Cholesterol Dependence of Collagen and Echovirus 1 Trafficking along the Novel α2β1 Integrin Internalization Pathway

et al. (2013) Cholesterol Dependence of Collagen and Echovirus 1 Trafficking along the Novel a2b1 Integrin Internalization Pathway. PLoS ONE 8(2): e55465. doi:10.1371/journal.pone.0055465 Cholesterol Dependence of Collagen and Echovirus 1 Trafficking along the Novel a2b1 Integrin Internalization Pathway Elina Siljama ki 0 Nina Rintanen 0 Maija Kirsi 0 Paula Upla 0 Wei Wang 0 Mikko Karjalainen 0 Elina Ikonen 0 Varpu Marjoma ki 0 Donald Gullberg, University of Bergen, Norway 0 1 Department of Biological and Environmental Science/Nanoscience Center, University of Jyva skyla , Jyva skyla , Finland, 2 Institute of Biomedicine, University of Helsinki , Helsinki , Finland , 3 Minerva Foundation Institute for Medical Research , Helsinki , Finland We have previously shown that soluble collagen and a human pathogen, echovirus 1 (EV1) cluster a2b1 integrin on the plasma membrane and cause their internalization into cytoplasmic endosomes. Here we show that cholesterol plays a major role not only in the uptake of a2b1 integrin and its ligands but also in the formation of a2 integrin-specific multivesicular bodies (a2-MVBs) and virus infection. EV1 infection and a2b1 integrin internalization were totally halted by low amounts of the cholesterol-aggregating drugs filipin or nystatin. Inhibition of cholesterol synthesis and accumulation of lanosterol after ketoconazole treatment inhibited uptake of collagen, virus and clustered integrin, and prevented formation of multivesicular bodies and virus infection. Loading of lipid starved cells with cholesterol increased infection to some extent but could not completely restore EV1 infection to control levels. Cold Triton X-100 treatment did not solubilize the a2-MVBs suggesting, together with cholesterol labeling, that the cytoplasmic endosomes were enriched in detergentresistant lipids in contrast to aV integrin labeled control endosomes in the clathrin pathway. Cholesterol aggregation leading to increased ion permeability caused a significant reduction in EV1 uncoating in endosomes as judged by sucrose gradient centrifugation and by neutral red-based uncoating assay. In contrast, the replication step was not dependent on cholesterol in contrast to the reports on several other viruses. In conclusion, our results showed that the integrin internalization pathway is dependent on cholesterol for uptake of collagen, EV1 and integrin, for maturation of endosomal structures and for promoting EV1 uncoating. The results thus provide novel information for developing anti-viral strategies and more insight into collagen and integrin trafficking. - Funding: This work was supported by grants from Academy of Finland (http://www.aka.fi/eng, grant numbers: 131429, 131489/Elina Ikonen; 140756, 218109/ Varpu Marjomaki) and National graduate school in Nanoscience (https://www.jyu.fi/science/muut_yksikot/nsc/en/studies/ngs). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The plasma membrane is a complex organelle composed of various glycerophospholipids, sphingolipids, cholesterol and a diverse set of proteins. The knowledge of plasma membrane organization has evolved from the first, homogenous fluid-mosaic model to the present model of membrane microdomains, each having their own protein and lipid composition [1]. Lipid rafts are small, dynamic microdomains enriched in sphingolipids, cholesterol and associated proteins, such as glycosylphosphatidylinisotol (GPI)-anchored proteins [2]. They have significant functions for example in sorting, signaling and endocytosis, and their dysfunction has been linked to pathological states [35]. Many pathogens, including bacteria, parasites and viruses, have been found to hijack lipid rafts during cell entry [6]. We have recently studied the cell entry pathway of a human picornavirus, echovirus 1 (EV1), as a model system to understand events that occur during integrin-dependent cargo uptake [710]. Our biochemical and imaging studies have shown that EV1 is internalized in complex with its receptor, a2b1 integrin into tubulovesicular structures, which then gradually mature into a2 integrin-specific multivesicular bodies (a2-MVBs) [9,11]. The early entry is independent of clathrin and caveolin-1 but is instead regulated by a set of typical macropinocytic regulators, namely PKCa [7,10], PLC, Rac1, Pak1 [11] and CtBP1/BARS [12]. This pathway differs from typical integrin recycling as virus-induced clustering directs a2b1 integrin into a non-recycling pathway [13,14]. Targeting of a2b1 integrin into the a2-MVBs drives enhanced turnover of integrin, which is blocked by inhibition of neutral calpain proteases [14]. Recently, we found out that soluble collagen, the physiological ligand for a2b1 integrin, also clusters integrin and is co-internalized to a (...truncated)