Echovirus 1 Entry into Polarized Caco-2 Cells Depends on Dynamin,Cholesterol, and Cellular Factors Associated with Macropinocytosis

Enteroviruses invade their hosts by crossing the intestinal epithelium. We have examined the mechanism by which echovirus 1 (EV1) enters polarized intestinal epithelial cells (Caco-2). Virus binds to VLA-2 on the apical cell surface and moves rapidly to early endosomes. Using inhibitory drugs, dominant negative mutants, and small interfering RNAs (siRNAs) to block specific endocytic pathways, we found that virus entry requires dynamin GTPase and membrane cholesterol but is independent of both clathrin- and caveolin-mediated endocytosis. Instead, infection requires factors commonly associated with macropinocytosis, including amiloride-sensitive Na⁺/H⁺ exchange, protein kinase C, and C-terminal-binding protein-1 (CtBP1); furthermore, EV1 accumulates rapidly in intracellular vesicles with dextran, a fluid-phase marker. These results suggest a role for macropinocytosis in the process by which EV1 enters polarized cells to initiate infection.     

Characterization of Human Astrovirus Cell Entry

Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle.     

Hazara virus and Crimean-Congo Hemorrhagic Fever Virus show a different pattern of entry in fully-polarized Caco-2 cell line

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) and Hazara virus (HAZV) belong to the same viral serotype and family. HAZV has lately been used as a model system and surrogate to CCHFV. However, virus-host cell interaction and level of pathogenicity for these viruses are not well investigated nor compared. In this study, we compared HAZV and CCHFV infection of human polarized epithelial cells to shed light on similarities and differences in virus-host cell interaction between these two viruses. We investigated the pattern of infection of CCHFV and HAZV in fully polarized human cells, the Caco-2 cell line. Polarization of Caco-2 cells lead to difference in expression level and pattern of proteins between the apical and the basolateral membranes. We found that CCHFV virus, in contrast to HAZV, is more likely infecting polarized cells basolaterally. In addition, we found that cytokines/pro-inflammatory factors or other viral factors secreted from CCHFV infected moDC cells enhance the entry of CCHFV contrary to HAZV. We have shown that CCHFV and HAZV early in infection use different strategies for entry. The data presented in this study also highlight the important role of cytokines in CCHFV-host cell interaction.     

Decay-accelerating factor binding determines the entry route of echovirus 11 in polarized epithelial cells

The interaction between echovirus 11 strain 207 (EV11-207) and decay-accelerating factor (DAF or CD55) at the apical surface of polarized Caco-2 cells results in rapid transport of the virus to tight junctions and in its subsequent uptake. A virus mutant (EV11-207R) which differs at 6 amino acids and whose affinity for DAF is apparently significantly lower remains at the apical surface, from where its uptake occurs. Binding of EV11-207 to DAF and its transport to tight junctions result in a loss of function of the junctions. In contrast, the mutant virus EV11-207R is not transferred to tight junctions, nor does it impair the integrity of these junctions. Cholesterol depletion from the apical membrane leads to DAF aggregation and, presumably, internalization and inhibits infection by EV11-207. However, infection by EV11-207R is significantly less sensitive to cholesterol depletion than infection by EV11-207, confirming the DAF requirement for EV11-207, but not EV11-207R, to infect cells. These data strongly indicate that in the case of infection of polarized epithelial cells by echovirus 11, DAF binding appears be a key determinant in the choice of entry pathway, at least in cell culture.     

Endocytic host cell machinery plays a dominant role in intracellular trafficking of incoming human immunodeficiency virus type 1 in human placental trophoblasts

Vertical transmission of human immunodeficiency virus type 1 (HIV-1) is the primary cause of infection by this retrovirus in infants. In this study, we report for the first time that there is a correlation between endocytic uptake of HIV-1 and virus gene expression in polarized trophoblasts. To shed light on the relationship between endocytosis and the fate of HIV-1 in polarized trophoblasts, the step-by-step movements of HIV-1 within the endocytic compartments were tracked by confocal imaging. Incoming virions were initially located in early endosomes. As time progressed, virions accumulated in late endosomes. HIV-1 was also found in apical recycling endosomes and at the basolateral pole. Experiments performed with indicator cells revealed that HIV-1 is recycled and transcytosed. These data indicate that the intracellular trafficking of HIV-1 upon entry into polarized human trophoblasts is a complex process which requires the active participation of the endocytic host cell machinery.     

Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells

Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm.     

Tetraspanin CD151 Mediates Papillomavirus Type 16 Endocytosis

Human papillomavirus type 16 (HPV16) is the primary etiologic agent for cervical cancer. The infectious entry of HPV16 into cells occurs via a so-far poorly characterized clathrin- and caveolin-independent endocytic pathway, which involves tetraspanin proteins and actin. In this study, we investigated the specific role of the tetraspanin CD151 in the early steps of HPV16 infection. We show that surface-bound HPV16 moves together with CD151 within the plane of the membrane before they cointernalize into endosomes. Depletion of endogenous CD151 did not affect binding of viral particles to cells but resulted in reduction of HPV16 endocytosis. HPV16 uptake is dependent on the C-terminal cytoplasmic region of CD151 but does not require its tyrosine-based sorting motif. Reexpression of the wild-type CD151 but not mutants affecting integrin functions restored virus internalization in CD151-depleted cells. Accordingly, short interfering RNA (siRNA) gene knockdown experiments confirmed that CD151-associated integrins (i.e., α3β1 and α6β1/4) are involved in HPV16 infection. Furthermore, palmitoylation-deficient CD151 did not support HPV16 cell entry. These data show that complex formation of CD151 with laminin-binding integrins and integration of the complex into tetraspanin-enriched microdomains are critical for HPV16 endocytosis.     

Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.     

Influenza entry pathways in polarized MDCK cells

In non-polarized cell culture models, influenza virus has been shown to enter host cells via multiple endocytic pathways, including classical clathrin-mediated endocytic routes (CME), clathrin- and caveolae-independent routes and macropinocytosis. However, little is known about the entry route of influenza virus in differentiated epithelia, in vivo site of infection for influenza virus. Here, we show that in polarized Madin–Darby canine kidney type II (MDCK II) cells, influenza virus has a specific utilization of the clathrin-mediated endocytic pathway and requires Eps15 for host cell entry.     

Effect of cell polarization on hepatitis C virus entry

The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.     

Vectorial release of poliovirus from polarized human intestinal epithelial cells.

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.     

Clathrin-mediated endocytosis in living host cells visualized through quantum dot labeling of infectious hematopoietic necrosis virus

Infectious hematopoietic necrosis virus (IHNV) is an important fish pathogen that infects both wild and cultured salmonids. As a species of the genus Novirhabdovirus, IHNV is a valuable model system for exploring the host entry mechanisms of rhabdoviruses. In this study, quantum dots (QDs) were used as fluorescent labels for sensitive, long-term tracking of IHNV entry. Using live-cell fluorescence microscopy, we found that IHNV is internalized through clathrin-coated pits after the virus binds to host cell membranes. Pretreatment of host cells with chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and clathrin light chain (LCa) depletion using RNA interference both resulted in a marked reduction in viral entry. We also visualized transport of the virus via the cytoskeleton (i.e., actin filaments and microtubules) in real time. Actin polymerization is involved in the transport of endocytic vesicles into the cytosol, whereas microtubules are required for the trafficking of clathrin-coated vesicles to early endosomes, late endosomes, and lysosomes. Disrupting the host cell cytoskeleton with cytochalasin D or nocodazole significantly impaired IHNV infectivity. Furthermore, infection was significantly affected by pretreating the host cells with bafilomycin A1, a compound that inhibits the acidification of endosomes and lysosomes. Strong colocalizations of IHNV with endosomes indicated that the virus is internalized into these membrane-bound compartments. This is the first report in which QD labeling is used to visualize the dynamic interactions between viruses and endocytic structures; the results presented demonstrate that IHNV enters host cells via clathrin-mediated endocytic, cytoskeleton-dependent, and low-pH-dependent pathways.     

Requirements for different components of the host cell cytoskeleton distinguish ecotropic murine leukemia virus entry via endocytosis from entry via surface fusion.

Murine ecotropic leukemia viruses use a common receptor for entry into host cells; however, the site of virus fusion appears to differ with the host cell. Entry in mouse NIH 3T3 fibroblasts is by endocytosis, whereas entry in rat XC sarcoma cells is by surface fusion. We report here the identification of a step common to both entry pathways, as well as of a step unique to the endocytic pathway. Recent demonstration of the clustering of the virus receptor on rat cells suggested a possible interaction of the receptor with the cellular cytoskeleton (M. H. Woodard, W. A. Dunn, R. O. Laine, M. Malandro, R. McMahon, O. Simell, E. R. Block, and M. S. Kilberg, Am. J. Physiol. 266:E817-E824, 1994). We tested the hypothesis that such an interaction might influence receptor function. We found that entry into NIH 3T3 and XC cells was greatly diminished by the disruption of the actin network before but not shortly after virus internalization, suggesting the actin network plays a critical role in an early step common to both entry pathways. Disruption of microtubules before and shortly after virus internalization markedly reduced entry in NIH 3T3 cells, while entry into XC cells remained efficient. These data suggest that intact microtubules are required in a postpenetration step unique to efficient virus entry via endocytosis. The physiological function of the receptor was not affected by disruption of either the actin network or the microtubules, as the uptake of cationic amino acids in NIH 3T3 and XC cells was comparable to that in control cells even when the cytoskeleton remained disrupted for as long as 3 h.     

Endocytic pathways in polarized Caco-2 cells: identification of an endosomal compartment accessible from both apical and basolateral surfaces

The enterocyte-like cell line Caco-2 forms a polarized epithelium when grown on filters. We have investigated the interaction of endocytic pathways from the apical and basolateral surfaces. The transferrin receptor was an appropriate marker for the basolateral route; uptake of radiolabeled transferrin was highly polarized, and recycling of this ligand back to the basolateral surface occurred with an efficiency of 95%, even after prolonged incubations with transferrin. Using a transferrin-peroxidase conjugate to delineate the morphological pathway, we have identified an early endocytic compartment in the basolateral cytoplasm of the cells. Longer incubations revealed a deeper endocytic compartment in the apical cytoplasm. Concanavalin A complexed to gold was used to simultaneously label the apical endocytic route. After 60 min, extensive mixing of the two labels was seen in endocytic elements throughout the apical cytoplasm, including in the Golgi area, but never in the basal cytoplasm. Using a second double labeling procedure in which antitransferrin receptor antibody complexed to gold was applied to the basolateral surface for up to 2 h and free peroxidase applied to the apical surface for shorter periods, we demonstrated that this apical marker rapidly (within 5 min) reached endosomes containing antibody-gold. Our results indicate that, in Caco-2 cells, the endocytic pathways from the apical and basolateral surfaces meet in an endosomal compartment from which transferrin can still be recycled.     

Par6B and atypical PKC regulate mitotic spindle orientation during epithelial morphogenesis

Cdc42 plays an evolutionarily conserved role in promoting cell polarity and is indispensable during epithelial morphogenesis. To further investigate the role of Cdc42, we have used a three-dimensional matrigel model, in which single Caco-2 cells develop to form polarized cysts. Using this system, we previously reported that Cdc42 controls mitotic spindle orientation during cell division to correctly position the apical surface in a growing epithelial structure. In the present study, we have investigated the specific downstream effectors through which Cdc42 controls this process. Here, we report that Par6B and its binding partner, atypical protein kinase C (aPKC), are required to regulate Caco-2 morphogenesis. Depletion or inhibition of Par6B or aPKC phenocopies the loss of Cdc42, inducing misorientation of the mitotic spindle, mispositioning of the nascent apical surface, and ultimately, the formation of aberrant cysts with multiple lumens. Mechanistically, Par6B and aPKC function interdependently in this context. Par6B localizes to the apical surface of Caco-2 cysts and is required to recruit aPKC to this compartment. Conversely, aPKC protects Par6B from proteasomal degradation, in a kinase-independent manner. In addition, we report that depletion or inhibition of aPKC induces robust apoptotic cell death in Caco-2 cells, significantly reducing both cyst size and number. Cell survival and apical positioning depend upon different thresholds of aPKC expression, suggesting that they are controlled by distinct downstream pathways. We conclude that Par6B and aPKC control mitotic spindle orientation in polarized epithelia and, furthermore, that aPKC coordinately regulates multiple processes to promote morphogenesis.     

Annexin VI stimulates endocytosis and is involved in the trafficking of low density lipoprotein to the prelysosomal compartment

Annexins are calcium-binding proteins with a wide distribution in most polarized and nonpolarized cells that participate in a variety of membrane-membrane interactions. At the cell surface, annexin VI is thought to remodel the spectrin cytoskeleton to facilitate budding of coated pits. However, annexin VI is also found in late endocytic compartments in a number of cell types, indicating an additional important role at later stages of the endocytic pathway. Therefore overexpression of annexin VI in Chinese hamster ovary cells was used to investigate its possible role in endocytosis and intracellular trafficking of low density lipoprotein (LDL) and transferrin. While overexpression of annexin VI alone did not alter endocytosis and degradation of LDL, coexpression of annexin VI and LDL receptor resulted in an increase in LDL uptake with a concomitant increase of its degradation. Whereas annexin VI showed a wide intracellular distribution in resting Chinese hamster ovary cells, it was mainly found in the endocytic compartment and remained associated with LDL-containing vesicles even at later stages of the endocytic pathway. Thus, data presented in this study suggest that after stimulating endocytosis at the cell surface, annexin VI remains bound to endocytic vesicles to regulate entry of ligands into the prelysosomal compartment.     

Measles virus preferentially transduces the basolateral surface of well-differentiated human airway epithelia

Measles virus (MV) is typically spread by aerosol droplets and enters via the respiratory tract. The progression of MV infection has been widely studied; yet, the pathway for virus entry in polarized human airway epithelia has not been investigated. Herein we report the use of a replication-competent Edmonston vaccine strain of MV expressing enhanced green fluorescent protein (MV-eGFP) to infect primary cultures of well-differentiated human airway epithelia. Previous studies with polarized Caco-2 cells (intestine-derived human epithelia) and MDCK cells (kidney-derived canine epithelia) demonstrated that MV primarily infected and exited the apical surface. In striking contrast, our results indicate that MV preferentially transduces human airway cells from the basolateral surface; however, virus release remains in an apical direction. When MV-eGFP was applied apically or basolaterally to primary cultures of airway epithelia, discrete foci of eGFP expression appeared and grew; however, the cell layer integrity was maintained for the duration of the study (7 days). Interestingly, utilizing immunohistochemistry and confocal microscopy, we observed widespread expression of the receptor for the vaccine strain of MV (CD46) at greatest abundance on the apical surface of the differentiated human airway epithelia as well as in human tracheal tissue sections. These data suggest that the progression of MV infection through the respiratory epithelium may involve pathways other than direct binding and entry through the apical surface of airway epithelia.     

Infection of polarized cultures of human intestinal epithelial cells with hepatitis A virus: vectorial release of progeny virions through apical cellular membranes

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract. We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30- to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% +/- 12.5% of the total virus yield compared with 0.23% +/- 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms.     

A novel cellular protein, VPEF, facilitates vaccinia virus penetration into HeLa cells through fluid phase endocytosis

Vaccinia virus is a large DNA virus that infects many cell cultures in vitro and animal species in vivo. Although it has been used widely as a vaccine, its cell entry pathway remains unclear. In this study, we showed that vaccinia virus intracellular mature virions bound to the filopodia of HeLa cells and moved toward the cell body and entered the cell through an endocytic route that required a dynamin-mediated pathway but not a clathrin- or caveola-mediated pathway. Moreover, virus penetration required a novel cellular protein, vaccinia virus penetration factor (VPEF). VPEF was detected on cell surface lipid rafts and on vesicle-like structures in the cytoplasm. Both vaccinia virus and dextran transiently colocalized with VPEF, and, importantly, knockdown of VPEF expression blocked vaccinia virus penetration as well as intracellular transport of dextran, suggesting that VPEF mediates vaccinia virus entry through a fluid uptake endocytosis process in HeLa cells. Intracellular VPEF-containing vesicles did not colocalize with Rab5a or caveolin but partially colocalized with Rab11, supporting the idea that VPEF plays a role in vesicle trafficking and recycling in HeLa cells. In summary, this study characterized the mechanism by which vaccinia virus enters HeLa cells and identified a cellular factor, VPEF, that is exploited by vaccinia virus for cell entry through fluid phase endocytosis.     

Laodelphax striatellus Atg8 facilitates Rice stripe virus infection in an autophagy-independent manner

Rice stripe virus(RSV)is the causative agent of rice stripe disease and is completely dependent on insect vectors for its plant-to-plant transmission.Laodelphax striatellus is the major insect vector for RSV.In this study,we explored the interactions be-tween RSV infection and L.striatellus autophagy,a potential intrinsic antiviral mechanism in insects.We found that L.striatellus autophagic activity did not affect RSV infection;however,the autophagy related-8(Atg8)gene significantly enhanced virus infection.Dur-ing RSV initial infection within the L.striatellus midgut,silencing of Atg8 expression significantly decreased the phosphorylation of c-Jun N-terminal kinase(p-JNK);however,when RSV infection is absent,silencing of Atg8 did not alter p-JNK levels.Thesc results indicated that Atg8 might activate the JNK machinery by allowing more virus infection into cells.We further revealed that Atg8-deficiency significantly decreased RSV accumu-lation on the surface of the insect midgut epithelial cells,suggesting a receptor trafficking function of the y-aminobutyric acid receptor-associated protein family.Using the RSV ovary entry as a model,in which vitellogenin receptor(V gR)mediates RSV cell entry,we clarified that Atg8-deficiency decreased the abundance of V gR localizing on the cytomem-brane and disturbed the attachment of RSV in the germarium zones.Collectively,these results revealed an autophagy-independent function of L.striatellus Atg8 that enhances RSV initial infection by increasing virus attachment on the infection sites.