Murine norovirus-1 entry into permissive macrophages and dendritic cells is pH-independent

Abstract

SummaryMurine norovirus (MNV) is a recently discovered mouse pathogen. Unlike the fastidious human noroviruses that cause the overwhelming majority of non-bacterial gastroenteritis worldwide, MNV readily infects cells in culture. Its replication in primary murine macrophages and dendritic cells and their derived cell lines allows the study of norovirus cell entry for the first time. In this study we determined the role of pH during MNV-1 infection since the low pH environment of endosomes often triggers uncoating of viruses. We demonstrated that MNV-1 viral titers by plaque assay and expression of the non-structural protein VPg by immunofluorescence were not affected by pH in cultured and primary macrophages and dendritic cells in the presence of two known endosome acidification inhibitors, bafilomycin A1 and chloroquine. These data indicate that MNV-1 enters permissive cells in a pH-independent manner. KeywordsRNA virus; norovirus; pH-independent entry; macrophages; dendritic cells; enteric virusNoroviruses are an understudied group of non-enveloped positive strand RNA viruses that belong to the Caliciviridae family (Green, 2007). Despite the significant impact of human noroviruses (HuNoV) on public health worldwide as the major agent of non-bacterial gastroenteritis (Green, 2007), no drug or vaccine exists to treat norovirus infections. This is partially due to the absence of a robust tissue culture system (Duizer et al., 2004;Straub et al., 2007). In contrast, murine norovirus (MNV), a highly prevalent agent in research mouse colonies (Hsu et al., 2005;Muller et al., 2007), readily infects murine macrophages and dendritic cells (DC) in culture and in vivo (Ward et al., 2006;Wobus et al., 2004;. Similar to HuNoV, MNV replicates in the gastrointestinal tract of its wild type or immunocompromised host, is shed in the feces, and is transmitted by the fecal-oral route (reviewed in: ). The ability to culture a norovirus has already led to insights into norovirus biology (Chaudhry et al., 2006;Daughenbaugh et al., 2006;Simmonds et al., 2008;Sosnovtsev et al., 2006) and inactivation (for example: (Baert et al., 2008;Belliot et al., 2008). However, no studies have yet addressed requirements for norovirus entry into cells. , email: E-mail: cwobus@umich.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. To gain access into host cells, viruses hijack cellular processes. The most commonly used endocytic pathway for virus entry is clathrin-mediated endocytosis. Viral entry can also occur via caveolin-mediated endocytosis, clathrin/caveolin-independent endocytosis, macropinocytosi...

Mouse Norovirus 1 Utilizes the Cytoskeleton Network To Establish Localization of the Replication Complex Proximal to the Microtubule Organizing Center

“…Two previous reports have suggested roles for actin in the replication cycle of Caliciviruses; however, the observations are conflicting. Perry et al (38) observed that treatment with cytochalasin D enhanced MNV-1 infection, whereas Stuart and Brown (48) observed that feline calicivirus infection was impaired. In each case, though, only virus-positive cells were enumerated and effects on virus replication were not quantitated.…”

High-Resolution Functional Profiling of the Norovirus Genome

“…BHK cells engineered to express T7 RNA polymerase (BSR-T7 cells, obtained from Karl-Klaus Conzelmann, Ludwig Maximilian University, Munich, Germany) were maintained in DMEM containing 10% FCS, penicillin (100 SI units/ml), streptomycin (100 g/ml), and 0.5 mg/ml G418. The cDNA clone pT7: MNV 3= RZ, containing the WT MNV-1 genome under the control of a truncated T7 polymerase promoter, and a modified version that contains a frameshift in the NS7 region of ORF1 (pT7:MNV 3= RZ F/S) were previously constructed (48).…”

“…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).…”

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.…”