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Comparative Study
. 1999 Nov;19(11):7305-13.
doi: 10.1128/MCB.19.11.7305.

Sensitivity of an epstein-barr virus-positive tumor line, Daudi, to alpha interferon correlates with expression of a GC-rich viral transcript

Y Gao  1 , S a Xue, B E Griffin
Affiliations
Comparative Study

Sensitivity of an epstein-barr virus-positive tumor line, Daudi, to alpha interferon correlates with expression of a GC-rich viral transcript

Y Gao et al. Mol Cell Biol. 1999 Nov.

Abstract

The exquisite sensitivity of the Burkitt's lymphoma (BL)-derived cell line Daudi to type I interferons has not previously been explained. Here we show that expression of an Epstein-Barr virus (EBV) transcript, designated D-HIT (Y. Gao et al., J. Virol. 71:84-94, 1997), correlates with the sensitivity of different Daudi cell isolates (or that of other EBV-carrying cells, where known) to alpha interferon (IFN-alpha). D-HIT, transcribed from a GC-rich repetitive region (IR4) of the viral genome, is highly structured, responding to RNase digestion in a manner akin to double-stranded RNA. Comparing EBV-carrying BL cell lines with differing responses to IFN-alpha, we found the protein levels of the dsRNA-activated kinase, PKR, to be similar, whereas the levels of the autophosphorylated active form of PKR varied in a manner that correlated with endogenous levels of D-HIT expression. In a classical in vitro kinase assay, addition of either poly(I)-poly(C) or an in vitro-transcribed D-HIT homolog stimulated the autophosphorylation activity of PKR from IFN-alpha-treated cells in both EBV-positive and EBV-negative B lymphocytes. By transfection experiments, these RNAs were shown to reduce cell proliferation and to sensitize otherwise relatively insensitive Raji cells to IFN-alpha. The data lead to a model wherein the D-HIT viral RNA also serves as a possible transcriptional activator of IFN-alpha or cellular genes regulated by this cytokine.

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Figures

FIG. 1
FIG. 1
Action of RNases A and V1 on the 2.8-kb D-HITs. Total RNA from induced Daudi/ATCC cells was treated with excess single (RNase A)- or double (RNase V1)-stranded RNA-specific RNases at 20°C for 30 min. Products were separated by electrophoresis and visualized after transfer to nitrocellulose and hybridization with a 32P-labelled EBV BamHI-Ia probe (A) or probes for rRNAs (B).
FIG. 2
FIG. 2
Interferon sensitivities of Daudi/ATCC cells. Cells were grown in the presence of various concentrations of IFN-α over a 28-day period, and the number of live cells (as a percentage of untreated cells) was plotted. The profile of Daudi/ATCC, showing viable cell counts as a percentage of cells grown in the absence of interferon (100%), is taken as a standard for medium IFN-α sensitivity. Superimposed on it are two curves from Daudi/ICRF cells (grown in the presence of 1 and 10 U of IFN-α/ml; high sensitivity), one from Raji cells (grown with 104 U; low sensitivity), and one from P3HR1 cells (102 U; high/medium sensitivity; data taken from the literature [2]), all EBV-positive lines.
FIG. 3
FIG. 3
Northern blots showing levels of endogenous D-HIT expression in parental Daudi cell lines ATCC and ICRF and two mutants, clone 1 and 100K, with different sensitivities to IFN-α (Fig. 2), compared with expression of homologs in two other BL-derived lines, Namalwa and Raji. Tracks 1 to 6 contain poly(A)+ RNA (7 μg) from Namalwa, Raji, Daudi/ATCC, Daudi/ICRF, Daudi/clone 1, and Daudi/100K cells, respectively. The upper blot was deliberately overexposed in an attempt to look for transcripts in apparently negative lines. A GAPDH probe was used as a control for RNA loading.
FIG. 4
FIG. 4
Northern blot analysis of D-HIT, IFN-α, or interferon-inducible (6-16) gene transcripts in B cells. Poly(A)+ RNA (5 μg) from uninduced or TPA- and SB-induced cells (as indicated) was loaded onto gels and detected by hybridization with 32P-radiolabelled probes from EBV IR4 DNA (A) or cDNAs for IFN-α (B) or 6-16 (B and C). The sources of the RNA and the nature of the inducing agents (TPA and SB), if used, are indicated, and predicted locations of the individual RNAs are marked. Although the levels of IFN-α in panel B are low, they mimic those observed for D-HIT (A) and 6-16 (B), where increased levels are observed with TPA-treated cells and even higher levels are detected with SB-treated cells.
FIG. 5
FIG. 5
Expression and activity of PKR. (A and B) Western blot analyses of PRK expression in untreated or IFN-α treated cells (A) and untreated or TPA- and SB-induced cells (B). Comparable levels of total protein were added to all tracks, and the presence of PKR was detected with monoclonal antibody 71/10. (C) Kinase activity of PKR in various B-cell lines. PKR was immunoprecipitated with 71/10, and kinase activity assessed in the presence of [γ-32P]ATP. The cells used in the assays were untreated or treated with IFN-α or with TPA and SB, respectively, as noted, prior to protein isolation. The data shown are from the same autoradiogram but reorganized so that the controls (PKR from Ramos and Raji cells) flank the Daudi data. (D) The same assay as in panel C, with either poly(I)-poly(C) or an in vitro-transcribed D-HIT homolog (both at 0.8 μg/ml) added to the phosphorylation reaction of PKR isolated from IFN-α-treated Daudi cells, as noted. Track 4 contains a 10-fold-higher concentration of viral RNA (8.0 μg/ml) than used in track 3. Arrowheads indicate the positions of PKR and phosphorylated PKR (PRK*). The abbreviations h (high), m (medium), and l (low or undetectable), and m/l (medium/low) represent the relative levels of D-HIT present in the EBV-positive cells (Table 1).
FIG. 6
FIG. 6
Proliferation assay. Raji cells were treated with either 16 μg (white bars) or 4 μg (grey bars) of poly(I)-poly(C) or with 4 μg of the EBV D-HIT homolog (black bars), and thymidine incorporation was measured 7 days posttransfection, using conditions 1 or 2 (see text for definition), as noted, in the presence of 104 U of IFN-α/ml. Cell survival is presented as percentage of control cells without added exogenous RNA.
FIG. 7
FIG. 7
The PKR interferon pathway. Sites in the pathway identified as, or postulated to be, stimulated by D-HIT expression are indicated by jagged arrows. Asterisks indicate phosphorylation. Parentheses are used for the cellular RNAs (6-16, 9-27 etc.) since work on another mRNA, 561, implies that these may not be dependent solely on interferon for activation but directly induced efficiently by dsRNA (42).
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