Clathrin- and caveolae-independent entry of feline infectious peritonitis virus in monocytes depends on dynamin

Abstract

Feline infectious peritonitis virus (FIPV), a coronavirus that causes a lethal chronic disease in cats, enters feline monocytes via endocytosis. In this study, the pathway of internalization is characterized by evaluating the effect of chemical inhibitors and/or expression of dominantnegative (DN) proteins on the percentage of internalized virions per cell and infection. Further, colocalization studies were performed to determine the involvement of certain cellular internalization proteins. FIPV is not internalized through a clathrin-mediated pathway, as chlorpromazine, amantadine and DN eps15 did not influence virus uptake and FIPV did not co-localize with clathrin. The caveolae-mediated pathway could be excluded based on the inability of genistein and DN caveolin-1 to inhibit virus uptake and lack of co-localization between FIPV and caveolin-1. Dynamin inhibitory peptide and DN dynamin effectively inhibited virus internalization. The inhibitor strongly reduced uptake to 20.3±1.1 % of uptake in untreated cells. In the presence of DN dynamin, uptake was 58.7±3.9 % relative to uptake in untransduced cells. Internalization of FIPV was slightly reduced to 85.0±1.4 and 87.4±6.1 % of internalization in control cells by the sterolbinding drugs nystatin and methyl-b-cyclodextrin, respectively. Rho GTPases were inhibited by Clostridium difficile toxin B, but no effect was observed. These results were confirmed with infection studies showing that infection was not influenced by chlorpromazine, amantadine and genistein, but was significantly reduced by dynamin inhibition and nystatin. In conclusion, these results indicate that FIPV enters monocytes through a clathrin-and caveolae-independent pathway that strongly depends on dynamin and is slightly sensitive to cholesterol depletion. INTRODUCTIONViral entry occurs through a number of successive steps allowing the virus to bring its genome inside the cell. The virus either fuses with the host cell membrane or utilizes the endocytosis machinery of the cell. Endocytosis can be divided in two categories: phagocytosis of large cargo and pinocytosis of smaller cargo (Conner & Schmid, 2003). Phagocytosis is an active and highly regulated process involving specific cell surface receptors and signalling cascades mediated by Rho GTPases (Hall & Nobes, 2000;Conner & Schmid, 2003). Pinocytosis can occur through several pathways. The cargo and its receptor determine which pinocytic pathway will be used (Conner & Schmid, 2003). A first possible route for pinocytosis is 'macropinocytosis' (Swanson & Watts, 1995). Other pathways are named after the main structural compound involved: 'clathrin-mediated endocytosis ' (Brodsky et al., 2001) and 'caveolae-mediated endocytosis' (Pelkmans & Helenius, 2002). Finally, there is a group called 'clathrin-and caveolae-independent pathways'. They are differentiated based on their dependency on cholesterol and/or association with lipid rafts, dynamin and Rho GTPases (Nichols & Lippincott-Schwartz, 2001;Conner & Schmid, 2003). Viruses are specialized in...

Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis

“…14 To determine whether this entry pathway has a role in the uptake of the mutants, we conducted drug inhibition studies with genistein, which blocks caveolinmediated uptake through the inhibition of protein tyrosine kinases. 19 As a positive control, we included rAAV5, a serotype capable of entering cells by both clathrin-and caveolin-mediated endocytosis. 20 Although rAAV5 transduction was inhibited by 40% in the presence of genistein, rAAV2-mediated transduction was unaffected and transduction of the capsid insertion mutants was even increased by up to 43% (D5) (Supplementary Figure 1).…”

Novel mechanism of IGF-binding protein-3 action on prostate cancer cells: inhibition of proliferation, adhesion, and motility

“…IGFBP-3 was shown to exert extracellular functions as well as functions that are mediated by internalization of the protein via distinct endocytic pathways, which can be blocked by chemical inhibitors of endocytosis (Lee et al 2004). To evaluate whether endocytosis is required for the inhibitory actions of IGFBP-3 on cell adhesion, we preincubated prostate cancer cells with chemical compounds that inhibit clathrin-mediated (chlorpromazine and amantadine) or caveolae-mediated (nystatin, cyclodextrin, and genistein) endocytosis (Van Hamme et al 2008) or combinations inhibiting both pathways. None of these inhibitors, alone or in combination, affected reduction in cellular attachment by IGFBP-3, suggesting that internalization of IGFBP-3 is not required for its inhibitory effects on cell adhesion (Fig.…”

Role of Membrane Rafts in Viral Infection

“…Studies on the expression of dominant-negative Eps15, which is required for the clathrin-dependent pathway, and caveolin-1, indicate that SARS-CoV and feline infectious peritonitis virus (FIPV), which causes a lethal chronic disease in cats, internalize into cells via a clathrin-and caveola-independent pathway. On the other hand, inhibition of virus infection by cholesterol depletion has demonstrated entry of these viruses into cells by raft-dependent endocytosis [83,85]. These findings indicate that raft-dependent endocytosis except clathrin-and caveola-dependent pathway is important for the virus infection.…”

“…In addition to its role in entry of non enveloped the role of membrane rafts in entry of enveloped viruses has been investigated in studies using influenza virus (Orthomyxoviridae) [57][58][59][60][61], human immunodeficiency virus type 1 (HIV-1; Retroviridae) [62][63][64][65][66][67], human T lymphotropic virus 1 (HTLV-1; Retroviridae) [68,69], Ebola virus (Filoviridae) [70], Marburg virus (Filoviridae) [70,71], Epstein-Barr virus (EBV; herpesviridae) [72,73], herpes simplex virus-1 (HSV-1; herpesviridae) [74] including porcine herpesvirus-1 pseudorabies virus [75], human herpesvirus-6 (HHV-6; Herpesviridae) [76], human herpesvirus-8 (HHV-8; Herpesviridae) [77], vaccinia virus (Poxviridae) [78], coronavirus including severe acute respiratory syndrome coronavirus (SARS-CoV; Coronaviridae) [79][80][81][82][83][84][85], West Nile virus (WNV; Flaviviridae) [86], dengue virus (DEN; Flaviviridae) [87][88][89], Japanese encephalitis virus (JEV; Flaviviridae) [89], human hepatitis C virus (HCV; Flaviviridae) [90,91], Semliki Forest virus (Togaviridae) [92][93][94][95], Sindbis virus (Togaviridae) …”