Mouse norovirus replication is associated with virus-induced vesicle clusters originating from membranes derived from the secretory pathway

Abstract

Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Despite the prevalence of these viruses within the community, the study of human norovirus has largely been hindered due to the inability to cultivate the viruses ex vivo and the lack of a small-animal model. In 2003, the discovery of a novel murine norovirus (MNV-1) and the identification of the tropism of MNV-1 for cells of a mononuclear origin led to the establishment of the first norovirus tissue culture system. Like other positive-sense RNA viruses, MNV-1 replication is associated with host membranes, which undergo significant rearrangement during infection. We characterize here the subcellular localization of the MNV-1 open reading frame 1 proteins and viral double-stranded RNA (dsRNA). Over the course of infection, dsRNA and the MNV-1 RNA-dependent RNA polymerase (NS7) were observed to proliferate from punctate foci located in the perinuclear region. All of the MNV-1 open reading frame 1 proteins were observed to colocalize with dsRNA during the course of infection. The MNV-1 replication complex was immunolocalized to virus-induced vesicle clusters formed in the cytoplasm of infected cells. Both dsRNA and MNV-1 NS7 were observed to localize to the limiting membrane of the individual clusters by cryo-immunoelectron microscopy. We show that the MNV-1 replication complex initially associates with membranes derived from the endoplasmic reticulum, trans-Golgi apparatus, and endosomes. In addition, we show that MNV-1 replication is insensitive to the fungal metabolite brefeldin A and consistently does not appear to recruit coatomer protein complex I (COPI) or COPII component proteins during replication. These data provide preliminary insights into key aspects of replication of MNV-1, which will potentially further our understanding of the pathogenesis of noroviruses and aid in the identification of potential targets for drug development.

FIG. 1.

Genome organization of MNV-1. The MNV-1 genome is organized into three ORFs (ORF1 to ORF3). ORF1 encodes for six nonstructural proteins indicated that are cleaved by a virus-encoded cysteine protease (NS6) to produce mature viral proteins. Adjacent to ORF1 and partially overlapping is ORF2 and ORF3, which encode the major (VP1) and minor (VP2) structural proteins, which are produced from a subgenomic RNA species. Some of the identified roles for these proteins are shown, whereas the descriptions in brackets (i.e., NTPase function for NS3) are postulated.

FIG. 2.

The MNV-1 RC localizes adjacent to the nucleus in infected cells. RAW264.7 were infected with MNV-1 at an MOI of 5; fixed at 0, 6, 12, 18, and 24 hpi; and dual labeled with antibodies to dsRNA and MNV-1 RNA polymerase (NS7). Replication was detected at 6 hpi, as indicated by the presence of coincidental punctuate labeling adjacent to the nucleus. As infection progressed, this area of coincidental labeling increased in both size and density, with signs of cytopathic effect being detected as early as 18 hpi.

FIG. 3.

MNV-1 nonstructural proteins all exhibit a similar subcellular localization pattern in infected cells. RAW264.7 cells were infected with MNV-1 at an MOI of 5; fixed at 12 hpi; and dual labeled with antibodies specific for dsRNA and for MNV-1 nonstructural proteins NS1-2, NS3, NS4, NS5, and NS6. Antibodies were subsequently visualized with species-specific IgG conjugated to Alexa Fluor 488 or 594.

FIG. 4.

The MNV-1 RC associates with membrane vesicles in infected cells. Cryosections of RAW264.7 cells infected with MNV-1 at an MOI of 5 and fixed at 12 hpi (A, C, E, and F) or 24 hpi (B and D) were immunolabeled with anti-dsRNA antibodies and 10-nm PAG (A and B) or anti-NS7 antibodies and 10-nm PAG (C and D). In panels E and F, the cryosections from MNV-1-infected RAW264.7 cells were dual labeled with antibodies to dsRNA (5-nm PAG) and NS7 (15-nm PAG). Arrows in panels E and F highlight the smaller 5-nm gold particles. Magnification bars represent 200 nm in all cases.

FIG. 5.

MNV-1 associates with markers for the ER, cis/medial-Golgi bodies, and endosomes in infection. RAW264.7 cells were infected with MNV-1 at an MOI of 5, fixed at 12 hpi, and dual labeled with markers for the MNV-1 RC (dsRNA or NS7, shown in green) and various cellular membrane markers (shown in red). The MNV-1 RC was found to partially colocalize with markers for the ER (calnexin), the cis/medial-Golgi apparatus (Giantin) and endosomes (EEA1), visualized as a yellow hue, but showed very little colocalization with the cis Golgi (GM130) and no colocalization with lysosomes (LAMP1).

FIG. 6.

The MNV-1 RC localizes adjacent to the nucleus near the Golgi apparatus. Ultrastructual analysis of the MNV-1-infected cells at 12 hpi shows the positioning of the MNV-1 RC adjacent to the Golgi apparatus. (A and B) Cryosections immunolabeled with antibodies to MNV-1 NS7 and 10-nm PAG. The identified RC appears to lie in close proximity to an apparently intact Golgi body. (C and D) Resin-embedded sections from MNV-1-infected RAW264.7 cells, again suggesting a close association of the Golgi apparatus with MNV-1 RNA replication.

FIG. 7.

MNV-1 replication is independent of COPI and COPII and insensitive to the action of BFA. (A) RAW264.7 cells were infected with MNV-1 at an MOI of 5, fixed at 12 hpi, and labeled with antibodies specific for dsRNA and for subunits of the COPI (p23; a to c) and COPII (Sec23; d to f) complexes. The antibodies were visualized with species-specific IgG conjugated to Alexa Fluor 488 or 594. No apparent colocalization was observed between dsRNA and either p23 or Sec23. (B) Western blot of lysates prepared from MNV-1-infected RAW264.7 cells that had been treated with BFA (1 μg/ml) prior to infection and at 0 and 6 hpi and cell lysates harvested at 12 hpi. The relative expression levels of MNV-1 NS7 and VP1 were assessed to determine the effect of BFA treatment on replication. None of the BFA-treated samples appear to show any significant difference in the level of either NS7 or VP1 expression. The expression level of the cellular protein actin served as a loading control. (C) Plaque assay of infected tissue culture fluid collected from MNV-1-infected RAW264.7 cells that were treated with BFA as described above at 24 hpi. No significant difference in the level of virus release was detected in any of the treated samples compared to the control.