Murine norovirus-1 cell entry is mediated through a non-clathrin-, non-caveolae-, dynamin- and cholesterol-dependent pathway

Abstract

For many viruses, endocytosis and exposure to the low pH within acidic endosomes is essential for infection. It has previously been reported that feline calicivirus uses clathrin-mediated endocytosis for entry into mammalian cells. Here, we report that infection of RAW264.7 macrophages by the closely related murine norovirus-1 (MNV-1) does not require the clathrin pathway, as infection was not inhibited by expression of dominant-negative Eps15 or by knockdown of the adaptin-2 complex. Further, infection was not inhibited by reagents that raise endosomal pH. RAW264.7 macrophages were shown not to express caveolin, and flotillin depletion did not inhibit infection, suggesting that caveolae and the flotillin pathway are not required for cell entry. However, MNV-1 infection was inhibited by methyl-b-cyclodextrin and the dynamin inhibitor, dynasore. Addition of these drugs to the cells after a period of virus internalization did not inhibit infection, suggesting the involvement of cholesterol-sensitive lipid rafts and dynamin in the entry mechanism. Macropinocytosis (MPC) was shown to be active in RAW264.7 macrophages (as indicated by uptake of dextran) and could be blocked by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), which is reported to inhibit this pathway. However, infection was enhanced in the presence of EIPA. Similarly, actin disruption, which also inhibits MPC, resulted in enhanced infection. These results suggest that MPC could contribute to virus degradation or that inhibition of MPC could lead to the upregulation of other endocytic pathways of virus uptake. INTRODUCTIONThe family Caliciviridae is divided into four genera: Vesivirus, Lagovirus, Sapovirus and Norovirus. The human noroviruses are the most common cause of acute viral gastroenteritis, especially in industrialized countries (Lopman et al., 2003); there is currently no treatment or vaccine for these viruses. In addition, there is still no routine tissue culture system for the propagation of human noroviruses, but the recent discovery of murine norovirus-1 (MNV-1) has provided an efficient model system for the study of norovirus replication, as this virus replicates efficiently in murine macrophages and dendritic cells (DCs) (Karst et al., 2003;Wobus et al., 2004). MNV-1 is highly prevalent in laboratory mice and has been shown to be lethal to mice with impaired innate immunity (Hsu et al., 2006;Karst et al., 2003;Muller et al., 2007).Noroviruses are non-enveloped and contain a singlestranded RNA genome of about 7.3 kb, which is linked to the viral VPg protein at the 59 end and is polyadenylated at the 39 end (Karst et al., 2003;Wobus et al., 2004). The genome is organized into four open reading frames (ORF-1-4). ORF-1 encodes the non-structural proteins, whereas ORF-2 and ORF-3 encode structural proteins (Sosnovtsev et al., 2006). ORF-4 was recently identified within the MNV-1 genome and encodes a single protein of unknown function (Thackray et al., 2007).For many viruses, infection requires virus uptake by endocytosis. A number of different endo...

Murine Norovirus Transcytosis across anIn VitroPolarized Murine Intestinal Epithelial Monolayer Is Mediated by M-Like Cells

“…MNV-1 and S99 binding to murine macrophages is dependent on terminal sialic acid residues of the ganglioside GD1a, N-linked, or O-linked glycoproteins, while CR3 binding requires only N-linked glycoproteins (21,22). Although multiple studies have elucidated aspects of the multistep process by which MNV enters permissive macrophages (21)(22)(23)(24)(25), how the virus crosses the intestinal epithelial barrier to reach susceptible macrophages and dendritic cells in the first place is unknown.…”

High-Resolution Functional Profiling of the Norovirus Genome

“…MNV has also been isolated from wild mice, confirming its widespread distribution (59). Studies with MNV have begun to probe various aspects of the norovirus replication cycle, including entry mechanisms and attachment factors (24,48,49,67), viral protein function (8,19,30,43,64,66), determinants of virulence (7), and host immune responses (14, 15, 42); however, our understanding of the molecular mechanisms of norovirus genome translation and replication lags far behind that for other RNA viruses.MNV is a positive-sense single-stranded RNA virus with a genome of approximately 7.4 kb covalently attached to a viral genome-linked protein (VPg) at the 5= end and polyadenylated at the 3= end (33). The 5= and 3= untranslated regions (UTRs) are extremely short, 5 and 78 nucleotides (nt), respectively, for MNV-1 (33).…”

Comparison of the cellular transport mechanism of cationic, star-shaped polymers and liposomes in HaCat cells

“…8 Many viruses, such as the influenza, Sendai, and echoviruses, were also found to infect cells utilizing non-clathrin-and non-caveolaemediated pathways for transport to the golgi apparatus and, subsequently, to the endoplasmic reticulum. 9,10 Previous research has reported that the mode of complex internalization might affect not only the intracellular processing kinetics but also the transfection efficiency. 11 Thus, we studied the intracellular processing of polyplexes and liposomes internalized by different endocytosis pathways We explored the colocalization of the two types of fluorescence using laser scanning confocal microscopy, which showed yellow particles in the cytoplasm.…”