RNA adenosine deaminase ADAR1 deficiency leads to increased activation of protein kinase PKR and reduced vesicular stomatitis virus growth following interferon treatment

Abstract

Two size forms of ADAR1 adenosine deaminase are known, one constitutively expressed (p110) and the other interferon (IFN)-induced (p150). To test the role of ADAR1 in viral infection, HeLa cells with ADAR1 stably knocked down and 293 cells overexpressing ADAR1 were utilized. Overexpression of p150 ADAR1 had no significant effect on the yield of vesicular stomatitis virus. Likewise, reduction of p110 and p150 ADAR1 proteins to less than approximately 10 to 15% of parental levels (ADAR1-deficient) had no significant effect on VSV growth in the absence of IFN treatment. However, inhibition of virus growth following IFN treatment was approximately 1 log(10) further reduced compared to ADAR1-sufficient cells. The level of phosphorylated protein kinase PKR was increased in ADAR1-deficient cells compared to ADAR1-sufficient cells following IFN treatment, regardless of viral infection. These results suggest that ADAR1 suppresses activation of PKR and inhibition of VSV growth in response to IFN treatment.

Figure 1. Stable knockdown of ADAR1 proteins

Western immunoblot analyses comparing ADAR1 expression in wild-type parental (CON^WT), ADAR1 stable knockdown (ADAR1^KD), puromycin-resistant control (CON^KD) and PKR stable knockdown (PKR^KD) HeLa cell clones. The large (150 kDa) and the small (110 kDa) size forms of ADAR1 are designated p150 and p110, respectively. Xrp, a non-specific cross-reacting protein migrating just above p150. (A) Whole cell extract analysis: Cells were mock treated or treated with either 1,000 units/ml of IFN-αA/D or IFN-β for 24 h. Whole-cell extract protein (10 μg) was analyzed in each lane on SDS-10% PAGE. (B) Analysis of fractionated cell lysates. Cytosolic and nuclear extract fractions were prepared from cells that had been treated with IFN-β 24 h and analyzed on SDS-7% PAGE. Membranes were probed using a polyclonal antibody against human ADAR1 and monoclonal antibodies against ß-actin and α-tubulin as loading and fractionation controls (Ahn et al., 2004).

Figure 2. Steady state levels of PKR and STAT1 proteins in HeLa clones

Western immunoblot analyses comparing PKR and STAT1 expression in ADAR1 stable knockdown (ADAR1^KD), wild-type parental (CON^WT), puromycin-resistant control (CON^KD, CON^KD2) and PKR stable knockdown (PKR^KD) HeLa cell clones. Cells were left untreated or treated with IFN-αA/D for 24 h, whole cell extracts were prepared, fractionated on SDS-10% PAGE (20 μg protein per lane), and membranes probed with antibodies against (A) STAT1 and (B) PKR. ß-actin was used as a loading control.

Figure 3. Effect of ADAR1 knockdown either stably or transiently on activation of PKR kinase

ADAR1 stable knockdown (ADAR1^KD), PKR stable knockdown (PKR^KD) and puromycin-resistant control (CON^KD) HeLa cell clones were subsequently transiently knocked down utilizing chemically synthesized siRNAs against luciferase (siLuc) as a control, ADAR1 (siAD1), or PKR (siPKR) as described under Materials and Methods. Following transient knockdown, the cells in monolayer culture were either mock-treated (−, lanes 1–10) or treated with 1000 units/ml IFN-β for 16 h (+, lanes 11–20) prior to infection with VSV. Whole cell extracts were prepared at 8 h after infection, fractionated on SDS-10% PAGE (30 μg protein per lane), and analyzed by western immunoblot analysis utilizing antibodies against PKR and phospho-threonine 446 PKR (P-PKR). ß-actin was used as a loading control.

Figure 4. Vesicular stomatitis virus infection does not alter the steady-state level of PKR or eIF-2α proteins in ADAR1^KD cells

Western immunoblot analyses comparing PKR and eIF-2α expression in wild-type parental (CON^WT), puromycin-resistant control (CON^KD), PKR stable knockdown (PKR^KD), and ADAR1 stable knockdown (ADAR1^KD) HeLa cell clones, either mock treated (−, lanes 1–8) or treated with 1000 units/ml IFN-β for 16 h (+, lanes 9–16) prior to infection with VSV (+) or were left uninfected (−). Whole cell extracts were prepared at 8 h after infection, fractionated on SDS-7% PAGE (20 μg protein per lane), and analyzed by western immunoblot utilizing antibodies against PKR, eIF-2α, and β-actin.

Figure 5. ADAR1 knockdown leads to increased phosphorylation of protein synthesis initiation factor eIF-2α

Wild-type parental (CON^WT), puromycin-resistant control (CON^KD), PKR stable knockdown (PKR^KD), and ADAR1 stable knockdown (ADAR1^KD) HeLa cell clones were treated with 1000 units/ml IFN-β for 16 h, and then infected with VSV for 8h. Whole cell extracts were prepared, fractionated on SDS-7% PAGE (30 μg protein per lane), and analyzed by western immunoblot analysis for the T446 phosphorylation of PKR (P-PKR), S51 phosphorylation of eIF-2α (P-eIF2α), and β-actin.