Activation of NF-kappaB in cells productively infected with HSV-1 depends on activated protein kinase R and plays no apparent role in blocking apoptosis - PubMed (original) (raw)

Activation of NF-kappaB in cells productively infected with HSV-1 depends on activated protein kinase R and plays no apparent role in blocking apoptosis

Brunella Taddeo et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Microarray data reported elsewhere indicated that herpes simplex virus 1 induces the up-regulation of nuclear factor kappaB (NF-kappaB)-regulated genes, including that of its inhibitor, IkappaBalpha, consistent with the reports that wild-type virus induces the activation of NF-kappaB. In this report we show that activation of NF-kappaB in infected cells is linked to the activation of protein kinase R (PKR). Specifically: (i) PKR is activated in infected cells although the effects of the activated enzyme on protein synthesis are negated by the viral gene gamma134.5, which encodes a protein phosphatase 1alpha accessory factor that enables the dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. NF-kappaB is activated in wild-type murine embryonic fibroblasts but not in related PKR-null cells. (ii) In cells infected with a replication-competent Deltagamma134.5 mutant (R5104), but carrying a US11 gene expressed early in infection, eukaryotic translation initiation factor 2alpha is not phosphorylated, and in in vitro assays, PKR bound to the US11 protein is not phosphorylated on subsequent addition of double-stranded RNA. Here we report that this mutant does not activate PKR, has no effect on the accumulation of IkappaBalpha, and does not cause the translocation of NF-kappaB in infected cells. (iii) One hypothesis advanced for the activation of NF-kappaB is that it blocks apoptosis induced by viral gene products. The replication-competent R5104 mutant does not induce the programmed cell's death. We conclude that in herpes simplex virus 1-infected cells, activation of NF-kappaB depends on activation of PKR and that NF-kappaB is not required to block apoptosis in productively infected cells.

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Figures

Fig. 1.

Fig. 1.

Accumulation of IκBα RNA and protein in SK-N-SH cells after HSV-1 infection. (A) SK-N-SH cells were mock-infected or infected with HSV-1(F). Total RNA was extracted from cells harvested at the indicated times after mock infection (lanes 1 and 4) or exposure to HSV-1(F) (lanes 5–8) and processed as described in the text. Ten micrograms of total RNA was loaded onto a denaturing formaldehyde gel and probed with a 32P-labeled fragment containing the entire coding sequence of IκBα. (B) SK-N-SH cells mock-infected or infected with HSV-1(F) were harvested at 3, 6, 9, 12, and 24 h after infection (lanes 2–6) and processed as described in Materials and Methods. The electrophoretically separated proteins were reacted with anti-IκBα antibody. (C and D) SK-N-SH cells were mock-infected or infected with HSV-1(F) or indicated mutant viruses. The cells were harvested at 8 h after infection and processed as described in Materials and Methods. The electrophoretically separated proteins were reacted with antibodies against IκBα, IKKα, or viral proteins US11 or ICP4.

Fig. 2.

Fig. 2.

Cytoplasmic localization of NF-κB in SK-N-SH cells does not result in apoptosis. (A) Localization of NF-κB. SK-N-SH cells were mock-infected (lanes 1, 2, 7, and 8) or infected with HSV-1(F) (lanes 3, 4, 9, and 10) or R5104 (lanes 5, 6, 11, and 12). Cytoplasmic (C) and nuclear (N) fractions were prepared from cells harvested at 4 h (lanes 1–6) and 6 h (lanes 7–12) after infection or mock infection and processed as described in Materials and Methods. Electrophoretically separated proteins were reacted with anti-NF-κB or anti-US11 antibodies. (B) Degradation of cellular DNA in infected cells. SK-N-SH cells were mock-infected (lane 1) or infected with HSV-1(F) (lane 2), R5104 (lane 3), or Δα27 mutant viruses (lane 4), and a DNA fragmentation assay was performed on cells collected at 12 h after infection as described in Materials and Methods.

Fig. 3.

Fig. 3.

IKKα activity in murine _PKR_-/- and PKR+/+ cells after HSV-1 infection. (A) Electrophoretically separated proteins from lysates of _PKR_-/- or PKR+/+ cells harvested at 7 h after infection with HSV-1(F) were reacted with anti-ICP27- or anti-US11-specific antibodies. (B) Electrophoretically separated proteins from lysates of _PKR_-/- or PKR+/+ cells harvested at 2, 4, 6, or 8 h after infection with HSV-1(F) were reacted with anti-IκBα antibody. (C) Autoradiographic images of GST-IκBα reacted with IKKα immunoprecipitated from _PKR_-/- or PKR+/+ cells 6 h after infection with HSV-1(F). The reaction conditions were as described in Materials and Methods.

Fig. 4.

Fig. 4.

PKR activation in SK-N-SH cells after HSV-1 infection. (A) PKR was immunoprecipitated from whole cell extracts prepared from SK-N-SH cells harvested at 4 or 6 h after mock infection or infection with HSV-1(F) or R51204 as described in Materials and Methods. The immune complex was subjected to an in vitro kinase assay in the presence of histone H1, analyzed on a denaturing 10% polyacrylamide gel, electrically transferred to a nitrocellulose sheet, and subsequently subjected to autoradiography to detect substrate phosphorylation. (B) Quantification of the amount of phosphorylated substrate relative to mock-infected cells obtained with the aid of the Molecular Dynamics Storm 860 PhosphorImager.

Fig. 5.

Fig. 5.

A schematic representation of a model of the activation of NF-κB in cells infected with a wild-type virus or R5104 mutant. In mutant virus-infected cells, the US11 protein made early blocks phosphorylation (P) and dimerization of PKR. As a consequence, NF-κB is not activated. In wild-type virus-infected cells, PKR is activated and the cascade of events schematically illustrated in this model leads to activation of NF-κB. dsRNA, double-stranded RNA.

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