Mouse norovirus 1 utilizes the cytoskeleton network to establish localization of the replication complex proximal to the microtubule organizing center

Abstract

Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Although Human noroviruses are significant enteric pathogens, there exists no reliable vaccine or therapy to treat infected individuals. To date, attempts to cultivate Human noroviruses within the laboratory have met with little success; however, the related murine norovirus mouse norovirus 1 (MNV-1) has provided an ideal model system to study norovirus replication due to the ease with which the virus is cultivated and the ability to infect a small animal model with this virus. Previously we have identified the association between MNV-1 and components of the host secretory pathway and proposed a role for the viral open reading frame 1 proteins in the replication cycle. Here we describe for the first time a role for cytoskeletal components in early MNV-1 replication events. We show that the MNV-1 utilizes microtubules to position the replication complex adjacent to the microtubule organizing center. Chemical disruption of the microtubule network disperses the sites of MNV-1 replication throughout the cell and impairs production of viral protein and infectious virus. Furthermore, we demonstrate the ability of MNV-1 to redistribute acetylated tubulin to the replication complex and that this association is potentially mediated via the MNV-1 major structural protein, VP1. Transient expression of MNV-1 VP1 exhibited extensive colocalization with both α-tubulin and acetylated tubulin and was observed to alter the distribution of acetylated tubulin in transfected cells. This study highlights the role of the cytoskeleton in early virus replication events and demonstrates the importance of this interaction in establishing the intracellular location of MNV-1 replication complexes.

Fig 1

The MNV-1 RC localizes around the MTOC and induces alterations in microtubule morphology. Raw264.7 cells were infected with MNV-1 at an MOI of 5, fixed at 1, 6, 12, 18, and 24 hpi, and labeled for the RC (NS7) (green) and β-tubulin (red). The MNV-1 RC was observed to localize to a perinuclear location proximal to the MTOC at 6 hpi and to induce alterations in the morphology of microtubules, namely, the formation of microtubule bundles, at 12, 18, and 24 hpi.

Fig 2

MNV-1 induces alterations in the distribution and morphology of vimentin. Raw264.7 cells were infected with MNV-1 at an MOI of 5, fixed at 1, 6, 12, 18, and 24 hpi, and labeled for the RC (NS6-7) (green) and vimentin (red). MNV-1 was observed to colocalize with vimentin at 12 hpi and to induce alterations in the distribution and morphology of vimentin at 18 and 24 hpi.

Fig 3

MNV-1 does not associate with actin during replication. Raw264.7 cells were infected with MNV-1 at an MOI of 5, fixed at 1, 6, 12, 18, and 24 hpi, and labeled for the RC (NS6-7) (red) and α-actin (green). No colocalization was observed between MNV-1 and actin at any of the time points analyzed. Minor changes in actin morphology were observed at 18 and 24 hpi, which may be attributed to the induction of apoptosis by MNV-1.

Fig 4

MNV-1 localizes proximal to the MTOC during replication. Raw264.7 cells were infected with MNV-1 at an MOI of 5, fixed at 12 hpi, and labeled for the RC (NS7/NS6-7) (red) and pericentrin or γ-tubulin (green). MNV-1 was observed to localize proximal to the MTOC but did not exhibit any colocalization with either pericentrin or γ-tubulin. The morphology and distribution of pericentrin and γ-tubulin did not alter during the course of infection.

Fig 5

Disruption of microtubules inhibits MNV-1 replication. Raw264.7 cells were infected with MNV-1 at an MOI of 5 and treated with nocodazole (Noz) at 1 h prior to infection, 1 hpi, and 6 hpi. (A) Lysates were collected at 12 hpi and analyzed by immunoblotting using antisera against NS7, VP1, and actin. Relative expression was quantitated using actin as the internal control and Quantity One software. (B) Production of viral RNA was assessed by real-time qPCR. The relative amounts of viral RNA are expressed as a percentage compared to the MNV-1 untreated sample. Error bars represent standard errors of the means (SEM), and a one-way analysis of variance (ANOVA) was performed to derive the statistical significance (n = 6). (C) Tissue culture fluid was collected at 12 hpi and analyzed for the production of infectious virus by plaque assay. For virus titer analyses, intracellular virus (n = 3) and extracellular virus (n = 6) were measured by plaque assay, and the titers are expressed as a percentage compared to the dimethyl sulfoxide (DMSO)-treated control. Error bars represent SEM, and a one-way ANOVA was performed to derive the statistical significance. (D) RAW264.7 cells were treated with Noz prior to infection were fixed at 12 hpi and analyzed by IF for the distribution of the MNV-1 RC (NS6-7) and pericentrin, α-tubulin, or β-tubulin. Noz treatment inhibited localization of the MNV-1 RC proximal to the MTOC and resulted in the distribution of the RC throughout the cytoplasm.

Fig 6

MNV-1 replication induces the redistribution of acetylated tubulin to the RC. (A) Raw264.7 cells were infected with MNV-1 at an MOI of 5, fixed at 1, 6, 12, 18, and 24 hpi, and labeled for the RC (NS6-7) and acetylated (ace) α-tubulin. Acetylated tubulin was observed to localize with the MNV-1 RC in infected cells. (B) Immunoblots of cell lysates collected at 1, 6, 12, 18, and 24 hpi were also analyzed for changes in the level of acetylated tubulin. The level of acetylated tubulin was observed to remain unchanged, as was the overall level of α-tubulin and actin. A paclitaxel-treated sample which showed an increase in tubulin acetylation was used as a control.

Fig 7

MNV-1 VP1 localizes with and induces the redistribution of acetylated tubulin. (A and B) Vero C1008 cells were transfected with MNV-1 ORF1 proteins, fixed at 24 hpt, and analyzed by IF using antisera specific for each ORF1 protein and β-tubulin (A) (red) and acetylated tubulin (ace-tubulin) (B) (green). Only minor amounts of the ORF1 proteins analyzed were observed to colocalize with either β-tubulin or ace-tubulin. (C) VP1-transfected cells were also analyzed by IF using antisera specific for VP1 and β-tubulin or ace-tubulin (green). In contrast to the ORF1 proteins, VP1 exhibited extensive colocalization with acetylated tubulin and was observed to alter the distribution of acetylated tubulin in transfected cells.

Fig 8

A proportion of MNV-1 VP1 protein associates with the RC during infection. A time course infection shows localization of the MNV-1 structural protein VP1 with the RC. Raw264.7 cells were infected with MNV-1 at an MOI of 5 and fixed at 0, 6, 12, and 18 hpi. Cells were labeled with antisera against the RC (dsRNA) and the MNV-1 capsid (VP1). A proportion of the MNV-1 VP1 was observed to localize within the RC at all time points analyzed, in addition to there being some diffuse labeling within the cytoplasm.