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. 2009 Dec;83(23):12462-72.
doi: 10.1128/JVI.01546-09. Epub 2009 Sep 23.

Inhibition of protein kinase R activation and upregulation of GADD34 expression play a synergistic role in facilitating coronavirus replication by maintaining de novo protein synthesis in virus-infected cells

Xiaoxing Wang  1 , Ying Liao, Pei Ling Yap, Kim J Png, James P Tam, Ding Xiang Liu
Affiliations

Inhibition of protein kinase R activation and upregulation of GADD34 expression play a synergistic role in facilitating coronavirus replication by maintaining de novo protein synthesis in virus-infected cells

Xiaoxing Wang et al. J Virol. 2009 Dec.

Abstract

A diversity of strategies is evolved by RNA viruses to manipulate the host translation machinery in order to create an optimal environment for viral replication and progeny production. One of the common viral targets is the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha). In this report, we show that phosphorylation of eIF-2alpha was severely suppressed in human and animal cells infected with the coronavirus infectious bronchitis virus (IBV). To understand whether this suppression is through inhibition of protein kinase R (PKR), the double-stranded-RNA-dependent kinase that is one of the main kinases responsible for phosphorylation of eIF-2alpha, cells infected with IBV were analyzed by Western blotting. The results showed that the level of phosphorylated PKR was greatly reduced in IBV-infected cells. Overexpression of IBV structural and nonstructural proteins (nsp) demonstrated that nsp2 is a weak PKR antagonist. Furthermore, GADD34, a component of the protein phosphatase 1 (PP1) complex, which dephosphorylates eIF-2alpha, was significantly induced in IBV-infected cells. Inhibition of the PP1 activity by okadaic acid and overexpression of GADD34, eIF-2alpha, and PKR, as well as their mutant constructs in virus-infected cells, showed that these viral regulatory strategies played a synergistic role in facilitating coronavirus replication. Taken together, these results confirm that IBV has developed a combination of two mechanisms, i.e., blocking PKR activation and inducing GADD34 expression, to maintain de novo protein synthesis in IBV-infected cells and, meanwhile, to enhance viral replication.

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Figures

FIG. 1.
FIG. 1.
Analysis of de novo protein synthesis in IBV-infected cells. IBV (I)- or mock (M)-infected Vero cell lysates harvested at 0, 8, 16, and 24 h p.i. were separated on an SDS-10% polyacrylamide gel and stained with Coomassie blue (upper panel). The lower panel shows metabolic labeling of IBV-infected cells. Vero cells infected with IBV at an MOI of approximately 2 were labeled with [35S]Met-Cys for 2 h before harvest at 0, 8, 16, and 24 h p.i. Cell lysates were separated with 10% SDS-PAGE and subjected to autoradiography. IBV structural proteins M and N are indicated on the right of the gels. Lane 1 represents the protein ladder.
FIG. 2.
FIG. 2.
Analysis of eIF-2α phosphorylation and its effect on IBV replication in IBV-infected Vero, H1299, and HUH7 cells. (a) Vero, H1299, and HUH7 cells infected with IBV at an MOI of approximately 2 were harvested and lysed at 0, 4, 8, 12, 16, 20, 24, and 28 h p.i. Lysates were separated on an SDS-12% polyacrylamide gel and examined by Western blotting using antibodies against p-eIF-2α, total eIF-2α, IBV M protein, and β-tubulin. H1299 cells were also infected with an equal amount of UV-IBV. Protein samples were separated on an SDS-12% polyacrylamide gel and probed with antibodies against p-eIF-2α (S51), total eIF-2α, IBV M protein, and β-tubulin. (b) H1299 cells were transfected with wild-type (wt) eIF-2α and its two mutants (S51A and S51D) for 16 h before infection with the recombinant IBV-Luc virus at an MOI of approximately 1. Cells were lysed in 1× PLB at 18 h p.i. and subjected to a firefly luciferase assay. The relative luciferase activity in cells expressing the S51A mutant was considered as 100%. Error bars indicate the SDs of the means from triplicate experiments. The asterisk indicates a P value corresponding to the difference between readings of wt eIF-2α and the S51D mutant (0.0459). (c) The same cell lysates were resolved on an SDS-12% polyacrylamide gel and examined by Western blotting using antibodies against total eIF-2α (top panel), Flag tag (F-eIF2α; middle panel), and β-tubulin (bottom panel).
FIG. 3.
FIG. 3.
Analysis of PKR phosphorylation and its effect on IBV replication in IBV-infected Vero and H1299 cells. (a) H1299 cells were infected with either IBV at an MOI of 1 or an equal amount of UV-inactivated virus and harvested at 0, 4, 8, 12, 16, and 20 h p.i. Protein samples were separated on an SDS-10% polyacrylamide gel and probed with antibodies against p-PKR (Thr451), total PKR, and β-tubulin. (b) Vero and H1299 cells were infected with IBV at an MOI of approximately 2 for 0, 8, 16, 24, 36, and 48 h before lysis. Cell lysates were separated on an SDS-12% polyacrylamide gel and examined by Western blotting with antibodies against p-PKR (Thr451), total PKR, and β-tubulin. (c) H1299 cells were transfected with wild-type PKR and the K296P mutant for 16 h before being infected with the recombinant IBV-Luc at an MOI of approximately 1. Cells were lysed in 1× PLB at 18 h p.i. and subject to a firefly luciferase assay. The relative luciferase activity in cells expressing empty vector was considered as 100%. Error bars indicate SDs of the means from triplicate experiments. (d) The same cell lysates from panel c were resolved on an SDS-10% polyacrylamide gel and examined by Western blotting with antibodies against Flag (top panel), total PKR (second panel), p-PKR (third panel), p-eIF-2α (fourth panel), total eIF-2α (fifth panel), and β-actin (bottom panel).
FIG. 4.
FIG. 4.
Restoration of PKR-mediated translation inhibition by IBV nsp2 protein. (a) H1299-RL cells were cotransfected with 0.5 μg firefly luciferase reporter plasmid, 1 μg PKR, and 2 μg of individual IBV proteins except nsp3, nsp4, and nsp6. All constructs except the reporter plasmid carried a Flag tag. An empty vector was used as a negative control to make the total DNA amounts equal for each transfection reaction (3.5 μg). At 18 h posttransfection, cells were lysed in 200 μl 1× PLB and subjected to a dual-luciferase assay. The dual-luciferase assay was carried out using a dual-luciferase assay kit (Promega). The firefly luciferase readings were normalized against Renilla luciferase readings, and the translation efficiency of reporter plasmid was considered as 100% in samples expressing the firefly luciferase only (vector). Error bars indicate SDs of the means from triplicate readings. S, spike protein; E, envelope protein. (b) IBV nsp2 and influenza A virus NS1 restored translation in a dose-dependent manner. H1299-RL cells were transfected with 0.5 μg pcDNA-FL, 1 μg PKR, and different amounts of NS1 or nsp2 plasmids (listed in micrograms) by use of Lipofectamine 2000 for 18 h before lysis in 1× PLB. The total amount of DNA in each transfection was kept constant by adding different amounts of the empty vector. The dual-luciferase assay was carried out using a dual-luciferase assay kit (Promega). The firefly luciferase readings were normalized against Renilla luciferase readings and the translation efficiency of reporter plasmid was considered as 100% in samples expressing the firefly luciferase only (vector). Values represent means ± SDs from triplicate experiments. (c) Cell lysates were separated on a SDS-10% polyacrylamide gel and detected by Western blotting with antibodies against Flag and β-actin.
FIG. 5.
FIG. 5.
Induction of GADD34 expression at mRNA and protein levels in IBV-infected Vero, H1299, and HUH7 cells. (a) Vero, H1299, and HUH7 cells were infected with live IBV at an MOI of approximately 2 or UV-IBV for 0, 4, 8, 12, 16, 20, 24, and 28 h. Total RNA was extracted and separated on an 0.9% agarose gel and examined by Northern blotting using probes for GADD34. 28S rRNA was used as a loading control. (b) H1299 cells infected with IBV at an MOI of approximately 2 for 0, 4, 8, 12, and 16 h were lysed, and total proteins were separated on an SDS-8% polyacrylamide gel. Western blotting was carried out with antibodies against GADD34 and β-tubulin.
FIG. 6.
FIG. 6.
Regulation of IBV replication by manipulation of GADD34 expression. (a) Facilitation of IBV replication by overexpression of GADD34 in HUH7 and H1299. (Upper panel) H1299 and HUH7 cells were transfected with pXJ40F-GADD34 (F-GADD34) or the empty vector for 12 h before infection with the recombinant IBV-Luc virus at an MOI of approximately 1 for 0, 8, 10, 12, 14, and 16 h before lysis in 1× PLB. Luciferase assay was carried out using a luciferase assay kit from Promega. Error bars indicate means ± SDs from triplicate experiments. (Lower panel) Proteins from the same lysates were resolved with 10% SDS-PAGE and examined by Western blotting with antibodies against GADD34, p-eIF-2α, total eIF-2α, and β-tubulin. M, cells transfected with empty vector and mock infected for 16 h before lysis. (b) GADD34 knockdown facilitated virus infection. Vero cells were stably transfected with short hairpin RNA of GADD34, and two knockdown clones were selected and examined by Western blotting (upper panel). Vero GADD34 knockdown cells (clone 2) and wild-type (wt) cells were infected with IBV for 0, 4, 8, 12, 16, 20, 24, and 28 h before RNA extraction. Equal amounts of total RNAs were separated on a 0.9% agarose gel and examined by Northern blotting using probes for IBV, GADD34, and GAPDH. The six RNA species of IBV are indicated on the right of the gels.
FIG. 7.
FIG. 7.
Reduction of IBV replication by inhibition of the PP1 activity with OA in IBV-infected Vero cells. (a) Light microscopy images of IBV-infected Vero cells either untreated or treated with different concentrations of OA. Vero cells were treated with DMSO or OA 1 h after infection with IBV-Luc. Images were taken at 18 h p.i. M, mock infection; I, IBV infection. (b) Cells from the experiment described above were harvested at 18 h p.i. Total proteins were resolved on an SDS-12% polyacrylamide gel and examined by Western blotting with antibodies against p-eIF-2α, total eIF-2α, IBV N, and β-tubulin. (c) Cell lysates were subjected to a luciferase activity assay. IBV infection in cells treated with DMSO was considered as 100%. Error bars indicate SDs of the means of triplicate experiments.
FIG. 8.
FIG. 8.
Working model. A low level of de novo host cell global translation was maintained in IBV-infected cells by reducing the eIF-2α phosphorylation via a combination of two mechanisms, i.e., inhibition of PKR activation and induction of GADD34. The light arrow indicates that the reaction is less active in cells under normal circumstances.
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