Protein kinase PKR plays a stimulus- and virus-dependent role in apoptotic death and virus multiplication in human cells

Abstract

The protein kinase regulated by double-stranded RNA (dsRNA), PKR, is implicated in a range of biologic processes, including apoptotic death and interferon antiviral responses, based in part on studies with mouse cells genetically deficient in Pkr. To test the role of the PKR protein in human cells, an RNA interference silencing strategy was used to generate stable HeLa cell lines with less than 2% of the PKR protein (PKR deficient) compared to either parental or control knockdown HeLa lines. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 on serine 51 was not detectably increased in response to dsRNA in PKR-deficient HeLa cells but was elevated severalfold in PKR-sufficient cells. PKR-deficient cells displayed reduced dsRNA-induced apoptosis compared to PKR-sufficient cell lines, whereas tumor necrosis factor alpha (TNF-alpha)-induced apoptosis was comparable between the HeLa lines. NF-kappaB was activated to a comparable extent in PKR-deficient and PKR-sufficient HeLa cells upon treatment with either dsRNA or TNF-alpha. The antiviral response against vesicular stomatitis virus was reduced in interferon-treated PKR-deficient compared to PKR-sufficient HeLa cells. However, the growth of two human viruses, adenovirus and reovirus, was unaffected by the PKR knockdown. Surprisingly, the yield of mutant adenovirus that fails to encode VAI RNA was not enhanced in PKR-deficient cells, indicating the importance of host factors in addition to PKR in conferring the VAI RNA phenotype.

FIG. 1.

Stable knockdown of PKR protein. (A) Western immunoblot analysis comparing PKR expression in wild-type parental (PKR⁺), PKR-deficient knockdown (PKR^kd), and PKR-sufficient control knockdown (PKR^kd-con) HeLa cell clones. Cells were mock treated or treated with 1,000 IU/ml of IFN-α A/D for 24 h. Whole-cell extract protein (10 μg) was analyzed in each lane; the membrane was probed with a polyclonal antibody against human PKR and a monoclonal antibody against β-actin as a loading control. (B) Quantification. Western blots were quantified using a VersaDoc (Bio-Rad) imaging system.

FIG. 2.

Double-stranded RNA-mediated apoptosis is impaired in PKR-deficient HeLa cells. (A) The effects of dsRNA on apoptosis induction in PKR⁺, PKR^kd, and PKR^kd-con HeLa cells were measured in 96-well plates, either mock transfected or transfected with 60 ng dsRNA. Apoptosis was measured using a Caspase-Glo 3/7 kit at 6 h posttransfection. Each experiment was repeated a minimum of three times. *, P < 0.0001, Student's t test, PKR^kd compared to the PKR⁺ parent; P was >0.4 for PKR^kd-con compared to the PKR⁺ parent. (B) Western immunoblot measurement of PARP cleavage at 8 h after dsRNA treatment. Ten μg of whole-cell extract protein was analyzed in each lane.

FIG. 3.

Time course of PARP cleavage following dsRNA treatment in PKR⁺ and PKR^kd HeLa cells. (A) Western blot analysis. Whole-cell extracts were prepared at 0, 2, 4, 6, and 8 h after transfection with dsRNA (+) or mock transfected (-). Ten μg of whole-cell extract protein was loaded in each lane. The membranes were probed with antibodies against PARP and PKR and against β-actin as a loading control. (B) Quantification of PARP. Western blots were quantified using a VersaDoc imager. Intact PARP116 (solid line) and the cleavage product PARP85 (broken line) quantitations from PKR⁺ (circles) and PKR^kd (squares) HeLa cells are relative to β-actin.

FIG. 4.

eIF-2α phosphorylation and protein expression following dsRNA treatment of PKR⁺ and PKR^kd cells. (A) Western blot analysis. Whole-cell extracts were made at 0, 2, 4, 6, and 8 h after transfection with dsRNA, and 30 μg protein was analyzed in each lane. The membranes were probed with antibodies against eIF-2α or phospho-eIF-2α(Ser 51) and against α-tubulin as a loading control. (B) Quantification. Western blots were quantified by scanning densitometry. The upper panel shows the eIF-2α protein profile, and the lower panel shows the phosphorylated eIF-2α protein profile. Circles, HeLa PKR⁺; squares, HeLa PKR^kd. (C) Protein expression. Luciferase reporter activity was measured in cells transfected in the absence (-) or presence (+) of dsRNA for 8 h, following transfection with pGL2-Control reporter DNA. *, P < 0.0001, Student's t test, PKR^kd compared to PKR⁺ parent or PKR^kd-con.

FIG. 5.

TNF-α-mediated apoptosis is normal in PKR-deficient HeLa cells. (A) The effect of TNF-α treatment on apoptosis induction in PKR⁺, PKR^kd, and PKR^kd-con HeLa cells was measured using the Caspase-Glo 3/7 kit at 18 h after treatment. Results shown are the means ± standard deviations determined from a minimum of six independent experiments. Based on Student's t test, P was >0.3 for TNF-α treatments between cell pairs. (B) Effects of TNF-α (right) and dsRNA (left) on eIF-2α phosphorylation. Whole-cell extracts were made at 4 h after transfection with dsRNA or treatment with TNF-α (10 ng/ml), and 30 μg protein was analyzed in each lane. The membranes were probed with antibodies against eIF-2α or phospho-eIF-2α(Ser 51) and against β-actin as a loading control.

FIG. 6.

Role of PKR in the NF-κB activation pathway. PKR⁺ and PKR^kd HeLa cells were transfected with an NF-κB-dependent firefly luciferase reporter plasmid. Transfected cells were mock treated or treated with either 10 ng/ml TNF-α or 100 μg/ml dsRNA and 50 μg/ml DEAE-dextran at 48 h after transfection. (A) TNF-α. Luciferase activity was measured at 4 h after TNF-α treatment (hatched bars) or in cells left untreated (open bars). (B) dsRNA. Luciferase activity was measured in dsRNA mock-treated and treated cells at 4, 6, and 8 h after dsRNA treatment of PKR⁺ (circles) and PKR^kd (squares) HeLa cells. Results shown are the means ± standard deviations determined from a minimum of six independent experiments.

FIG. 7.

Single-cycle multiplication of vesicular stomatitis virus in PKR⁺, PKR^kd, and PKR^kd-con HeLa cells. Cells were mock treated (-) or treated with 1,000 units/ml IFN-α A/D (+) for 24 h and then infected with VSV. Cells were harvested at 16 h p.i., and virus yields were determined by plaque titration. Based on Student's t test, P was >0.05 for PKR^kd compared to PKR⁺ parent and PKR^kd-con without IFN-α A/D pretreatment. *, P < 0.0001, PKR^kd compared to PKR⁺ parent and PKR^kd-con with IFN-αA/D pretreatment.

FIG. 8.

Single-cycle multiplication of adenovirus in PKR⁺ and PKR^kd HeLa cells. Cells were either mock treated (-) or treated with 1,000 units/ml IFN-α A/D (+) for 24 h and then infected with either wild-type adenovirus (A) or VAI deletion mutant adenovirus (B). At 36 h p.i., cells were harvested and virus yields determined by plaque titration. Based on Student's t test, P was >0.18 between infected cell pairs.