SARS-CoV-2 infects cells after viral entry via clathrin-mediated endocytosis.
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ABSTRACT:
SUBMITTER: Bayati A
PROVIDER: S-EPMC7816624 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with high morbidity and mortality in domestic pigs and wild boars. The disease has become a global threat to the pig production industry and has caused enormous economic losses in many countries in recent years. However, the molecular mechanism underlying ASF virus (ASFV) entry of the host cells is not fully understood, which restricts the development of vaccines and antiviral-drugs of ASFV. In this study, we found that the host protein CD1d acts as a host factor, which mediates ASFV entry into the host cells. As the main capsid protein on the surface of ASFV virions, p72 can mediate viral entry. Using IP-MS assay, CD1d was identified as a binding partner of p72 on surface of ASFV virions. Knockdown of CD1d expression and blocking the cells with anti-pCD1d antibody, or incubating ASFV virions with soluble CD1d protein could significantly inhibit ASFV infection. CD1d is located on the membrane surface of primary porcine alveolar macrophages (PAMs) and mediates the virus entry via binding to p72. CD1d knockout or CD1d knockdown assay showed that CD1d could facilitate ASFV virions internalization via clathrin-mediated endocytosis (CME). Furthermore, CD1d interacts with EPS15 to mediate ASFV entry via clathrin-mediated endocytosis. Overall, our findings revealed that CD1d is a novel host-entry factor involved in ASFV internalization via the EPS15-clathrin endocytosis axis and a potential target for antiviral intervention.
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Project description:BackgroundJapanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes acute viral encephalitis in humans. Pigs are important amplifiers of JEV. The entry mechanism of JEV into porcine cells remains largely unknown. In this study, we present a study of the internalization mechanism of JEV in porcine kidney epithelial PK15 cells.ResultsWe demonstrated that the disruption of the lipid raft by cholesterol depletion with methyl-β-cyclodextrin (MβCD) reduced JEV infection. We also found that the knockdown of clathrin by small interfering RNA (siRNA) significantly reduced JEV-infected cells and JEV E-glycoprotein levels, suggesting that JEV utilizes clathrin-dependent endocytosis. In contrast, the knockdown of caveolin-1, a principal component of caveolae, had only a small (although statistically significant) effect on JEV infection, however, JEV entry was not affected by genistein. These results suggested that JEV entry was independent of caveolae.ConclusionsTaken together, our results demonstrate that JEV enters porcine kidney epithelial PK15 cells through cholesterol- and clathrin-mediated endocytosis.
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Project description: Not available
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Project description:Emergence of SARS-CoV-2 variants warrants sustainable efforts to upgrade both the diagnostic and therapeutic protocols. Understanding the details of cellular and molecular basis of the virus-host cell interaction is essential for developing variant-independent therapeutic options. The internalization of SARS-CoV-2, into lung epithelial cells, is mediated by endocytosis, especially clathrin-mediated endocytosis (CME). Although vaccination is the gold standard strategy against viral infection, selective inhibition of endocytic proteins, complexes, and associated adaptor proteins may present a variant-independent therapeutic strategy. Although clathrin and/or dynamins are the most important proteins involved in CME, other endocytic mechanisms are clathrin and/or dynamin independent and rely on other proteins. Moreover, endocytosis implicates some subcellular structures, like plasma membrane, actin and lysosomes. Also, physiological conditions, such as pH and ion concentrations, represent an additional factor that mediates these events. Accordingly, endocytosis related proteins are potential targets for small molecules that inhibit endocytosis-mediated viral entry. This review summarizes the potential of using small molecules, targeting key proteins, participating in clathrin-dependent and -independent endocytosis, as variant-independent antiviral drugs against SARS-CoV-2 infection. The review takes two approaches. The first outlines the potential role of endocytic inhibitors in preventing endocytosis-mediated viral entry and its mechanism of action, whereas in the second computational analysis was implemented to investigate the selectivity of common inhibitors against endocytic proteins in SARS-CoV-2 endocytosis. The analysis revealed that remdesivir, methyl-β-cyclodextrin, rottlerin, and Bis-T can effectively inhibit clathrin, HMG-CoA reductase, actin, and dynamin I GTPase and are more potent in inhibiting SARS-CoV-2 than chloroquine. CME inhibitors for SARS-CoV-2 infection remain understudied.
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Project description:Biomarkers have important roles in various physiological functions and disease pathogenesis. As a nucleocytoplasmic DNA virus, Singapore grouper iridovirus (SGIV) causes high economic losses in the mariculture industry. Aptamer-Q5-complexed major capsid protein (MCP) in the membrane of SGIV-infected cells can be used as a specific molecular probe to investigate the crucial events of MCP endocytosis into SGIV-infected host cells during viral infection. Chlorpromazine blocks clathrin-mediated endocytosis, and MCP endocytosis into SGIV-infected cells decreased significantly when the cells were pretreated with chlorpromazine. The disruption of cellular cholesterol by methyl-β-cyclodextrin also significantly reduced MCP endocytosis. In contrast, inhibitors of key regulators of caveolae/raft-dependent endocytosis and macropinocytosis, including genistein, Na+/H+ exchanger, p21-activated kinase 1 (PAK1), myosin II, Rac1 GTPase, and protein kinase C (PKC), had no effect on MCP endocytosis. The endocytosis of the biomarker MCP is dependent on low pH and cytoskeletal actin filaments, as shown with various inhibitors (chloroquine, ammonia chloride, cytochalasin D). Therefore, MCP enters SGIV-infected host cells via clathrin-mediated endocytosis, which is dependent on dynamin, cholesterol, low pH, and cytoskeletal actin filaments. This is the first report of a specific aptamer-based probe used to analyze MCP endocytosis into SGIV-infected host cells during viral infection. This method provides a convenient strategy for exploring viral pathogenesis and facilitates the development of diagnostic tools for and therapeutic approaches to viral infection.
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Project description:JC polyomavirus (JCPyV) establishes a persistent, lifelong, asymptomatic infection within the kidney of the majority of the human population. Under conditions of severe immunosuppression or immune modulation, JCPyV can reactivate in the central nervous system (CNS) and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease. Initiation of infection is mediated through viral attachment to α2,6-sialic acid-containing lactoseries tetrasaccharide c (LSTc) on the surface of host cells. JCPyV internalization is dependent on serotonin 5-hydroxytryptamine subfamily 2 receptors (5-HT2Rs), and entry is thought to occur by clathrin-mediated endocytosis (CME). However, the JCPyV entry process and the cellular factors involved in viral internalization remain poorly understood. Treatment of cells with small-molecule chemical inhibitors and RNA interference of 5-HT2R endocytic machinery, including β-arrestin, clathrin, AP2, and dynamin, significantly reduced JCPyV infection. However, infectivity of the polyomavirus simian virus 40 (SV40) was not affected by CME-specific treatments. Inhibition of clathrin or β-arrestin specifically reduced JCPyV internalization but did not affect viral attachment. Furthermore, mutagenesis of a β-arrestin binding domain (Ala-Ser-Lys) within the intracellular C terminus of 5-HT2AR severely diminished internalization and infection, suggesting that β-arrestin interactions with 5-HT2AR are critical for JCPyV infection and entry. These conclusions illuminate key host factors that regulate clathrin-mediated endocytosis of JCPyV, which is necessary for viral internalization and productive infection.IMPORTANCE Viruses usurp cellular factors to invade host cells. Activation and utilization of these proteins upon initiation of viral infection are therefore required for productive infection and resultant viral disease. The majority of healthy individuals are asymptomatically infected by JC polyomavirus (JCPyV), but if the host immune system is compromised, JCPyV can cause progressive multifocal leukoencephalopathy (PML), a rare, fatal, demyelinating disease. Individuals infected with HIV or taking prolonged immunomodulatory therapies have a heightened risk for developing PML. The cellular proteins and pathways utilized by JCPyV to mediate viral entry are poorly understood. Our findings further characterize how JCPyV utilizes the clathrin-mediated endocytosis pathway to invade host cells. We have identified specific components of this pathway that are necessary for the viral entry process and infection. Collectively, the conclusions increase our understanding of JCPyV infection and pathogenesis and may contribute to the future development of novel therapeutic strategies for PML.
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Project description:Agrobacterium tumefaciens can cause crown gall tumors on a wide range of host plants. As a natural genetic engineer, the bacterium can transfer both single-stranded DNA (ssDNA) [transferred DNA (T-DNA)] molecules and bacterial virulence proteins into various recipient cells. Among Agrobacterium-delivered proteins, VirE2 is an ssDNA binding protein that is involved in various steps of the transformation process. However, it is not clear how plant cells receive the T-DNA or protein molecules. Using a split-green fluorescent protein approach, we monitored the VirE2 delivery process inside plant cells in real time. We observed that A. tumefaciens delivered VirE2 from the bacterial lateral sides that were in close contact with plant membranes. VirE2 initially accumulated on plant cytoplasmic membranes at the entry points. VirE2-containing membranes were internalized through clathrin-mediated endocytosis to form endomembrane compartments. VirE2 colocalized with the early endosome marker SYP61 but not with the late endosome marker ARA6, suggesting that VirE2 escaped from early endosomes for subsequent trafficking inside the cells. Dual endocytic motifs at the carboxyl-terminal tail of VirE2 were involved in VirE2 internalization and could interact with the μ subunit of the plant clathrin-associated adaptor AP2 complex (AP2M). Both the VirE2 cargo motifs and AP2M were important for the transformation process. Because AP2-mediated endocytosis is well conserved, our data suggest that the A. tumefaciens pathogen hijacks conserved endocytic pathways to facilitate the delivery of virulence factors. This might be important for Agrobacterium to achieve both a wide host range and a high transformation efficiency.
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Project description:Bacterial toxins possess specific mechanisms of binding and uptake by mammalian cells. Mycoplasma pneumoniae CARDS (Community Acquired Respiratory Distress Syndrome) toxin is a 68 kDa protein, which demonstrates high binding affinity to human surfactant protein-A and exhibits specific biological activities including mono-ADP ribosylation and vacuolization. These properties lead to inflammatory processes in the airway and a range of cytopathologies including ciliostasis, loss of tissue integrity and injury, and cell death. However, the process by which CARDS toxin enters target cells is unknown. In this study, we show that CARDS toxin binds to mammalian cell surfaces and is internalized rapidly in a dose and time-dependent manner using a clathrin-mediated pathway, as indicated by inhibition of toxin internalization by monodansylcadaverine but not by methyl-β-cyclodextrin or filipin. Furthermore, the internalization of CARDS toxin was markedly inhibited in clathrin-depleted cells.
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