraction kit (GeneDireX) and ligated together with a T4 ligation kit (ThermoFisher). The ligated product was subsequently transformed into the E. coli DH5α strain and screened using kanamycin. The constructed pET30a(+) plasmids containing the IQGAP1-CH, IQ, and GRD/RGCT segments were then transformed into E. coli BL21 (Medical Supply Company Ltd) and induced for target protein expression with 0.6 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) in Luria-Bertani (LB) medium containing kanamycin at a final concentration of 50 μg/ml, incubated at 25 °C for 16 hours. His-IQGAP1-CH, IQ, or GRD/RGCT recombinant proteins were purified using column chromatography on Ni Sepharose™ Six Fast Flow (GE Healthcare, Chicago, Illinois, USA). The induction and purification of the recombinant proteins followed the manufacturer’s protocol and previous studies. Isolation of extracellular vesicles Nine 10-cm plates of HeLa cells, EV-A71 infected cells (infected for two days) and EV-A71-infected IQGAP1 knockdown cells (transfected with shIQGAP1 into HeLa cells for three days, followed by infection with EV-A71 for two days), were cultured until cell density reached saturation. The medium was then replaced with DMEM without FBS and cultured for an additional two days (8 mL per plate; a total of 72 mL). The supernatant was collected and centrifuged at 500 g for 10 minutes at 4°C. The supernatant was transferred to new centrifuge tubes and centrifuged at 10,000 g for 30 minutes at 4°C. The collected supernatant was concentrated using an Amicon® Ultra-15 Centrifugal Filter Unit (NMWL: 3 kDa) by centrifuging at 6,000 g for 1 hour at 4°C until a final volume of 500 µL was achieved, after which it was resuspended in 9 mL of 1x PBS. The resuspended solution was subjected to ultracentrifugation at 110,000 g for 2 hours at 4°C. Following this, the supernatant was removed and washed once with 9 mL of 1x PBS, followed by another round of ultracentrifugation at 110,000 g for 2 hours at 4°C. The extracellular vesicles were then resuspended in 200 µL of 1x PBS. Their size and quantity were analyzed using Nanoparticle Tracking Analysis, while Calnexin (a negative marker), CD81, and Tsg101 (extracellular vesicles markers), as well as IQGAP1 and EV-A71 VP1 protein expression, were assessed using Western blotting. The morphology of the extracellular vesicles was observed using transmission electron microscopy (TEM). Analysis of Autophagy and the Formation of Autophagosomes The expression changes of LC3I and LC3II were analyzed using Western blotting. The cell lines examined included HeLa cells, EV-A71-infected HeLa cells, EV-A71-infected IQGAP1 knockdown HeLa cells, and EV-A71-infected IQGAP1 HeLa cells treated with dihydroartemisinin. We also examined HAP1 cells, EV-A71-infected HAP1 cells, and EV-A71-infected IQGAP1 knockout HAP1 cells. Furthermore, transmission electron microscopy (TEM) was utilized to examine the process of autophagy, or the formation of autophagy vesicles, within these cells. Identification of Dihydroartemisinin in the treatment of mice infected with EV-A71 Seven-day-old ICR newborn mice were purchased from the National Institutes of Applied Research in Taipei, Taiwan, and were raised in the laboratory animal center at National Taiwan Ocean University in Keelung, Taiwan. The mice administered an intraperitoneal (IP) injection of With or without 50 µL of 10 6 PFU MP4, the subjects were treated with 10 µL of PBS (positive control) or DHA (20 mg kg−1) every 12 hours for 5 consecutive days. Important parameters, such as survival rate, clinical score, and body weight, were recorded daily for 14 days or longer. The clinical score was used to grade symptoms, with 0 representing health, 1 indicating slow movement, 2 indicating single hind-limb paralysis, 3 indicating bilateral hind-limb paralysis, and 4 indicating death. Body weight was determined and normalized using the following equation: Normalized body weight = Wn / Wo Where Wn and Wo represent the body weights of the treated mice on Day n and Day 0, respectively. RESULTS Proteomics analyses of host proteins interacting with viral 3C, 3D and 3CD proteins Numerous studies have confirmed that picornaviruses utilize their own viral 3A protein to induce phosphatidylinositol 4-kinase (PI4K) to activate the synthesis of phosphatidylinositol 4-phosphate (PI4P), subsequently altering the membrane composition to form replication organelles 28-34 . However, research has indicated that poliovirus, a member of the Picornaviridae family, can also induce the synthesis of PI4P through the viral 3CD protein. This alters the distribution of PI4P within the cell and promotes the formation of replication organelles 35 . Therefore, the objective of this study is to further investigate the hypothesis that, after the infection of cells by EV-A71, the viral 3CD protein regulates the biosynthesis of PI4P, thereby facilitating the formation of replication organelles to assist in viral replication. In order to simulate the conditions of viral infection in cells, we synthesized EV-A71 viral protein RNA using the research methods for subsequent experiments. Initially, messenger RNA (mRNA) was synthesized using laboratory-established plasmids pCMV8-3flag-3C (C147G, protease dead mutant), 3D, and the 3CD (C147G, protease dead mutant). The synthesized mRNA for 3C, 3D, and 3CD (which contains the Flag sequence) was introduced into HeLa cells for eight hours, after which western blotting was utilized to ascertain the expression of the viral proteins. The results indicated that after eight hours of transfection, the viral proteins 3C, 3D, and 3CD were all expressed (3C: 20 kDa; 3D: 53 kDa; 3CD: 73 kDa) (Figure 1A). Silver staining revealed numerous distinct protein bands in the samples expressed with viral 3C, 3D, and 3CD proteins. For instance, band 1 in the 3CD sample exhibited significant expression, while bands 3D and 3C did not; conversely, band 2 in the 3D sample showed significant expression, whereas 3CD did not. Subsequently, specific protein bands were selected for excision and separation, and were analyzed using mass spectrometry, with the cutting positions indicated in the figure (Figure 1B). The results were then compared against a human protein database (Swiss-Prot) using the following filtering criteria: The first step in the procedure is the removal of all keratin and actin proteins. Next, the results are sorted in each band by score, and the proteins with the highest scores are selected. Finally, the number of identified peptide chains is compared to the number of unique peptide chains that can only be attributed to a specific protein. The greater the number of unique peptides identified, the higher the likelihood that the protein is correctly identified. The assessment of the coverage of the identified peptide chains for each protein is also crucial, as higher coverage indicates that the protein is more easily identifiable. Finally, it is essential to confirm that the protein size corresponds to the position of the band. A total of 20 protein bands from the 3CD sample, 23 protein bands from the 3C group, and 19 protein bands from the 3D group were identified through a comprehensive analysis of the mass spectrometry results (Table 1). To this end, samples transfected with pCMV8-3flag-3CD underwent duplicate analyses followed by mass spectrometry. The protein data resulting from the two analyses were then compared, revealing a total of 98 intersecting proteins. Subsequently, the Gene Ontology consortium (GO) was utilized to analyze the biological processes, molecular functions, and cellular components associated with these proteins. In the cellular component category, it was observed that many proteins were related to vesicles or organelles (see Supplemental Table S1). Within the molecular function category, nine proteins were identified as being associated with phospholipid binding (see Supplemental Table S2). Within the membrane-bounded organelle category, 92 proteins were found to interact with 3CD, and among these, 11 were determined to be functionally or process-wise related to phospholipids (Supplemental Table S3). Mass Spectrometry analysis of membrane and non-membrane proteins To identify the proteins on the cell membrane that interact with the EV-A71 3CD protein, a detergent (1% NP40) was first utilized to separate membrane proteins from non-membrane proteins (Figure 2A). Subsequently, we employed SDS-PAGE to separate all proteins from both the membrane and non-membrane fractions, followed by Coomassie blue staining. The results revealed significant differences between all cellular proteins and those exclusively found on the membrane compared to non-membrane proteins (Figure 2B). Subsequently, the outcomes of the three groups that had been stained with Coomassie blue were separated into 14 fractions, ranging from small to large molecular weight. Thereafter, mass spectrometry analysis was conducted. A cross-referencing process was then initiated, whereby the results from the whole cell protein group were compared with the previously mentioned 3CD mass spectrometry results, with the exclusion of the control group. The objective of this cross-referencing process was to select proteins associated with lipids. A total of 37 proteins were identified as interacting with 3CD, and further Gene Ontology (GO) analysis identified 10 proteins linked to lipids, of which three were specifically related to lipid binding (Table 2). Through the consolidation of findings from mass spectrometry and GO analysis, the protein IQGAP1 was identified as a key player in this interaction, exhibiting the highest lipid binding values. It is imperative to investigate further whether IQGAP1 exerts a significant influence on the infection cycle of EV-A71 upon infected into host cells. Translocation of cellular IQGAP1 to the membranes of cellular organelles upon EV-A71 infection A comprehensive analysis involving mass spectrometry and GO analysis was conducted to determine the protein most likely to interact with the EV-A71 3CD protein. The results indicated that IQGAP1 is the most likely candidate for this interaction. Consequently, we hypothesize that IQGAP1 may play a role in the formation of the replication complex for EV-A71, thereby facilitating its replication. To demonstrate that IQGAP1 translocate to organelles following EV-A71 infection, HeLa cells were infected with EV-A71 at a multiplicity of infection (MOI) of 1 for 48 hours. Membrane fractionation experiments were conducted for analysis. The results indicated that after EV-A71 infection, the expression of IQGAP1 in the membrane protein samples was significantly higher than that in the control group. Conversely, the expression of IQGAP1 in cytosolic (non-membrane) protein samples was significantly lower than that in the control group, suggesting that EV-A71 infection promotes the translocation of IQGAP1 from the cytoplasm to the organelles (Figure 3A). This translocation may be associated with the facilitation of the formation of the EV-A71 replication complex. To further investigate this interaction, an examination by immunofluorescence staining was conducted on both untransfected and pCMV8-3flag-3CD-transfected HeLa cells. The results indicated that following transfection with pCMV8-3flag-3CD, a significant 3CD-Flag signal was detected within the cells. In the merged images, the observation of overlapping signals of 3CD-Flag and IQGAP1 was indicative of an interaction between 3CD-Flag and IQGAP1 (Figure 3B & Figure S1). Furthermore, the signal from Calnexin was employed to determine the localization of IQGAP1. Calnexin, a protein associated with the endoplasmic reticulum (ER), exhibited overlapping signals with 3CD-Flag and IQGAP1, suggesting a direct interaction between 3CD and IQGAP1 within the ER (Figure 3C). The subsequent step was to analyze the specific region of IQGAP1 to which EV-A71 3CD binds. IQGAP1 is comprised of six domains: the CH domain, CC domain, WW domain, IQ domain, GRD domain, and RGCT domain. The CH, IQ, and GRD/RGCT domains are of relevance. Initially, DNA fragments corresponding to the CH domain, IQ domain, and GRD/RGCT domain were inserted into the pET-30a(+) vector (His-tag). Subsequently, recombinant protein experiments were conducted using the pET-30a-CH, pET-30a-IQ, and pET-30a-GRD/RGCT plasmids to produce recombinant proteins for the CH domain-His, IQ domain-His, and GRD/RGCT domain-His of IQGAP1. Furthermore, the pCMV8-3flag-3CD plasmid was transfected into HeLa cells. Following a 48-hour period, the cells were harvested, and total protein was extracted using cell lysis buffer. Subsequently, a pull-down assay was performed to analyze the results. The findings indicated that the recombinant protein of IQGAP1’s IQ domain-His was successfully detected, while the recombinant proteins of IQGAP1’s CH domain-His and GRD/RGCT domain-His were not detected. This finding indicates that the primary binding site for EV-A71 3CD is the IQ domain of IQGAP1 (Figure 3D). IQGAP1 impacts the release of EV-A71 viral particles The results presented above demonstrate that EV-A71 3CD interacted with IQGAP1 and binds to its IQ domain. Subsequently, we aim to confirm whether the suppression of IQGAP1 expression in cells affects EV-A71 replication. To achieve this objective, three short hairpin RNAs (shRNAs) targeting IQGAP1 (#83, #85 and #86) were introduced into HeLa cells for 72 hours. Subsequently, western blotting was employed to analyze the expression levels of IQGAP1. The results indicated that transfection with three shIQGAP1 plasmids led to a significant decrease in IQGAP1 expression, with plasmid #85 demonstrating the most substantial reduction (Figure 4A). Subsequently, a knockdown experiment was conducted using plasmid #85 shIQGAP1. The initial transfection of HeLa cells with shIQGAP1 was conducted for a duration of 48 hours. This was followed by an infection with EV-A71 at a multiplicity of infection (MOI) of 1, after which a subsequent 24-hour culture period was permitted. The expression of viral RNA within the cells was analyzed, and the viral titer in the culture medium was measured. The findings demonstrated that a decrease in IQGAP1 expression in EV-A71-infected cells (Figure 4B) was associated with a substantial increase in viral RNA levels compared to the infection group (Figure 4C). In contrast to the anticipated outcome, the progeny viral titers in the conditioned medium of the EV-A71-infected cells demonstrated a substantial decrease (Figure 4D). This finding suggests that IQGAP1 does not appear to play a direct role in EV-A71 RNA replication. However, it is possible that IQGAP1 contributes to the release of EV-A71 viral particles. IQGAP1 promotes the formation of extracellular vesicles in the EV-A71 infected cells Many studies indicated that EV-A71 not only releases viral particles through lytic release but also utilizes extracellular vesicles to encapsulate enterovirus particles or viral RNA for release into the extracellular space 36-38 . Extracellular vesicles containing EV-A71 particles have been found to be larger than those secreted by uninfected cells 36-38 . Furthermore, the infection rate and immune response resistance of extracellular vesicles containing EV-A71 particles are higher than those of the EV-A71 particles themselves 37,38 . Furthermore, research has demonstrated that IQGAP1 facilitates the formation of extracellular vesicles by cells 39 . Hence, in this study, we would like to ascertain whether EV-A71 utilizes IQGAP1 to form extracellular vesicles containing EV-A71 particles, thereby enabling viral release. Initially, HeLa cells were subjected to IQGAP1 knockdown experiments, followed by infection with EV-A71. HeLa cells (mock control), EV-A71-infected cells, and EV-A71-infected cells with IQGAP1 knockdown were then cultured in FBS-free DMEM medium for 48 hours. The subsequent step involved the collection of the above-mentioned samples for the purpose of analyzing the expression of extracellular vesicle markers (Tsg101 and CD81), a negative marker (calnexin, an ER marker), IQGAP1, and EV-A71 VP1 proteins. As shown in Figure 5A, there were no significant expression levels different between viral VP1, calnexin, Tsg101 and CD81 from the intracellular cell lysates (Figure 5A). Notably, the extracellular vesicles released from EV-A71-infected cells with IQGAP1 knockdown exhibited diminished expression of both IQGAP1 and EV-A71 VP1 proteins, accompanied by a substantial decrease in Tsg101 expression (Figure 5B). Furthermore, nanoparticle tracking analysis (NTA) was employed to assess the size and quantity of extracellular vesicles released from HeLa cells, EV-A71-infected cells, and EV-A71-infected cells with IQGAP1 knockdown. The results indicated that there was no significant difference in the number of extracellular vesicles among the three groups; however, extracellular vesicles released from EV-A71-infected cells were significantly larger compared to the control group, while extracellular vesicles released from EV-A71-infected cells with IQGAP1 knockdown were significantly smaller than those from the EV-A71-infected cells group (Figure 5B-D). Transmission electron microscopy (TEM) analyses demonstrated that extracellular vesicles released from EV-A71-infected cells contained EV-A71 particles (Figure 5E & 5F), whereas extracellular vesicles released from EV-A71-infected cells with IQGAP1 knockdown showed no evidence of EV-A71 particles (Figure 5G). In summary, the results indicate that EV-A71 utilizes IQGAP1 to assemble extracellular vesicles containing EV-A71 particles, thereby promoting EV-A71 release. EV-A71 co-opts IQGAP1 facilitating the formation of autophagosomes and promoting the non-lytic release of viral particles Some reports suggested that the poliovirus can migrate to phagosomes by binding to the viral 3CD protein and the LC3 II protein. Subsequently, the phagosome closes and transforms into an autophagosome, allowing the poliovirus to be released from the cell in a non-lytic manner 14 . Furthermore, studies demonstrate that EV-A71 induces the formation of autophagosomes, which, upon accumulation, promote their fusion with multivesicular bodies (MVBs). This process culminates in the packaging of EV-A71 RNA into exosomal vesicles, which are subsequently released into the extracellular space 36 . Furthermore, it has been reported that IQGAP1 interacts with intracellular ATG9A and CHMP2A, facilitating the closure of phagosomes and their transformation into autophagosomes 40 . Consequently, the objective of this study is to further analyze whether the formation of autophagy and autophagosomes in EV-A71-infected cells is influenced by the inhibition functional activities of IQGAP1 or the complete knockout of IQGAP1 expression level in the cells. Initially, we conducted IQGAP1 knockdown experiments on the HeLa cells, followed by EV-A71 infection, while collecting both HeLa cells and EV-A71-infected cells. Furthermore, HAP1 cells (a near-haploid cell line frequently utilized in biomedical and genetic research, which facilitates gene knockout and the preparation of cells with specific gene deletions) and HAP1 IQGAP1 knockout cells were employed to be infected by EV-A71 and collect samples. Subsequent western blot analysis was performed to examine the expression changes of LC3I and LC3II, and transmission electron microscopy was employed to assess the formation of autophagosomes within the cells. The results indicated that following EV-A71 infection of HeLa or HAP1 cells, the expression of LC3II increased significantly compared to the control group. However, when IQGAP1 was knocked down or knocked out, the expression of LC3II decreased significantly relative to the infected group (Figure 6A & 6B). Furthermore, transmission electron microscopy revealed the formation of autophagosomes following EV-A71 infection in the HeLa (Figures 6C & 6D) or in the HAP1 cells (Figures 6F & 6G). In contrast, when IQGAP1 was genetically silenced, unclosed phagosomes (termed ”phagophores”) were observed within the cells (see Figures 6E & 6H). The present findings indicate that IQGAP1 exerts a promoting effect on autophagy and facilitates the closure of phagosomes, thereby leading to their transformation into autophagosomes in the context of EV-A71 infection. As demonstrated in the preceding results, following the infection of cells by EV-A71, the activation of autophagy is initiated through the engagement of IQGAP1. This process leads to the subsequent transportation of EV-A71 into the phagophore and the facilitation of the closure of autophagosomes, culminating in the formation of an autophagosome. Subsequently, the release of viral particles into the extracellular space occurs in a non-lytic manner. Dihydroartemisinin, an IQGAP1 inhibitor, impedes the non-lytic release of EV-A71 Next, the potential impact of IQGAP1 function inhibition on the non-lytic release of EV-A71 was examined. To identify a predicted inhibitor that targets IQGAP1 and suppresses its protein function, known as Artenimol (also referred to as dihydroartemisinin; DHA), the GeneCard and DrugBank websites were utilized. Initially, a series of in vitro tests were conducted to assess the toxicity of dihydroartemisinin at varying concentrations. The findings demonstrated that at a concentration of 10 µg/ml, the cell viability of HeLa cells diminished to 60%. The median lethal concentration (LC50) of dihydroartemisinin was calculated to be 12 µg/ml, and 5 µg/ml of dihydroartemisinin was subsequently employed for the experiments (Figure 7A). In the course of the experimental procedure, the objective was to determine the effect of IQGAP1 inhibition on the expression of viral RNA within infected cells and the viral titer in the extracellular environment. As illustrated in Figure 7B, the inhibition of IQGAP1 function in EV-A71-infected cells resulted in a substantial augmentation in viral RNA expression, in comparison to the infection group. Concurrently, a significant decrease in the extracellular viral titer was observed (Figure 7C). The results from the extracellular vesicle experiments demonstrated that the expression of IQGAP1 and EV-A71 VP1 proteins in extracellular vesicles released from EV-A71-infected cells significantly increased. Conversely, the expression levels of IQGAP1 and EV-A71 VP1 proteins in extracellular vesicles released from EV-A71-infected cells with inhibited IQGAP1 function exhibited a significant decrease, accompanied by a substantial reduction in Tsg101 expression (Figure 7D). Furthermore, nanoparticle tracking analysis (NTA) was utilized to evaluate the size and quantity of extracellular vesicles released from EV-A71-infected cells with inhibited IQGAP1 function. The results indicated that the number of extracellular vesicles did not show significant differences compared to the previous HeLa cells and EV-A71-infected cells; however, the size of the extracellular vesicles was significantly smaller than that of the previous infection group (Figure 7E). In experiments involving autophagosomes, it was found that inhibiting IQGAP1 function resulted in a significant decrease in LC3II expression compared to the infection group (Figure 7F). Furthermore, transmission electron microscopy revealed the presence of phagophores within the cells when IQGAP1 function was inhibited (Figure 7G). Dihydroartemisinin can effectively reduce EV-A71 infection in mice and alleviate the onset of symptoms As demonstrated by the results of the in vitro test, dihydroartemisinin (DHA) has been shown to effectively inhibit the non-lytic release of viral particles from EV-A71. Consequently, further research was conducted to investigate whether DHA has an inhibitory effect on EV-A71 infection in 7-day-old ICR mice. As demonstrated in the results section, the infected group demonstrated bilateral hind limb paralysis in over 50% of the subjects by the third day, followed by death on the fifth day post-infection. Conversely, the DHA treatment group did not exhibit hind limb paralysis until the third day, and death occurred on the sixth day post-infection (Figure 8A & 8B). The resultant data demonstrate that the body weight of the infected mice began to decline after the second day, while the body weight of the DHA treatment group started to decrease only on the fourth day (Figure 8C). The clinical score assessments indicated that DHA effectively reduced the symptoms associated with EV-A71 infection in mice (Figure 8D). Moreover, the resultant analysis of brain tissue from mice in each group reveals that after DHA treatment, the expression of viral RNA in the brains of EV-A71-infected mice significantly decreased compared to the infected group (Figure 8E). The results obtained from this study provide substantial evidence that DHA is a highly effective agent in reducing the susceptibility of mice to EV-A71 infection. DISCUSSION Non-enveloped viruses, upon infecting host cells, undergo protein translation and genome replication, ultimately assembling into viral particles. These mature viral particles are released into the extracellular space through cell lysis, categorizing them as lytic viruses 41,42 . However, a substantial body of research has indicated that non-enveloped viruses can also release viral particles through non-lytic mechanisms in addition to cell lysis. The process entails the use of extracellular vesicles to encapsulate viral particles, thereby enabling their release from within the cell into the external environment. These infectious extracellular vesicles have the capacity to effectively evade the host’s immune response, thereby enhancing the efficiency of infecting additional host cells. For instance, following infection with the encephalomyocarditis virus (EMCV) 43,44 , the rotavirus 45,46 , the astrovirus 47 , and the hepatitis A virus (HAV) 48 , there is a significant increase in the production of extracellular vesicles. With respect to research on enteroviruses, it is widely accepted that their release typically occurs via a lytic mechanism. However, recent studies have demonstrated that enteroviruses can also release viral particles or genomes into the extracellular space through a non-lytic mechanism. For instance, echovirus 16, after infecting human beta EndoC-βH1 cells, produces infectious extracellular vesicles that are not inhibited by virus-specific neutralizing antibodies 49 . Furthermore, numerous reports have indicated that EV-A71 can also release viral particles through a non-lytic mechanism. For instance, following the infection of NSC-34 cells or differentiated C2BBe1 cells with EV-A71, the morphology of these cells remains indistinguishable from that of RD cells, and there is an absence of evidence of cytopathic effects (CPE). In addition, the presence of infectious viral particles has been detected in the culture medium of infected cells 50,51 . Following a 9 to 15-hour infection period with Enterovirus A71 in RD cells, the cell membranes of the infected cells remain intact, and the presence of extracellular vesicles measuring approximately 101.8 nanometers has been detected in the extracellular space. Extracellular vesicles have been shown to contain EV-A71 particles with a diameter ranging from 27 to 30 nanometers 38 . Research has also demonstrated that extracellular vesicles secreted by EV-A71-infected cells are infectious and can induce more severe symptoms in mice compared to EV-A71 particles 52 . A number of studies have revealed that the primary discovery of this article is that the poliovirus, which belongs to the enterovirus family, can bind to assembled viral particles via the viral 3CD protein. It has been observed to interact with the LC3 II protein and subsequently translocate to the phagophore. There, it is converted into an autophagosome and released extracellularly from the cell through a non-lytic mechanism 14 . Furthermore, research has confirmed that EV-A71 releases viral particles via a non-lytic mechanism involving autophagy. For instance, EV-A71-induced double-membrane autophagosomes can serve as a replication environment for viral RNA. The subsequent accumulation of these autophagosomes promotes their fusion with multivesicular bodies (MVBs), a process that allows EV-A71 RNA to be packaged into extracellular vesicles and released into the extracellular space 36 . At present, research on the mechanisms by which viruses encapsulate viral particles within extracellular vesicles to release progeny viruses into the extracellular environment remains limited. However, extant research has demonstrated that viruses facilitate the encapsulation of viral particles within extracellular vesicles by interacting with tetraspanins associated with these vesicles. For instance, the HIV Gag protein has been observed to bind to CD63 and CD81 on extracellular vesicles, thereby encapsulating HIV within them 53 . Furthermore, the HHV-6 glycoprotein gB has been demonstrated to interact with CD63, a process that may facilitate the assembly of HHV-6 within extracellular vesicles 54 . In a similar manner, the HCV envelope glycoprotein E2 has been observed to engage with CD81, a process that seems to facilitate the encapsulation of HCV within extracellular vesicles 55,56 . In addition to interacting with tetraspanins, some studies have indicated that viruses can also engage with the ESCRT complex within extracellular vesicles, enabling their encapsulation, as observed with the respiratory syncytial virus (RSV) 57 . It is well established that EV-A71 releases viruses into the extracellular space through non-lytic release via extracellular vesicles 37,38 . However, there are currently no reports detailing how EV-A71 encapsulates progeny viruses within these extracellular vesicles. In this study, we ascertained that EV-A71 utilizes the host protein IQGAP1 to encapsulate progeny viruses within extracellular vesicles. Inhibition of IQGAP1 results in the absence of EV-A71 detection within the extracellular vesicles. Although IQGAP1’s primary function is in the regulation of the cell cycle and cell signaling pathways, studies have demonstrated a significant association between IQGAP1 and the formation of two types of membrane-bound structures, namely, autophagosomes and extracellular vesicles 39,40 . Research on autophagy has revealed that during autophagy, cells promote the formation of phagophores by binding ATG2A and ATG9A. These proteins subsequently interact with IQGAP1, which, in turn, indirectly binds to the ESCRT-III protein CHMP2A. This interaction plays a pivotal role in the process of phagophore closure, which in turn promotes the formation of autophagosomes. The release of extracellular vesicles into the extracellular space is the final outcome of this complex process 40 . In studies focused on extracellular vesicles, IQGAP1 serves as a crucial connector, linking GSDMD and the associated IL-1β complex to components of the ESCRT complex, specifically Tsg101. This interaction enhances the packaging of GSDMD and IL-1β into extracellular vesicles, which are subsequently released into the extracellular environment 39 . CONCLUSION In summary, the host protein IQGAP1, which is associated with phospholipid metabolism and identified through mass spectrometry and GO analysis, is utilized by EV-A71 to produce extracellular vesicles that contain progeny viruses. This process not only facilitates viral release but also enhances viral infection while allowing the virus to evade the host’s immune response. This research is of significant value for future studies related to other viruses and extra cellular vesicles, as it encourages a deeper exploration of how viruses generate infectious extracellular vesicles through interactions between viral proteins and host proteins. Additionally, it facilitates the examination of disparities in internal protein and microRNA expression between infectious and non-infectious extracellular vesicles. AUTHOR CONTRIBUTIONS H-H Chen & G-H Young: Conceptualization, Methodology, Software C-J Lin & H-I Su : Data curation, Writing- Original draft preparation. R-Y Wang & C-K Chen : Visualization, Investigation. R-Y Wang : Supervision. : T-C Ho : Software, Validation.: C C-C Huang & Y-C Liou: Writing- Reviewing and Editing ACKNOWLEDGMENTS The authors would like to thank National Science and Technology Council, Taiwan (NSTC 112-2221-E-182-023 and NSTC 113-2320-B-182-007) and the Chang Gung Memorial Hospital Research Fund (CMRPD1M0423) to RW, and the APC was funded by Chang Gung University (BMRBP16) CONFLICT OF INTEREST STATEMENT The authors have declared no conflict of interest. DATA AVAILABILITY STATEMENT The data will be available upon reader request . ORCID Tzu-Chieh Ho ORCID: 0000-0002-2913-9319 Yih-Cherng Liou ORCID: 0000-0003-0149-6856 Robert Y.L. Wang ORCID: 0000-0002-1322-3890 Reference: doi: 10.1093/infdis/129.3.304 doi: 10.1038/s41591-019-0613-1 doi: 10.1371/journal.pone.0090624 10.1586/14737159.7.4.419 doi: 10.1099/0022-1317-77-8-1699 doi: 10.1016/S1473-3099(10)70194-8 doi: 10.1016/s0168-1702(99)00019-2 doi: 10.1056/NEJM199909233411301 doi: 10.1128/JVI.05063-11 doi: 10.1128/JVI.73.12.9969-9975.1999 doi: 10.1016/j.micinf.2010.03.006 doi: 10.1128/mbio.03276-24 doi: 10.1038/sj.embor.embor867 doi: 10.1016/j.febslet.2009.05.007 doi: 10.1038/ncb1757 doi: 10.1074/jbc.M104315200 doi: 10.1016/j.yexmp.2008.06.001 doi: 10.1128/jvi.01899-23 doi: 10.1128/JVI.00470-13 doi: 10.1371/journal.ppat.1004463 doi: 10.1016/j.virol.2010.12.060 doi: 10.1016/j.cell.2010.03.050 doi: 10.1016/j.devcel.2006.06.005 doi: 10.1128/JVI.00791-17 doi: 10.1128/JVI.02830-14 doi: 10.1128/JVI.03650-13 doi: 10.1128/JVI.06778-11 doi: 10.1038/emboj.2011.429 doi: 10.1371/journal.ppat.1007086 doi: 10.1016/j.micpath.2022.105875 doi: 10.1128/mSphere.00377-20 doi: 10.1080/21505594.2019.1705022 doi: 10.15252/embj.2022110780 doi: 10.1083/jcb.202404047 doi: 10.1099/jgv.0.001557 doi: 10.1016/j.coviro.2011.05.002 doi: 10.1038/s41467-022-31181-y doi: 10.1371/journal.ppat.1007594 doi: 10.1007/s12192-015-0597-9 doi: 10.3390/v12070763 doi: 10.1128/jvi.00848-22 doi: 10.3390/v7122967 doi: 10.3390/microorganisms8111753 doi: 10.1038/srep36983 doi: 10.1093/infdis/jiaa174 doi: 10.1007/s11262-016-1292-3 doi: 10.1083/jcb.200508014 doi: 10.1111/j.1600-0854.2008.00796.x doi: 10.1002/eji.200424887 doi: 10.1073/pnas.1221899110 doi: 10.1038/s41598-017-18672-5 1. Pallansch M, Roos R. Enterovirus: Polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses, 4th eds. 2001.2. Schmidt NJ, Lennette EH, Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis. 1974;129(3):304-309. 3. Schubert RD, Hawes IA, Ramachandran PS, et al. Pan-viral serology implicates enteroviruses in acute flaccid myelitis. Nat Med. 2019;25(11):1748-1752. 4. Bessaud M, Razafindratsimandresy R, Nougairede A, et al. Molecular comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity. PloS one . 2014;9(3):e90624. 5. Nasri D, Bouslama L, Pillet S, Bourlet T, Aouni M, Pozzetto B. Basic rationale, current methods and future directions for molecular typing of human enterovirus. Expert Rev Mol Diagn. 2007;7(4):419-434. doi: 6. Pöyry T, Kinnunen L, Hyypiä T, et al. Genetic and phylogenetic clustering of enteroviruses. J Gen Virol . 1996;77(8):1699-1717. 7. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis . 2010;10(11):778-790. 8. AbuBakar S, Chee H-Y, Al-Kobaisi MF, Xiaoshan J, Chua KB, Lam SK. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res . 1999;61(1):1-9. 9. Ho M, Chen E-R, Hsu K-H, et al. An epidemic of enterovirus 71 infection in Taiwan. N Engl J Med . 1999;341(13):929-935. 10. Shih S-R, Stollar V, Li M-L. Host factors in enterovirus 71 replication. J Virol . 2011;85(19):9658-9666. 11. Shindarov L, Chumakov M, Voroshilova M, et al. Epidemiological, clinical, and pathomorphological characteristics of epidemic poliomyelitis-like disease caused by enterovirus 71. J Hyg Epidemiol Microbiol Immunol . 1979;23(3):284-295. 12. Brown BA, Oberste MS, Alexander Jr JP, Kennett ML, Pallansch MA. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol . 1999;73(12):9969-9975. 13. Weng K-F, Chen L-L, Huang P-N, Shih S-R. Neural pathogenesis of enterovirus 71 infection. Microbes Infect . 2010;12(7):505-510. 14. Aponte-Diaz D, Harris JM, Kang TE, et al. Non-lytic spread of poliovirus requires the nonstructural protein 3CD. mBio . 2025;16(1):e03276-24. 15. Weissbach L, Settleman J, Kalady MF, et al. Identification of a human rasGAP-related protein containing calmodulin-binding motifs. J Biol Chem . 1994;269(32):20517-20521. 16. Hart MJ, Callow MG, Souza B, Polakis P. IQGAP1, a calmodulin‐binding protein with a rasGAP‐related domain, is a potential effector for cdc42Hs. EMBO J . 1996;15(12):2997-3005. 17. Briggs MW, Sacks DB. IQGAP proteins are integral components of cytoskeletal regulation. EMBO Rep . 2003;4(6):571-574. 18. White CD, Brown MD, Sacks DB. IQGAPs in cancer: a family of scaffold proteins underlying tumorigenesis. FEBS Lett . 2009;583(12):1817-1824. 19. Nojima H, Adachi M, Matsui T, Okawa K, Tsukita S, Tsukita S. IQGAP3 regulates cell proliferation through the Ras/ERK signalling cascade. Nat Cell Biol . 2008;10(8):971-978. 20. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res . 1989;49(17):4682-4689. 21. Tan X, Egami H, Ishikawa S, et al. Relationship between the expression of extracellular signal-regulated kinase 1/2 and the dissociation of pancreatic cancer cells: Involvement of ERK1/2 in the dissociation status of cancer cells. Int J Oncol . 2004;24(4):815-820. 22. Briggs MW, Li Z, Sacks DB. IQGAP1-mediated stimulation of transcriptional co-activation by β-catenin is modulated by calmodulin. J Biol Chem. 2002;277(9):7453-7465. 23. Wang Y, Wang A, Wang F, et al. IQGAP1 activates Tcf signal independent of Rac1 and Cdc42 in injury and repair of bronchial epithelial cells. Experimental and molecular pathology . 2008;85(2):122-128. 24. Dai Y-C, Yeh S-Y, Cheng Y-Y, et al. Bglf4 kinase regulates the formation of the ebv cytoplasmic assembly compartment and the recruitment of cellular iqgap1 for virion release. J Virol . 2024;98(2):e01899-23. 25. Lu J, Qu Y, Liu Y, et al. Host IQGAP1 and Ebola virus VP40 interactions facilitate virus-like particle egress. J Virol . 2013;87(13):7777-7780.26. Dolnik O, Kolesnikova L, Welsch S, Strecker T, Schudt G, Becker S. Interaction with Tsg101 is necessary for the efficient transport and release of nucleocapsids in marburg virus-infected cells. PLoS Pathog. 2014;10(10):e1004463. 27. Gladue D, Holinka L, Fernandez-Sainz I, et al. Interaction between Core protein of classical swine fever virus with cellular IQGAP1 protein appears essential for virulence in swine. Virology . 2011;412(1):68-74. 28. Hsu N-Y, Ilnytska O, Belov G, et al. Viral reorganization of the secretory pathway generates distinct organelles for RNA replication. Cell . 2010;141(5):799-811. 29. Wessels E, Duijsings D, Niu T-K, et al. A viral protein that blocks Arf1-mediated COP-I assembly by inhibiting the guanine nucleotide exchange factor GBF1. Dev Cell . 2006;11(2):191-201.30. Xiao X, Lei X, Zhang Z, et al. Enterovirus 3A facilitates viral replication by promoting phosphatidylinositol 4-kinase IIIβ–ACBD3 interaction. J Virol . 2017;91(19):10.1128/jvi. 00791-17. 31. Dorobantu CM, Ford-Siltz LA, Sittig SP, et al. GBF1-and ACBD3-independent recruitment of PI4KIIIβ to replication sites by rhinovirus 3A proteins. J Virol. 2015;89(3):1913-1918. 32. Dorobantu CM, van der Schaar HM, Ford LA, et al. Recruitment of PI4KIIIβ to coxsackievirus B3 replication organelles is independent of ACBD3, GBF1, and Arf1. J Virol . 2014;88(5):2725-2736.33. Greninger AL, Knudsen GM, Betegon M, Burlingame AL, DeRisi JL. The 3A protein from multiple picornaviruses utilizes the golgi adaptor protein ACBD3 to recruit PI4KIIIβ. J Virol . 2012;86(7):3605-3616. 34. Sasaki J, Ishikawa K, Arita M, Taniguchi K. ACBD3‐mediated recruitment of PI4KB to picornavirus RNA replication sites. EMBO J. 2012;31(3):754-766.35. Banerjee S, Aponte-Diaz D, Yeager C, et al. Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein. PLoS Pathog . 2018;14(5):e1007086. 36. Zhang R, Chen J, Zi R, et al. Enterovirus 71-induced autophagosome fusion with multivesicular bodies facilitates viral RNA packaging into exosomes. Microb Pathog . 2022;173:105875. 37. Gu J, Wu J, Cao Y, et al. A mouse model for infection with enterovirus A71 in small extracellular vesicles. Msphere . 2020;5(4):10.1128/msphere. 00377-20. 38. Gu J, Wu J, Fang D, et al. Exosomes cloak the virion to transmit Enterovirus 71 non-lytically. Virulence . 2020;11(1):32-38. 39. Liao Y, Chen X, Miller‐Little W, et al. The Ras GTPase‐activating‐like protein IQGAP1 bridges Gasdermin D to the ESCRT system to promote IL‐1β release via exosomes. EMBO J . 2023;42(1):e110780. 40. Javed R, Mari M, Trosdal E, et al. ATG9A facilitates the closure of mammalian autophagosomes. J Cell Biol . 2025;224(2):e202404047. 41. Owusu IA, Quaye O, Passalacqua KD, Wobus CE. Egress of non-enveloped enteric RNA viruses. J Gen Virol . 2021;102(3):001557. 42. Moyer CL, Nemerow GR. Viral weapons of membrane destruction: variable modes of membrane penetration by non-enveloped viruses. Curr Opin Virol . 2011;1(1):44-49. 43. van der Grein SG, Defourny KA, Rabouw HH, et al. The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy. Nat Commun . 2022;13(1):3625. 44. van der Grein SG, Defourny KA, Rabouw HH, et al. Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential. PLoS Pathog . 2019;15(2):e1007594. 45. Bautista D, Rodríguez L-S, Franco MA, Angel J, Barreto A. Caco-2 cells infected with rotavirus release extracellular vesicles that express markers of apoptotic bodies and exosomes. Cell Stress Chaperones. 2015;20(4):697-708. 46. Iša P, Pérez-Delgado A, Quevedo IR, López S, Arias CF. Rotaviruses associate with distinct types of extracellular vesicles. Viruses . 2020;12(7):763. 47. Baez-Navarro C, Quevedo IR, López S, Arias CF, Iša P. The Association of Human Astrovirus with Extracellular Vesicles Facilitates Cell Infection and Protects the Virus from Neutralizing Antibodies. J Virol. 2022;96(14):e00848-22. 48. Longatti A. The dual role of exosomes in hepatitis A and C virus transmission and viral immune activation. Viruses . 2015;7(12):6707-6715. 49. Netanyah E, Calafatti M, Arvastsson J, Cabrera-Rode E, Cilio CM, Sarmiento L. Extracellular vesicles released by enterovirus-infected EndoC-βH1 cells mediate non-lytic viral spread. Microorganisms . 2020;8(11):1753. 50. Too IHK, Yeo H, Sessions OM, et al. Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway. Sci Rep . 2016;6(1):36983.51. Huang H-I, Lin J-Y, Chiang H-C, Huang P-N, Lin Q-D, Shih S-R. Exosomes facilitate transmission of enterovirus A71 from human intestinal epithelial cells. J Infect Dis. 2020;222(3):456-469. 52. Mao L, Wu J, Shen L, Yang J, Chen J, Xu H. Enterovirus 71 transmission by exosomes establishes a productive infection in human neuroblastoma cells. Virus genes . 2016;52:189-194. 53. Booth AM, Fang Y, Fallon JK, Yang J-M, Hildreth JE, Gould SJ. Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. J Cell Biol . 2006;172(6):923-935. 54. Mori Y, Koike M, Moriishi E, et al. Human herpesvirus‐6 induces MVB formation, and virus egress occurs by an exosomal release pathway. Traffic . 2008;9(10):1728-1742. 55. Masciopinto F, Giovani C, Campagnoli S, et al. Association of hepatitis C virus envelope proteins with exosomes. Eur J Immunol . 2004;34(10):2834-2842.56. Ramakrishnaiah V, Thumann C, Fofana I, et al. Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7. 5 cells. Proc Natl Acad Sci U S A . 2013;110(32):13109-13113. 57. Chahar HS, Corsello T, Kudlicki AS, Komaravelli N, Casola A. Respiratory syncytial virus infection changes cargo composition of exosome released from airway epithelial cells. Sci Rep . 2018;8(1):387. Supplementary Material File (eva71-iqgap1-jmv figures.docx) Download 5.04 MB File (table 2025-07-11.docx) Download 25.68 KB Information & Authors Information Version history V1 Version 1 16 July 2025 Peer review timeline Published Journal of Medical Virology Version of Record 18 Nov 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Journal of Medical Virology Keywords bioautography enterovirus immune responses phagocytosis research and analysis methods virus classification Authors Affiliations Han-Hsiang Chen Chang Gung University College of Medicine View all articles by this author Guang-Huar Young Chang Gung University College of Medicine View all articles by this author Chin-Jung Lin National Taiwan Ocean University View all articles by this author Heng-I Su Chang Gung University College of Medicine View all articles by this author Tzu-Chieh Ho Indiana University Department of Pediatrics View all articles by this author Yih-Cherng Liou National University of Singapore Department of Biological Sciences View all articles by this author Chih-Ching Huang National Taiwan Ocean University View all articles by this author Robert Y.-L. Wang 0000-0002-1322-3890 [email protected] Chang Gung University College of Medicine View all articles by this author Chin-Kuo Chen Keelung Chang Gung Memorial Hospital View all articles by this author Metrics & Citations Metrics Article Usage 394 views 171 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Han-Hsiang Chen, Guang-Huar Young, Chin-Jung Lin, et al. Enterovirus A71 co-opts the IQGAP1 for a non-lytic release of viral particles. Authorea . 16 July 2025. DOI: https://doi.org/10.22541/au.175263847.75476434/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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