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.2007 Sep;9(9):2218-29.
doi: 10.1111/j.1462-5822.2007.00951.x. Epub 2007 May 8.

Mouse hepatitis coronavirus replication induces host translational shutoff and mRNA decay, with concomitant formation of stress granules and processing bodies

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Mouse hepatitis coronavirus replication induces host translational shutoff and mRNA decay, with concomitant formation of stress granules and processing bodies

Matthijs Raaben et al. Cell Microbiol.2007 Sep.

Abstract

Many viruses, including coronaviruses, induce host translational shutoff, while maintaining synthesis of their own gene products. In this study we performed genome-wide microarray analyses of the expression patterns of mouse hepatitis coronavirus (MHV)-infected cells. At the time of MHV-induced host translational shutoff, downregulation of numerous mRNAs, many of which encode protein translation-related factors, was observed. This downregulation, which is reminiscent of a cellular stress response, was dependent on viral replication and caused by mRNA decay. Concomitantly, phosphorylation of the eukaryotic translation initiation factor 2alpha was increased in MHV-infected cells. In addition, stress granules and processing bodies appeared, which are sites for mRNA stalling and degradation respectively. We propose that MHV replication induces host translational shutoff by triggering an integrated stress response. However, MHV replication per se does not appear to benefit from the inhibition of host protein synthesis, at least in vitro, since viral replication was not negatively affected but rather enhanced in cells with impaired translational shutoff.

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Figures

Figure 1
Figure 1
Kinetics of the MHV‐induced host translational shutoff.
A. LR7 cells were infected with MHV (moi 10) and metabolically labelled for 15 min starting at the indicating time points. Cell lysates were processed and subjected to SDS‐PAGE as described inExperimental procedures. Positions of the viral proteins S, N and M are indicated.
B. The amount of radioactivity in the gel was quantified with a PhosphorImager. For each time point, the amount of radioactivity in the MHV structural proteins S, N and M was combined (viral expression). For the host proteins, the amount of radioactivity in the regions between the MHV proteins was quantified (host expression). The data are presented as relative expression (2 h post infection = 1).
C and D. Genomic viral RNA levels in MHV‐infected LR7 cells (moi 10) were measured by quantitative RT‐PCR at the indicated time points post infection. The data are presented as relative viral RNA levels (12 h post infection = 1) in C, or as the fold change increase relative to the previous time point in D. Error bars indicate the standard deviations (n = 3).
Figure 2
Figure 2
Replication‐dependent mRNA decay in MHV‐infected cells as determined by microarray analyses. LR7 cells were infected with MHV (moi 10). Total RNA was isolated from MHV‐infected and mock‐infected cells and processed for microarray analysis as described inExperimental procedures. The scatter plots display the average expression values from independent dye‐swap hybridizations for each gene present on the arrays. Red spots represent upregulated gene transcripts while green spots represent downregulated transcripts. The dashed lines indicate the twofold change cut‐off. Transcripts downregulated at 6 h post infection (in B) are represented by the yellow dots throughout the figure. Note that these transcripts are significantly changed at 6 h post infection according to SAM, applying a false‐discovery rate of 1% and a cut‐off at a twofold change (described inExperimental procedures).
A. The expression profile of MHV‐infected cells compared to mock‐infected cells at 4 h post infection (average of four arrays; two independent dye‐swaps;n = 4).
B. The expression profile of MHV‐infected cells compared with mock‐infected cells at 6 h post infection (n = 6).
C. The expression profile of MHV‐infected cells compared with mock‐infected cells at 6 h post infection (n = 4). Both MHV‐ and mock‐infected cells were treated with ActD for 7 h (from −1 till 6 h post infection).
D. The expression profile of ActD‐treated cells compared with mock‐treated cells. Non‐infected cells were treated or mock treated with ActD for 7 h (n = 1).
E. The expression profile of cells treated with UV‐inactivated viral particles compared with mock‐treated cells at 6 h post inoculation (n = 1).
Figure 3
Figure 3
MHV induces phosphorylation of eIF2α at Ser51, and the subsequent formation of SGs and P bodies.
A. The phosphorylation state of eIF2α in MHV‐infected LR7 cells at 4 and 6 h post infection (indicated by 4 and 6) or after mock infection was determined by Western blotting using eIF2αP‐specific antibodies (top) and related to total eIF2α levels by stripping and reprobing of the membrane (bottom). The fold change differences between MHV‐infected and mock‐infected cells are indicated above each lane. As a positive control, LR7 cells were treated with 0.5 mM sodium arsenite (indicated as S.A.) for 30 min. This experiment was repeated twice with similar results.
B. LR7 cells were mock‐infected, treated with S.A. for 30 min, or infected with MHV. Cells were fixed and processed for immunofluorescence using TIAR (αTIAR) and GW182 (αGW182) antibodies according toExperimental procedures. MHV‐infected cells were fixed at 4 and 6 h post infection (indicated by 4 and 6). Representative images are shown for each condition. The scaled bar corresponds to 20 μM.
Figure 4
Figure 4
MHV replication is not negatively affected in MEF cells with impaired host translational shutoff and reduced SG assembly. Confluent monolayers of MEF cells were infected with MHV‐EFLM. Cells were lysed at the indicated time points post infection and the intracellular luciferase levels were determined (in RLU). As the number of infected cells differed to some extent between the different MEFs, the obtained RLU values for each cell line were normalized for the number of infected cells as determined by immunocytochemistry using a polyclonal anti‐MHV serum (Rottieret al., 1981). Error bars indicate the standard deviations in all graphs (n = 3).
A. Luciferase expression in MEF cells expressing wild‐type eIF2α (MEF eIF2α wt) or mutant eIF2α (MEF eIF2αS51A).
B. Luciferase expression in wild type (MEF wt), TIA‐1 knockout (MEF TIA‐1–/–) and TIAR knockout (MEF TIAR–/–) cells.
Figure 5
Figure 5
Model for MHV‐induced host translational shutoff. A cellular stress response is elicited upon infection with MHV, which results in increased phosphorylation of eIF2α (eIF2αP). Subsequently, SGs containing stalled translational preinitiation complexes are formed and host translational shutoff is induced. In addition, the stress response also induces the formation of P bodies, which are cytoplasmic sites of mRNA decay. Degradation of many mRNAs encoding protein translation‐related factors is observed, which may contribute to the MHV‐induced host translational shutoff. Overall, the initiated stress response appears to inhibit MHV replication.
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