Latently expressed human herpesvirus 8-encoded interferon regulatory factor 2 inhibits double-stranded RNA-activated protein kinase

Abstract

Human herpesvirus 8 (HHV-8; Kaposi's sarcoma herpesvirus) encodes four open reading frames with homology to cellular proteins of interferon regulatory factor (IRF) family. Three of them, viral IRF-1 (vIRF-1), vIRF-2, and vIRF-3, have been cloned and found, when overexpressed, to down-regulate the transcriptional activity of interferon type I gene promoters in infected cells by interfering with the transactivating activity of cellular IRFs. In this study, we have further characterized vIRF-2 and shown that it is a nuclear protein which is constitutively expressed in HHV-8-positive pleural effusion lymphoma cell lines. Nuclear localization of vIRF-2 was confirmed by in situ detection of ectopically expressed enhanced green fluorescent protein/vIRF-2 fusion protein. We found that the expression of vIRF-2 in HEK293 cells inhibited the antiviral effect of interferon and rescued translation of vesicular stomatitis virus mRNA from interferon-induced translational block. To provide insight into the mechanism of this effect we have demonstrated that vIRF-2 physically interacts with PKR consequently inhibiting autophosphorylation of double-stranded RNA-activated protein kinase (PKR) and blocking phosphorylation of PKR substrates histone 2A and eukaryotic translation initiation factor 2alpha. These results suggest that the latently expressed vIRF-2 has a role in viral mimicry which targets the activity of interferon-induced PKR kinase. By inhibiting the kinase activity of PKR and consequent down-modulation of protein synthesis, HHV-8 has evolved a mechanism by which it can overcome the interferon-mediated antiviral effect. Thus, the anti-interferon functions of vIRF-2 may contribute to the establishment of a chronic or latent infection.

FIG. 1

Expression of vIRF-2 and K11 ORFs in BCBL-1 cells. (A) Scheme of the 89- to 95-kbp region of the HHV-8 genome showing the cluster of vIRF-2 (K11.1), K11, and vIRF-3 (K10.5 and K10.6) homologues of cellular IRFs. (B) RT-PCR analysis from control and TPA (50 ng/ml)-induced BCBL-1 cells for 24 h. Constitutive expression of vIRF-2 (V2) is in contrast with inducible expression of K11. No specific product could be detected using a combination of 5′ vIRF-2 and 3′ K11 primers (V2 + K11), detecting, a theoretical common transcript. Control PCR products amplified from the BCBL-1 genomic library are shown. RT-PCR amplification of GPDH mRNA is shown as a control.

FIG. 2

Detection of vIRF-2 protein in cell lysates from HHV-8-positive lymphomas. (A) Specificity of the His₆/vIRF-2-immunopurified antibody is demonstrated by detection of 5 and 500 ng of vIRF-2/GST fusion protein (lanes 1 and 3). No cross-reaction with the same amount of vIRF-1/GST protein was observed (lanes 2 and 4). (B) Detection of ectopically expressed vIRF-2 in NIH 3T3 cells. Cells were transfected with 4 μg of empty or vIRF-2-expressing pcDNA vector. Whole-cell lysates (20 μg) were prepared 48 h later and immunoblotted with vIRF-2 antibody. (C) Detection of vIRF-2 in nuclear and cytoplasmic fractions from BCBL-1 cells. BCBL-1 and control HHV-8-negative Louckes cells were treated with TPA (50 ng/ml) for 24 h. Cytoplasmic and nuclear fractions (20 μg) were prepared from control or TPA-stimulated cells by differential centrifugation and immunoblotted with vIRF-2 antibody. The only immunoreactive protein, with an apparent mobility of 20 kDa, is marked. (D) Immunoblot analysis of nuclear extracts (20 μg) from different cell lines using immunopurified anti-vIRF-2 antibody. The region around 20 kDa is shown.

FIG. 3

Nuclear localization of EGFP/vIRF-2 fusion protein. HeLa cells were transfected with 4 μg of EGFP/vIRF-2 expression vector. Cells were fixed 36 h later and counterstained with DNA stain (Hoechst). (A) Visualization of EGFP/vIRF-2 using a GFP-specific filter. (B) Computer-generated overlay of sequentially captured images from the same field, using GPF- and Hoechst-specific filters. Dark blue shows nuclei; bright blue indicates colocalization of nuclear and EGFP signals.

FIG. 4

Expression of vIRF-2 rescues IFN-induced block of viral protein synthesis. (A) Autoradiography analysis of metabolically labeled cells transfected with empty (pcDNA3.1) or FLAG/vIRF-2-expressing (vIRF-2F/pcDNA3.1) vector. Cells were mock infected (−) or infected with VSV (+) and 5 h later pulse-labeled with ³⁵S-labeled amino acid mixture as described in Materials and Methods. Positions of early VSV proteins are marked on the right. (B) Relative levels of 29-kDa VSV matrix protein (M) in pcDNA- or vIRF-2F/pcDNA-transfected cells and its dependency on increased concentrations of IFN. Amounts of viral proteins in cells pretreated with increased concentrations of IFN were quantified from autoradiograms by using a PhosphorImager densitometer and data analysis software. Error bars represent standard errors from three independent experiments. (C) Immunoblot detection of FLAG-tagged vIRF-2 in representative samples of pcDNA- or vIRF-2F/pcDNA-transfected cells by using anti-FLAG antibody.

FIG. 5

vIRF-2 binds to PKR in vitro. (A) In vitro-translated, ³⁵S-labeled PKR was incubated with GST-tagged vIRF-2 or vIRF-1 coupled to glutathione-agarose beads as described in Materials and Methods. Lane 1 represents 10% of the input volume of in vitro-translated PKR (p68) used for the binding reactions. (B) Absence of binding of in vitro-translated, ³⁵S-labeled luciferase (Lucif) or IRF-3 to GST/vIRF-2 beads. (C) Whole-cell lysates from IFN (500 U/ml, 16 h)-treated or untreated HeLa cells were incubated with GST-tagged vIRF-2 or vIRF-1 coupled to glutathione-agarose beads. Bound proteins were separated by SDS-PAGE and subjected to immunoblot analysis with anti-PKR antiserum. Lanes 1 and 2 show the presence of p68 in 10% of the input volume of cell lysates used for the binding reactions.

FIG. 6

vIRF-2 inhibits PKR autophosphorylation and phosphorylation of histone 2A (H2A) and eIF-2α in vitro. (A) Protein kinase assay of PKR (p68) immunoprecipitated by rabbit polyclonal antibody from control HeLa cells in the absence of H2A substrate (lane 1) and in the presence of H2A and ATF2/GST (2 μg) or vIRF-2/GST (400 ng). (B) Phosphorylation of H2A by PKR immunoprecipitated from mock-infected (−) or VSV-infected (+) cells. Increased amounts of purified vIRF-2/GST protein were added to each reaction as indicated. (C) Phosphorylation of eIF-2α by PKR immunoprecipitated by monoclonal antibody from control and poly(I-C) (pIC)-stimulated HeLa cells. Reactions were performed at the absence or presence of 500 ng of of each recombinant protein indicated at the top. The levels of phosphorylated PKR and eIF-2α were quantitated and are plotted below in the corresponding panel. Representative samples of at least three independent experiments are shown.