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. 2008 May;82(9):4492-501.
doi: 10.1128/JVI.00017-08. Epub 2008 Feb 27.

Coronavirus infection modulates the unfolded protein response and mediates sustained translational repression

John Bechill  1 , Zhongbin Chen, Joseph W Brewer, Susan C Baker
Affiliations

Coronavirus infection modulates the unfolded protein response and mediates sustained translational repression

John Bechill et al. J Virol. 2008 May.

Abstract

During coronavirus replication, viral proteins induce the formation of endoplasmic reticulum (ER)-derived double-membrane vesicles for RNA synthesis, and viral structural proteins assemble virions at the ER-Golgi intermediate compartment. We hypothesized that the association and intense utilization of the ER during viral replication would induce the cellular unfolded protein response (UPR), a signal transduction cascade that acts to modulate translation, membrane biosynthesis, and the levels of ER chaperones. Here, we report that infection by the murine coronavirus mouse hepatitis virus (MHV) triggers the proximal UPR transducers, as revealed by monitoring the IRE1-mediated splicing of XBP-1 mRNA and the cleavage of ATF6alpha. However, we detected minimal downstream induction of UPR target genes, including ERdj4, ER degradation-enhancing alpha-mannosidase-like protein, and p58(IPK), or expression of UPR reporter constructs. Translation initiation factor eIF2alpha is highly phosphorylated during MHV infection, and translation of cellular mRNAs is attenuated. Furthermore, we found that the critical homeostasis regulator GADD34, which recruits protein phosphatase 1 to dephosphorylate eIF2alpha during the recovery phase of the UPR, is not expressed during MHV infection. These results suggest that MHV modifies the UPR by impeding the induction of UPR-responsive genes, thereby favoring a sustained shutdown of the synthesis of host cell proteins while the translation of viral proteins escalates. The role of this modified response and its potential relevance to viral mechanisms for the evasion of innate defense signaling pathways during coronavirus replication are discussed.

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Figures

FIG. 1.
FIG. 1.
Analysis of the activation status of the IRE1/XBP-1 and ATF6 pathways during MHV infection. Cells were incubated in the absence or presence of 2 μg/ml of tunicamycin (Tun) or infected with MHV. (A) RT-PCR analysis of XBP-1u and XBP-1s mRNAs during MHV infection. RNA was extracted from cells at the times indicated and subjected to RT-PCR analysis using primers flanking the XBP-1 splice site. The PCR products were resolved on a 5% polyacrylamide gel and visualized by staining with ethidium bromide. The PCR products generated from the XBP-1u and XBP-1s mRNAs are indicated. M, molecular-size markers. (B and C) Western blot analysis of XBP-1(S) and ATF6α proteins from MHV-infected cells. Whole-cell lysates or nuclear (N) and cytosolic (C) fractions were prepared from untreated (Mock), tunicamycin-treated, or MHV-infected cells and subjected to Western blot analysis using antisera specific to XBP-1 or ATF6α. For ATF6α, both precursor (P) and cleaved (C) products are indicated in panel C. Detection of the resident ER protein calnexin and MHV nonstructural protein 8 (nsp8) was used as a loading control and a marker for MHV infection, respectively.
FIG. 2.
FIG. 2.
Analysis of induction of UPRE and ERSE promoters during MHV infection. DBT cells were transfected with either UPR reporter plasmid pUPRE-GL3 or ERSE reporter plasmid pERSE-GL3 DNA and, 24 h later, infected with MHV or treated with tunicamycin (Tun; 2 μg/ml). Cell lysates were prepared at the times indicated and assayed in triplicate using a dual-luciferase assay as described in Materials and Methods. Error bars show standard deviations.
FIG. 3.
FIG. 3.
Northern blot analysis of UPR target genes during MHV infection. DBT cells were infected with MHV or treated with tunicamycin (Tun; 2 μg/ml), and RNA was extracted and subjected to Northern blot analysis using probes against the ERdj4, EDEM, p58IPK, and MHV nucleocapsid gene sequences. The seven MHV mRNAs and multiple p58IPK mRNAs are indicated. Ethidium bromide stain of 28S rRNA is visualized as a loading control.
FIG. 4.
FIG. 4.
Analysis of XBP-1 and ATF6α mRNAs during MHV infection. (A) Results of quantitative RT-PCR analysis of XBP-1 mRNA. RNA was isolated from MHV-infected, untreated, or tunicamycin-treated cells and subjected to quantitative RT-PCR analysis using rRNA as a control, as described in Materials and Methods. Error bars show standard deviations. M, mock-treated cells. (B) Northern blot analysis of ATF6α and XBP-1 mRNA during MHV infection. RNA was extracted and subjected to Northern blot analysis using probes against XBP-1 or ATF6α. Dashes indicate the mRNAs detected with the probe to ATF6α. Ethidium bromide staining of 28S rRNA is shown as a loading control. Tun, tunicamycin.
FIG. 5.
FIG. 5.
Analysis of reporter induction by XBP-1-GFP and ATF6α-GFP during MHV infection. HeLa-MHVR cells were transfected with either pUPRE-GL3, pRL, and XBP-1(S)-GFP plasmid DNAs (A) or pERSE-GL3, pRL, and ATF6α(373)-GFP plasmid DNAs (B) using Lipofectamine 2000 (Invitrogen). Twenty-four hours later, cells were either mock infected (M) or infected with MHV (I), and lysates were prepared at 3, 6, and 9 hpi. The pEGFP plasmid (Invitrogen) was used as the vector control (Vect). The lysates were analyzed in triplicate using a dual-luciferase assay. Error bars show standard deviations.
FIG. 6.
FIG. 6.
Analysis of the eIF2α pathway in MHV-infected cells. (A) Lysates were generated from either MHV-infected cells or cells treated with tunicamycin (Tun) (2 μg/ml), and Western blot analysis was performed using antibodies directed against phosphorylated eIF2α (P-eIF2α) and eIF2α. (B) Analysis of protein synthesis in MHV-infected cells. DBT cells were either treated with tunicamycin or infected with MHV at an MOI of 10 and incubated for the indicated periods of time. A half hour before harvest, cells were starved in medium without methionine for 15 min and then pulsed with [35S]methionine for 15 min. Cell lysates were separated by electrophoresis on a 12% polyacrylamide gel and imaged using a phosphoimager. MHV structural proteins are indicated: S, spike; N, nucleocapsid; M, membrane. (C) Western blot analysis of lysates prepared from MHV-infected or tunicamycin-treated cells probed with antibodies specific to ATF4, CHOP, and GADD34.
FIG. 7.
FIG. 7.
Models depicting the UPR during pharmacologically induced ER stress versus MHV infection. (Top) During pharmacological induction of the UPR, IRE1-mediated splicing of XBP-1 mRNA, cleavage of ATF6α, and PERK-mediated phosphorylation of eIF2α are triggered. Translational attenuation is induced, but translational recovery initiates with the production of ATF4 which, with ATF6α, activates the transcription of CHOP and GADD34. GADD34 recruits PP1 to the ER to dephosphorylate eIF2α and promote the return to homeostasis. XBP-1 and ATF6α activate the transcription of UPR targets, which are produced and further modulate recovery and homeostasis. (Bottom) During MHV infection, IRE1-mediated splicing of XBP-1 mRNA occurs and ATF6α is cleaved, but very little induction of UPR target genes is realized. eIF2α is phosphorylated and ATF4 is produced; however, CHOP and GADD34 are not expressed. Phosphorylation of eIF2α persists and this correlates with the ongoing repression of most cellular protein synthesis. eIF2α-P, phosphorylated eIF2α.
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