EMCV Replication in BHK-21 in Vitro is Mediated by Caveolin-1, Actin, Dynamin-Dependent Endocytic Pathway

2020 ◽  
Author(s):  
Qiongyi Li ◽  
Yang Liu ◽  
Shujuan Xu ◽  
Kexue Zhao ◽  
Ying Ling ◽  
...  
Keyword(s):  
Virus Infection ◽  
Early Stage ◽  
Infection Process ◽  
Confocal Imaging ◽  
Endocytic Pathway ◽  
Virus Infectivity ◽  
Caveolin 1 ◽  
Chemical Inhibitors ◽  
Microscopy Analysis

Abstract Background: Encephalomycarditis virus is a member of Cardiovirus, belongs to the family Picornaviridae, and can infect different domestic and wild animals. However, the endocytic pathway by which EMCV infected BHK-21 cells remains unclear. In this study, endocytic pathway used by EMCV replication in BHK-21 cells was elucidated.Methods: The function of numerous cellular key factors implicated in the various endocytic mechanisms known to date were systematic detected using chemical inhibitors. Furthermore, RNA interference (RNAi) silencing, the overexpression of dominant protein combined to virus infectivity assays, and confocal imaging to examine which cellular molecules involved in the infection process were also analyzed.Results: The results indicated that the EMCV replication was related to endocytosis. However, neither clathrin nor macropinocytosis pathway was involved in virus infection. QRT-PCR and WB analyses showed that caveolin-1 were significantly up-regulated in EMCV infected BHK-21 cells. Immune-fluorescent confocal microscopy analysis showed that caveolin-1 was temporally co-localized with EMCV VP1 at the early stage of EMCV infection. Overexpressed caveolin-1 or downregulated caveolin-1 expression influenced the EMCV infection. Furthermore, EMCV infection was found to depend on dynamin and actin by chemical inhibitors resulted in diminished of virus infection.Conclusions: EMCV replication in BHK-21 cells via caveolin-1, dynamin, and actin-dependent endocytosis pathways.

scholarly journals Caveolin-1 Mediated Endocytic Pathway is Involved in Encephalomyocarditis Virus Replication in BHK-21 Cells

2020 ◽  
Author(s):  
Qiongyi Li ◽  
Yang Liu ◽  
Shujuan Xu ◽  
Kexue Zhao ◽  
Ying Ling ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Early Stage ◽  
Encephalomyocarditis Virus ◽  
Endocytic Pathway ◽  
Virus Infectivity ◽  
Key Factors ◽  
Caveolin 1 ◽  
Chemical Inhibitors ◽  
Microscopy Analysis

Abstract Background: Encephalomyocarditis virus, member of Cardiovirus genus within Picornaviridae family, is an important pathogen that infects different domestic and wild animals. However, the molecular mechanism of its entry remains unclear. In this study, we investigated the mechanism of EMCV infectivity in relation to endocytic pathway using BHK-21 cells. Methods: The function of numerous cellular key factors implicated in the various endocytic mechanisms were systematically explored using chemical inhibitors. Furthermore, RNA interference (RNAi) as well as the overexpression of dominant protein combined to virus infectivity assays, and confocal microscopy was used to examine EMCV infection in details. Results: The results indicated that the EMCV entry into BHK-21 cells depends on caveolin, dynamin, and actin but not clathrin nor macropinocytosis pathways. The effects of overexpression and knockdown of caveolin-1, one components of the caveolae, was examined on EMCV infection. The results showed that EMCV infection was positive correlation with caveolin-1 expression. Confocal microscopy analysis and internalization assay showed that caveolin-1 is required at the early stage of EMCV infection. Conclusions: Caveolin-1, dynamin, and actin-dependent endocytosis pathways are necessary for EMCV infection in vitro.

scholarly journals Caveolin-1 is involved in encephalomyocarditis virus replication in BHK-21 cells

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Qiongyi Li ◽  
Yang Liu ◽  
Shujuan Xu ◽  
Kexue Zhao ◽  
Ying Ling ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Early Stage ◽  
Encephalomyocarditis Virus ◽  
Endocytic Pathway ◽  
Virus Infectivity ◽  
Key Factors ◽  
Caveolin 1 ◽  
Chemical Inhibitors ◽  
Microscopy Analysis

Abstract Background Encephalomyocarditis virus, member of Cardiovirus genus within Picornaviridae family, is an important pathogen that infects different domestic and wild animals. However, the molecular mechanism of its entry remains unclear. In this study, we investigated the mechanism of EMCV infectivity in relation to endocytic pathway using BHK-21 cells. Methods The function of numerous cellular key factors implicated in the various endocytic mechanisms were systematically explored using chemical inhibitors. Furthermore, RNA interference (RNAi) as well as the overexpression of dominant protein combined to virus infectivity assays, and confocal microscopy was used to examine EMCV infection in details. Results The results indicated that the EMCV entry into BHK-21 cells depends on caveolin, dynamin, and actin but not clathrin nor macropinocytosis pathways. The effects of overexpression and knockdown of caveolin-1, one components of the caveolae, was examined on EMCV infection. The results showed that EMCV infection was positive correlation with caveolin-1 expression. Confocal microscopy analysis and internalization assay showed that caveolin-1 is required at the early stage of EMCV infection. Conclusions Caveolin-1, dynamin, and actin-dependent endocytosis pathways are necessary for EMCV infection in vitro.

scholarly journals Study on the Role of Cytc in Response to BmNPV Infection in Silkworm, Bombyx mori (Lepidoptera)

2019 ◽  
Vol 20 (18) ◽  
pp. 4325 ◽  
Author(s):  
Xue-Yang Wang ◽  
Kang-Hui Wu ◽  
Hui-Lin Pang ◽  
Ping-Zhen Xu ◽  
Mu-Wang Li ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Early Stage ◽  
Malpighian Tubule ◽  
Infection Process ◽  
Insect Vector ◽  
Apoptosis Pathway ◽  
Expression Levels ◽  
Mitochondrial Apoptosis Pathway

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens of the silkworm. Cytochrome c (cytc) showed a significant response to BmNPV infection in our previous transcriptome study. However, little is known about the role of Bombyx mori cytc (Bmcytc) in resistance to BmNPV infection. In this study, the expression levels analysis of Bmcytc showed stable expression levels in selected tissues of the resistant strain AN following BmNPV infection, while there was downregulation in the susceptible strain p50, except in the malpighian tubule. To further study the role of Bmcytc in viral infection, Bmcytc was knocked down with siRNA in vitro, resulting in significant downregulation of selected downstream genes of the mitochondrial pathway, including Bmapaf, Bmcaspase-Nc, and Bmcaspase-1; this was also confirmed by overexpression of Bmcytc using the pIZT/V5-His-mCherry insect vector, except Bmcaspase-1. Moreover, knockdown of Bmcytc significantly promoted the infection process of BmNPV in vitro, while the infection was inhibited by overexpression of Bmcytc at the early stage and subsequently increased rapidly. Based on these results, we concluded that Bmcytc plays a vital role in BmNPV infection by regulating the mitochondrial apoptosis pathway. Our work provides valuable data for the clarification of the mechanism of silkworm resistance to BmNPV infection.

scholarly journals Cell-extracellular matrix interactions in the fluidic phase direct the topology and polarity of self-organized epithelial structures

2020 ◽  
Author(s):  
Mingxing Ouyang ◽  
Jiun-Yann Yu ◽  
Yenyu Chen ◽  
Linhong Deng ◽  
Chin-Lin Guo
Keyword(s):  
Extracellular Matrix ◽  
Large Scale ◽  
Type I Collagen ◽  
Mdck Cells ◽  
Early Stage ◽  
Single Cells ◽  
Confocal Imaging ◽  
Type I

AbstractIn vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as a large-scale scaffold to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell-ECM interactions to spatiotemporally coordinate their positioning and differentiation to different ECM at the whole-tissue scale is not fully understood. Here, using in vitro assay with engineered MDCK cells co-expressing H2B-mCherry (nucleus) and gp135 (Podocalyxin)-GFP (apical marker), we show that such spatiotemporal coordination for epithelial morphogenesis and polarization can be initiated and determined by cell-soluble ECM interaction in the fluidic phase. The coordination depends on the native topology of ECM components such as sheet-like basement membrane (BM, mimicked by Matrigel in experiments) and linear fiber-like type I collagen (COL). Two types of coordination are found: scaffold formed by BM (COL) facilitates a close-ended (open-ended) coordination that leads to the formation of lobular (tubular) epithelium, where polarity is preserved throughout the entire lobule/tubule. During lobular formation with BM, polarization of individual cells within the same cluster occurs almost simultaneously, whereas the apicobasal polarization in the presence of COL can start at local regions and proceed in a collective way along the axis of tubule, which might suggest existence of intercellular communications at the cell-population level. Further, in the fluidic phase, we found that cells can form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side. Based on reconstructions from time-lapse confocal imaging, this is likely derived from polarization occurring at early stage and being maintained through growth of the epithelial structures. Under suspension culture with COL, the polarization was impaired with formation of multi-lumens on the tubes, implying the importance of ECM microenvironment for tubulogenesis. Our results suggest a mechanism for cells to form polarity and coordinate positioning in vivo, and a strategy for engineering epithelial structures through cell-soluble ECM interaction and self-assembly in vitro.

Mechanisms of H2O2-mediated injury to type II cell surfactant metabolism and protection with PEG-catalase

1991 ◽  
Vol 261 (5) ◽  
pp. C751-C757 ◽  
Author(s):  
B. A. Holm ◽  
B. B. Hudak ◽  
L. Keicher ◽  
C. Cavanaugh ◽  
R. R. Baker ◽  
...  
Keyword(s):  
Early Stage ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lung Injuries ◽  
Type Ii Cell ◽  
Microscopy Analysis ◽  
Choline Phosphate ◽  
Dose Dependent Decrease

Alterations in type II pneumocyte function, including surfactant biosynthesis, may play a significant role in the development and pathophysiology of oxidant-induced lung injury. The results of this study showed that type II cells exposed to 50-300 microM H2O2 demonstrated a dose-dependent decrease in phosphatidylcholine (PC) synthesis with only minimal changes in cell viability. The activities of the choline-phosphate cytidyltransferase and cholinephosphotransferase, specific enzymes of PC synthesis, were not significantly decreased by the exposure. However, the activity of glycerol-3-phosphate acyltransferase, a sulfhydryl-dependent enzyme involved in an early stage of phospholipid synthesis, was decreased by the exposures in a manner that was similar to that seen for PC synthesis. Further studies showed that incubation of type II cells with polyethylene glycol-conjugated catalase for 1 h resulted in an increase in the cell-associated catalase activity (53 +/- 5 vs. 6.7 +/- 1.5 units/mg protein for controls). Confocal microscopy analysis showed that a significant portion of this activity was located intracellularly. More importantly, these cells were protected from changes in PC synthesis rates when subsequently incubated with 300 microM H2O2. These results indicate that the deleterious effects of H2O2 on type II cell surfactant synthesis may be pharmacologically modified in vitro, a concept that may have utility with regard to the modulation of in vivo lung injuries.

scholarly journals The Protective Role of TLR2 Mediates Impaired Autophagic Flux by Activating the mTOR Pathway During Neospora caninum Infection in Mice

2021 ◽  
Vol 11 ◽  
Author(s):  
Jielin Wang ◽  
Xiaocen Wang ◽  
Pengtao Gong ◽  
Fu Ren ◽  
Xin Li ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Neospora Caninum ◽  
Early Stage ◽  
Infection Process ◽  
Autophagic Flux ◽  
Caninum Infection ◽  
Early Stages

Autophagy has been shown to play an essential role in defending against intracellular bacteria, viruses, and parasites. Mounting evidence suggests that autophagy plays different roles in the infection process of different pathogens. Until now, there has been no conclusive evidence regarding whether host autophagy is involved in Neospora caninum infection. In the current study, we first monitored the activation of autophagy by N. caninum, which occurred mainly in the early stages of infection, and examined the role of host autophagy in N. caninum infection. Here, we presented evidence that N. caninum induced an increase in autophagic vesicles with double-membrane structures in macrophages at the early stage of infection. LC3-II expression peaked and decreased as infection continued. However, the expression of P62/SQSTM1 showed significant accumulation within 12 h of infection, indicating that autophagic flux was blocked. A tandem fluorescence protein mCherry-GFP-LC3 construct was used to corroborate the impaired autophagic flux. Subsequently, we found that N. caninum infection induced the activation of the TLR2–AKT–mTOR pathways. Further investigation revealed that TLR2–mTOR, accompanied by the blockade of autophagic flux, was responsible for impaired autophagy but was not associated with AKT. In vitro and in vivo, N. caninum replication was strongly blocked by the kinase inhibitor 3-methyladenine (3-MA, autophagy inhibitor). In contrast, rapamycin (Rapa, an autophagy inducer) was able to promote intracellular proliferation and reduce the survival rate of N. caninum-infected mice. On the other hand, the accumulation of autophagosomes facilitated the proliferation of N. caninum. Collectively, our findings suggest that activation of host autophagy facilitates N. caninum replication and may counteract the innate immune response of the host. In short, inhibition of the early stages of autophagy could potentially be a strategy for neosporosis control.

scholarly journals Multi-Omics Sequencing Provides Insights Into Age-Dependent Susceptibility of Grass Carp (Ctenopharyngodon idellus) to Reovirus

2021 ◽  
Vol 12 ◽  
Author(s):  
Libo He ◽  
Denghui Zhu ◽  
Xinyu Liang ◽  
Yongming Li ◽  
Lanjie Liao ◽  
...  
Keyword(s):  
Virus Infection ◽  
Grass Carp ◽  
Early Stage ◽  
Nucleotide Metabolism ◽  
Hemorrhagic Disease ◽  
Ctenopharyngodon Idellus ◽  
Intergroup Comparison ◽  
Age Dependent

Grass carp (Ctenopharyngodon idellus) is an important aquaculture species in China that is affected by serious diseases, especially hemorrhagic disease caused by grass carp reovirus (GCRV). Grass carp have previously shown age-dependent susceptibility to GCRV, however, the mechanism by which this occurs remains poorly understood. Therefore, we performed transcriptome and metabolome sequencing on five-month-old (FMO) and three-year-old (TYO) grass carp to identify the potential mechanism. Viral challenge experiments showed that FMO fish were susceptible, whereas TYO fish were resistant to GCRV. RNA-seq showed that the genes involved in immune response, antigen presentation, and phagocytosis were significantly upregulated in TYO fish before the GCRV infection and at the early stage of infection. Metabolome sequencing showed that most metabolites were upregulated in TYO fish and downregulated in FMO fish after virus infection. Intragroup analysis showed that arachidonic acid metabolism was the most significantly upregulated pathway in TYO fish, whereas choline metabolism in cancer and glycerophospholispid metabolism were significantly downregulated in FMO fish after virus infection. Intergroup comparison revealed that metabolites from carbohydrate, amino acid, glycerophospholipid, and nucleotide metabolism were upregulated in TYO fish when compared with FMO fish. Moreover, the significantly differentially expressed metabolites showed antiviral effects both in vivo and in vitro. Based on these results, we concluded that the immune system and host biosynthesis and metabolism, can explain the age-dependent viral susceptibility in grass carp.

scholarly journals The Rabies Virus Glycoprotein Receptor p75NTR Is Not Essential for Rabies Virus Infection

2007 ◽  
Vol 81 (24) ◽  
pp. 13622-13630 ◽  
Author(s):  
Christine Tuffereau ◽  
Klaus Schmidt ◽  
Christelle Langevin ◽  
Florence Lafay ◽  
Georg Dechant ◽  
...  
Keyword(s):  
Virus Infection ◽  
Rabies Virus ◽  
Neurotrophin Receptor ◽  
Virus Infectivity ◽  
Primary Neurons ◽  
Drg Neurons ◽  
Rabies Virus Glycoprotein ◽  
Virus Glycoprotein

ABSTRACT Rabies virus glycoprotein (RVG) is known to be the only factor that mediates rabies infection. The neurotrophin receptor (p75NTR), through its cysteine-rich domain 1, is a specific receptor for RVG and neutralizes virus infectivity, but its role in virus infection has remained obscure. We used adult mouse dorsal root ganglion (DRG) neurons as a model to study the role of p75NTR in RV infection of primary neurons. We show that RV infects around 20% of DRG neurons, of which more than 80% are p75NTR positive, have large diameters, and are capsaicin insensitive. Surprisingly, RV binding and infection are absent in about half of the p75NTR-expressing DRG neurons which have small diameters and are often capsaicin sensitive. This indicates that p75NTR is not sufficient to mediate RV interaction in sensory neurons. The rate and specificity of neural infection are unchanged in RV-infected p75NTRExonIV−/− mice that lack all extracellular receptor domains and in wild-type mice infected with two independent RV mutants that lack p75NTR binding. Accordingly, the mortality rate is unchanged in the absence of RV-p75NTR interaction. We conclude that although p75NTR is a receptor for soluble RVG in transfected cells of heterologous expression systems, an RVG-p75NTR interaction is not necessary for RV infection of primary neurons. This means that other receptors are required to mediate RV infection in vivo and in vitro.

scholarly journals In Silico Selection and In Vitro Evaluation of New Molecules That Inhibit the Adhesion of Streptococcus mutans through Antigen I/II

2020 ◽  
Vol 22 (1) ◽  
pp. 377
Author(s):  
Raúl E. Rivera-Quiroga ◽  
Néstor Cardona ◽  
Leonardo Padilla ◽  
Wbeimar Rivera ◽  
Cristian Rocha-Roa ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Early Stage ◽  
Dynamics Simulation ◽  
Tooth Surface ◽  
Binding Affinities ◽  
Cariogenic Bacteria ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis

Streptococcus mutans is the main early colonizing cariogenic bacteria because it recognizes salivary pellicle receptors. The Antigen I/II (Ag I/II) of S. mutans is among the most important adhesins in this process, and is involved in the adhesion to the tooth surface and the bacterial co-aggregation in the early stage of biofilm formation. However, this protein has not been used as a target in a virtual strategy search for inhibitors. Based on the predicted binding affinities, drug-like properties and toxicity, molecules were selected and evaluated for their ability to reduce S. mutans adhesion. A virtual screening of 883,551 molecules was conducted; cytotoxicity analysis on fibroblast cells, S. mutans adhesion studies, scanning electron microscopy analysis for bacterial integrity and molecular dynamics simulation were also performed. We found three molecules ZINC19835187 (ZI-187), ZINC19924939 (ZI-939) and ZINC19924906 (ZI-906) without cytotoxic activity, which inhibited about 90% the adhesion of S. mutans to polystyrene microplates. Molecular dynamic simulation by 300 nanoseconds showed stability of the interaction between ZI-187 and Ag I/II (PDB: 3IPK). This work provides new molecules that targets Ag I/II and have the capacity to inhibit in vitro the S. mutans adhesion on polystyrene microplates.

scholarly journals Internalization of Coxsackievirus A9 Is Mediated by β2-Microglobulin, Dynamin, and Arf6 but Not by Caveolin-1 or Clathrin

2010 ◽  
Vol 84 (7) ◽  
pp. 3666-3681 ◽  
Author(s):  
Outi Heikkilä ◽  
Petri Susi ◽  
Tuire Tevaluoto ◽  
Heidi Härmä ◽  
Varpu Marjomäki ◽  
...  
Keyword(s):  
Virus Infection ◽  
Cell Surface ◽  
A549 Cells ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Human Enterovirus ◽  
Caveolin 1 ◽  
Β2 Microglobulin ◽  
Lung Carcinoma Cells ◽  
Coxsackievirus A9

ABSTRACT Coxsackievirus A9 (CAV9) is a member of the human enterovirus B species within the Enterovirus genus of the family Picornaviridae. It has been shown to utilize αV integrins, particularly αVβ6, as its receptors. The endocytic pathway by which CAV9 enters human cells after the initial attachment to the cell surface has so far been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung carcinoma cells. The small interfering RNA (siRNA) silencing of integrin β6 subunit inhibited virus proliferation, confirming that αVβ6 mediates the CAV9 infection. However, siRNAs against integrin-linked signaling molecules, such as Src, Fyn, RhoA, phosphatidylinositol 3-kinase, and Akt1, did not reduce CAV9 proliferation, suggesting that the internalization of the virus does not involve integrin-linked signaling events. CAV9 endocytosis was independent of clathrin or caveolin-1 but was restrained by dynasore, an inhibitor of dynamin. The RNA interference silencing of β2-microglobulin efficiently inhibited virus infection and caused CAV9 to accumulate on the cell surface. Furthermore, CAV9 infection was found to depend on Arf6 as both silencing of this molecule by siRNA and the expression of a dominant negative construct resulted in decreased virus infection. In conclusion, the internalization of CAV9 to A549 cells follows an endocytic pathway that is dependent on integrin αVβ6, β2-microglobulin, dynamin, and Arf6 but independent of clathrin and caveolin-1.

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