1 Departamento de Biología Celular y Molecular, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain
2 Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal sn, 28040 Madrid, Spain
Correspondence
C. Rivas
mdcrivas{at}farm.ucm.es
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ABSTRACT |
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INTRODUCTION |
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Since the IFN-induced cellular antiviral response is the primary defence mechanism against virus infections, many viruses have evolved mechanisms to counteract the effects of IFN (Vilcek & Sen, 1996; Goodbourn et al., 2000
). In this sense, viruses have developed multiple strategies to avoid the deleterious effects of the PKR and 2-5A system at different levels (Gale & Katze, 1998
), such as inhibition of PKR activation (Katze et al., 1987
), sequestration of dsRNA (Lloyd & Shatkin, 1993
; Lu et al., 1995
), inhibition of PKR dimerization (Gale et al., 1997
, 1998
), synthesis of PKR pseudosubstrates (Dever et al., 1998
; Taylor et al., 1999
), activation of antagonist phosphatases (He et al., 1997
) or PKR degradation (Black et al., 1989
).
Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus-8 (HHV-8), is recognized as the essential infectious agent linked to the development of Kaposi's sarcoma, primary effusion lymphoma and some forms of Castleman's disease, an atypical B cell lymphoproliferative disorder (Chang et al., 1994; Soulier et al., 1995
). The genome of KSHV contains cellular homologues that permit the manipulation of the local environment for efficient virus replication and evasion of the immune response and may also contribute to host proliferation and cell transformation. These cellular homologues include a cluster of open reading frames (ORFs) encoding four proteins with homology to the cellular transcription factors of the IRF family. vIRF1 inhibits virus-mediated transcriptional activation of the IFNA gene promoter and IFN-stimulated activation of ISG promoters (Gao et al., 1997
; Li et al., 1998
; Zimring et al., 1998
; Buryseck et al., 1999
). Furthermore, expression of vIRF1 also activates KSHV genes such as vIL6 and inhibits radiation and adriamycin-induced apoptosis in KSHV-infected cells (Sarid et al., 1999
). Another KSHV IRF-like protein, vIRF2, inhibits the antiviral effect of IFN and rescues translation of vesicular stomatitis virus mRNA from the IFN-induced translation block. vIRF2 interacts physically with PKR and inhibits its autophosphorylation, thus blocking phosphorylation of eIF-2
(Burysek & Pitha, 2001
). Finally, the ORFK10.5 encodes the LANA2 protein (previously called vIRF3) with homology to cellular IRF-4 and KSHV vIRF2 (Rivas et al., 2001
). LANA2 functions as a dominantnegative mutant of both IRF-3 and IRF-7 and inhibits virus-mediated transcriptional activity of the IFNA promoter (Lubyova & Pitha, 2000
).
KSHV has thus developed strategies to counteract IFN through the synthesis of several viral proteins with homology to the IRF-like proteins. However, only one KSHV protein (vIRF2) has been identified so far that specifically counteracts PKR action, the most common target of virus action to block IFN signalling pathways. In this investigation, we have examined the ability of KSHV LANA2 to modulate the dsRNA-activated pathways, PKR and the 2-5A system. We found that LANA2 was specifically able to inhibit apoptosis and the blockade in translation mediated by PKR. These effects may be mediated by a decrease in eIF-2 phosphorylation but not by direct interaction between LANA2 and PKR.
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METHODS |
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Cells and viruses.
African green monkey kidney cell line BSC-40 was grown in Dulbecco's modified medium (DMEM) supplemented with 10 % newborn calf serum. Mouse fibroblasts derived from homozygous PKR knockout mice were obtained from C. Weissmann (Imperial College, London, UK) and grown using the same media. Cells were cultivated at 37 °C with 5 % CO2. The recombinant vaccinia viruses (VV) VV-RL, VV-N and VV-2-5AS have been described previously (Díaz-Guerra et al., 1997a
). The recombinant virus VV-PKR expresses PKR under lacI repressor/operator controlling elements (Lee & Esteban, 1993
). A VV recombinant expressing the p36/LACK protein of Leishmania infantum VVp36 has been described previously (Gonzalo et al., 2001
). LANA2 was extracted from the EGFP-LANA2 plasmid and subcloned into the SmaI site of the haemagglutinin insertional VV vector PHLZ (Vazquez & Esteban, 1999
). The EGFP-LANA2 plasmid was generated by cloning the DNA encoding LANA2 excised from the pcDNA-LANA2 plasmid (kindly provided by Y. Chang and P. Moore) and inserted into the EGFP vector (Clontech) as a KpnIApaI fragment. VV-LANA2 was generated by homologous recombination of PHLZ-LANA2 with wild-type VV Western Reserve strain (WR) in BSC-40 cells and selected by blue plaque formation in response to addition of X-Gal. Virus was subjected to five rounds of plaque purification to generate homogeneous population of recombinants, grown and titrated in BSC-40 cells.
Analysis of low molecular mass DNA.
Low molecular mass DNA was isolated from cultured cells as described (Hirt, 1967), analysed by agarose gel electrophoresis and stained with ethidium bromide.
Measurement of the extent of apoptosis.
The Cell Death Detection ELISA kit from Boehringer Mannheim was used according to the manufacturer's instructions. This assay, based on the quantitative sandwich-enzyme-immunoassay principle using mouse monoclonal antibodies directed against DNA and histones, respectively, estimates the amount of cytoplasmic histone-associated DNA.
Total RNA isolation.
Total cellular RNA from mock-infected or infected cells was isolated using the Ultraspec-II Resin Purification System (Biotecx). For analysis, denatured RNA was fractionated on 1 % formaldehyde/agarose gels and stained using ethidium bromide (Díaz-Guerra et al., 1997b).
Analysis of protein synthesis.
For immunoblot analysis, protein samples were fractionated by SDS-PAGE, transferred to nitrocellulose paper and analysed by immunoperoxidase staining after reactivity with different sera.
Metabolic labelling of proteins.
BSC-40 cells cultured in 24-well plates were infected with the viruses indicated and rinsed three times with methionine/cysteine (Met/Cys)-free DMEM 30 min prior to labelling. After incubation for an additional 30 min at 37 °C with Met/Cys-free DMEM, medium was removed and 25 µCi [35S]Met-Cys Pro-mix (Amersham) ml-1 in Met/Cys-free DMEM was added for an additional 30 min. After three washes with PBS, cells were harvested in lysis buffer followed by SDS-PAGE and autoradiography. Experiments were repeated at least twice.
Immunoprecipitation.
For in vivo co-immunoprecipitations, BSC-40 cells were grown in 10 cm plates and infected for 24 h with VV-LANA2 and/or VV-PKR. Cells were scraped and the clarified supernatant was mixed with 150 µl of protein ASepharose, previously incubated with specific antibodies directed against LANA2 or PKR, and further incubated overnight at 4 °C. Immunocomplexes were resolved by SDS-PAGE followed by immunoblot analysis with anti-PKR and anti-LANA2 antibodies.
Measurement of -Gal activity.
Confluent BSC-40 cells seeded in 24-well plates were infected with the indicated viruses and 5 mM IPTG was added to induce PKR expression. Cells were collected 24 h post-infection (p.i.), resuspended in 100 µl 0·25 M Tris, pH 7·8, and lysed by three freezethaw cycles. Lysis extracts were diluted to 1 ml with water, centrifuged and 10 µl of supernatant were used for -Gal determination. Cell lysate supernatants were mixed with 150 µl of CPRG solution (1 mM MgCl2, 45 mM
-mercaptoethanol, 0·1 M sodium phosphate, pH 7·5, 5 mM CPRG) in a 96-well plate, incubated at 37 °C for 1 h and the absorbance at 540 nm determined.
Caspase 3 activity assay.
Cells (2x106) were collected in lysis buffer (150 mM KCl, 10 % glycerol, 1 mM DTT, 5 mM magnesium acetate, 0·5 % Nonidet P-40) and clarified by centrifugation. Equal amounts of supernatant and 2x reaction buffer (100 mM HEPES, pH 7·5, 20 % glycerol, 5 mM DTT, 0·5 mM EDTA) were mixed and assayed for caspase 3 activity using 200 µM DEVD-pNA (Calbiochem) as substrate. Free pNA produced by caspase activity was determined by measuring absorbance at 405 nm.
NFB activation assay.
Mouse fibroblasts derived from homozygous PKR knockout mice were mock-infected or double-infected with 2 p.f.u. per cell of VV-PKR and up to 6 p.f.u. per cell of WR or VV-LANA2. At 24 h p.i., whole-cell extracts were prepared and processed following the manufacturer's instructions for the NFB p65 Transcription Factor Assay kit (Active Motif Europe).
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RESULTS |
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LANA2 affects translational control induced by PKR
One of the major biological functions of PKR is to control translation through phosphorylation of eIF-2 (reviewed by Gale & Katze, 1998
). Since LANA2 appears to affect apoptosis induced by PKR, we next determined the effect of LANA2 on the inhibition of protein synthesis induced by PKR. BSC-40 cells were infected with VV-PKR in combination with WR or VV-LANA2 and de novo protein synthesis was determined by labelling with [35S]methionine at 24 h p.i. As shown in Fig. 4
(a), PKR severely inhibited protein synthesis in the presence of IPTG and this inhibition was partially prevented after co-infection with the recombinant expressing LANA2. To provide further evidence for the rescue of the translational control, we measured expression of
-Gal. This was achieved due to the presence of the lacZ gene in the viral genome of the recombinant viruses VV-PKR or VV-LANA2, which provides a visual marker for the isolation of the recombinant vectors. Thus, to quantify the effect of LANA2 on the extent of inhibition of viral protein synthesis by PKR,
-Gal activity was measured in extracts from cells infected with VV-PKR in combination with the recombinant virus VV-LANA2 or the recombinant virus VVp36, which also contains the lacZ gene in the viral genome and was used as a control. As shown in Fig. 4(b)
, inhibition of
-Gal activity by PKR expression was rescued by increasing amounts of LANA2. Thus, LANA2 was able to overcome the translational control induced by PKR over a single protein such as
-Gal.
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LANA2 does not interact with PKR
To study whether the effect of LANA2 on the eIF-2 phosphorylation induced by PKR was mediated by a direct interaction between LANA2 and PKR, in vivo binding assays were performed as described in Methods. We could not detect any direct interaction between the proteins, suggesting there is no physical interaction between LANA2 and PKR (data not shown).
LANA2 inhibits caspase 3 but not caspase 9 activation
PKR induces apoptosis by activation of the FADD/caspase 8 pathway. In addition, although dispensable, caspase 9 is also a mediator of PKR-induced cell death (Gil et al., 2002). Both apoptotic pathways converge in the final activation of terminator caspases, such as caspase 3. In view of the relevance of caspases in PKR-induced cell death, we decided to study whether LANA2 was able to block the activation of caspases 3 and 9 induced by PKR. Increasing amounts of LANA2 did not reduce the activation of caspase 9 triggered by PKR, as observed by the appearance of a smaller-sized protein (denoted with an arrow in Fig. 5
a). However, increasing amounts of LANA2 decreased the levels of caspase 3 activation as shown in Fig. 5(b)
. These results suggest that only the FADD/caspase 8 pathway is affected by LANA2.
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DISCUSSION |
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KSHV contains several homologues of cellular IRFs (Moore et al., 1996), having evolved a mechanism by which it directly subverts the functions of IRFs and down-regulates the induction of the IFN genes that are important components of the innate immunity (Lubyova & Pitha, 2000
). However, only one of the KSHV IRF-like proteins, vIRF2, has been demonstrated to target the PKR protein specifically (Burysek & Pitha, 2001
). The aim of this study was to investigate whether LANA2, another IRF-like protein from KSHV, with homology to vIRF2 (Rivas et al., 2001
), had some effect on the two IFN-induced pathways, the 2-5A synthetase/RNase L system and PKR.
Activation of the IFN-induced enzyme RNase L causes RNA breakdown as well as apoptosis of animal cells, an effect that is enhanced by co-expression of 2-5A synthetase (Castelli et al., 1997; Díaz-Guerra et al., 1997b
; Zhou et al., 1997
). Analysis of RNA stability or apoptosis induced by activation of the 2-5A system in the presence of LANA2 indicated that LANA2 did not interfere with this IFN-induced pathway.
PKR conditions cellular apoptosis in response to activation by various stimuli such as dsRNA accumulated as a by-product of virus replication (Der et al., 1997), or when it is overexpressed (Lee & Esteban, 1994
). Studies on the effect of LANA2 on PKR activity demonstrated that LANA2 inhibited apoptosis induced by PKR activation. Two of the cellular substrates of PKR are eIF-2
, which on phosphorylation abrogates translation initiation (Rowlands et al., 1988
), and I
B
, the inhibitor of the transcription factor NF
B (reviewed by Baldwin, 1996
). Activation of NF
B by PKR was not altered after LANA2 expression, suggesting that the activity of LANA2 did not affect this pathway. However, analysis of the levels of the phosphorylated form of eIF-2
after PKR activation in the presence of increasing amounts of LANA2 showed an inhibitory effect of LANA2 on eIF-2
phosphorylation. The decrease in the levels of phosphorylated eIF-2
may explain how LANA2 relieves the block of translation induced by PKR. However, we could not detect direct interaction between the proteins, suggesting that LANA2 activity is not executed through direct interaction with PKR. It is of interest to note that MC159L protein from molluscum contagiosum virus inhibits apoptosis induced by PKR in the absence of direct interaction but cannot block phosphorylation of eIF-2
(Gil et al., 2001
), indicating different modes of action of these viral proteins that antagonize PKR activity.
Two apoptotic pathways in mammalian cells are employed to destroy virus-infected cells: the extrinsic death-receptor pathway and the intrinsic mitochondrial pathway. Both pathways converge to activate caspases, in particular the effector caspase 3 (Thornberry & Lazebnik, 1998). PKR induces apoptosis by activation of the FADD/caspase 8 pathway. In addition, although dispensable, caspase 9 is also a mediator of PKR-induced cell death (Gil et al., 2002
). Our experiments measuring the effect of LANA2 on PKR-induced caspase activation revealed that LANA2 could inhibit PKR-induced activation of caspase 3 but not caspase 9, suggesting that only the FADD/caspase 8 pathway is affected by LANA2.
In conclusion, our findings show that KSHV encodes, in addition to the described vIRF2, another IRF-like protein, LANA2, that targets PKR. In contrast to vIRF2 (Burysek & Pitha, 2001), LANA2 is not a direct PKR inhibitor; however, it is able to target the eIF-2
phosphorylation pathway with the ability to revert the translational block imposed by PKR. Since both LANA2 and vIRF2 are KSHV latent proteins (Burysek & Pitha, 2001
; Rivas et al., 2001
), their co-operation to inhibit PKR activity could be a key mechanism in KSHV persistence.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Black, T., Safer, B., Hovanessian, A. & Katze, M. G. (1989). The cellular 68,000 Mr protein kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection: implications for translational regulation. J Virol 63, 22442251.[Medline]
Burysek, L. & Pitha, P. M. (2001). Latently expressed human herpesvirus 8-encoded interferon regulatory factor 2 inhibits double-stranded RNA-activated protein kinase. J Virol 75, 23452352.
Burysek, L., Yeow, W. S., Lubyova, B., Kellum, M., Schafer, S. L., Huang, Y. Q. & Pitha, P. M. (1999). Functional analysis of the HHV-8 encoded vIRF-1 and its association with cellular IRFs and p300. J Virol 73, 73347342.
Castelli, J. A., Hassel, B. A., Wood, K. A., Li, X.-L., Amemiya, K., Dalakas, M. C., Torrence, P. & Youle, R. J. (1997). A study of the interferon antiviral mechanism: apoptosis activation of the 2-5A system. J Exp Med 186, 967972.
Chang, Y., Cesarman, E., Pessin, M. S., Lee, F., Culpepper, J., Knowles, D. M. & Moore, P. S. (1994). Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science 266, 18651869.[Medline]
Chong, K. L., Feng, L., Schappert, K., Meurs, E., Donahue, T. F., Friesen, J. D., Hovanessian, A. G. & Williams, B. R. (1992). Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2. EMBO J 11, 15531562.[Abstract]
Der, S. D., Yang, Y., Weissmann, C. & Williams, B. R. G. (1997). A double stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis. Proc Natl Acad Sci U S A 94, 32793283.
Dever, T. E., Sripriya, R., McLachlin, J. R., Lu, J., Fabian, J. R., Kimball, S. R. & Miller, L. K. (1998). Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2 kinase homolog. Proc Natl Acad Sci U S A 95, 41644169.
Díaz-Guerra, M., Rivas, C. & Esteban, M. (1997a). Inducible expression of the 2-5A-synthetase/RNaseL system results in inhibition of vaccinia virus replication. Virology 227, 220228.[CrossRef][Medline]
Díaz-Guerra, M., Rivas, C. & Esteban, M. (1997b). Activation of the IFN-inducible enzyme RNaseL causes apoptosis of animal cells. Virology 236, 354363.[CrossRef][Medline]
Gale, M., Jr & Katze, M. G. (1998). Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol Ther 78, 2946.[CrossRef][Medline]
Gale, M., Jr, Korth, M. J., Tang, N. M., Tan, S. L., Hopkins, D. A., Dever, T. E., Polyak, S. J., Gretch, D. R. & Katze, M. G. (1997). Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 230, 217227.[CrossRef][Medline]
Gale, M., Jr, Blakely, C. M., Kwieciszewski, B. & 7 other authors (1998). Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation. Mol Cell Biol 18, 52085218.
Gao, S. J., Boshoff, C., Jayachandra, S., Weiss, R. A., Chang, Y. & Moore, P. S. (1997). KSHV ORFK9 (vIRF) is an oncogene which inhibits the interferon signaling pathway. Oncogene 15, 19791985.[CrossRef][Medline]
Gil, J., Alcami, J. & Esteban, M. (1999). Induction of apoptosis by double-stranded-RNA dependent protein kinase (PKR) involves the subunit of eukaryotic translation initiation factor 2 and NF-
B. Mol Cell Biol 19, 46534663.
Gil, J., Alcami, J. & Esteban, M. (2000). Activation of NF-B by the dsRNA dependent protein kinase, PKR involves the I
B kinase complex. Oncogene 19, 13691378.[CrossRef][Medline]
Gil, J., Rullas, J., Alcami, J. & Esteban, M. (2001). MC159L protein from the poxvirus molluscum contagiosum virus inhibits NF-B activation and apoptosis induced by PKR. J Gen Virol 82, 30273034.
Gil, J., Garcia, M. A. & Esteban, M. (2002). Caspase 9 activation by the dsRNA-dependent protein kinase, PKR: molecular mechanism and relevance. FEBS Lett 529, 249255.[CrossRef][Medline]
Gonzalo, R. M., Rodríguez, J. R., Rodríguez, D., Gonzalez-Aseguinolaza, G., Larraga, V. & Esteban, M. (2001). Protective immune response against cutaneous leishmaniasis by prime-booster immunization regimens with vaccinia virus recombinants expressing Leishmania infantum p36/LACK and IL-12 in combination with purified p36. Microbes Infect 3, 701711.[CrossRef][Medline]
Goodbourn, S., Didcock, L. & Randall, R. E. (2000). Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. J Gen Virol 81, 23412364.
Harada, H., Taniguchi, T. & Taneka, N. (1998). The role of interferon regulatory factors in the interferon system and cell growth control. Biochimie 88, 641650.[CrossRef]
He, B., Gross, M. & Roizman, B. (1997). The 134·5 protein of herpes simples virus 1 complexes with protein phosphatase 1
to dephosphorylate the
subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A 94, 843848.
Hirt, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 27, 365369.
Katze, M. G., Decorato, D., Safer, B., Galabru, J. & Hovanessian, A. G. (1987). Adenovirus VAI RNA complexes with the 68,000 Mr protein kinase to regulate its autophosphorylation and activity. EMBO J 6, 689697.[Abstract]
Kumar, A., Haque, J., Lacoste, J., Hiscott, J. & Williams, B. R. G. (1994). Double-stranded RNA-dependent protein kinase activates transcription factor NF-B by phosphorylating I
B. Proc Natl Acad Sci U S A 91, 62886292.[Abstract]
Kunzi, M. S. & Pitha, P. M. (1998). The role of cytokines in viral infections. In Topley and Wilson's Microbiology and Microbial Infections, 9th edn, vol. 1, p. 193. Edited by L. Collier, A. Balows & M. Sussman. London: Arnold.
Lee, S. B. & Esteban, M. (1993). The interferon-induced double-stranded RNA-activated human p68 protein kinase inhibits the replication of vaccinia virus. Virology 193, 10371041.[CrossRef][Medline]
Lee, S. B. & Esteban, M. (1994). The interferon-induced double-stranded RNA-activated protein kinase induces apoptosis. Virology 199, 491496.[CrossRef][Medline]
Lee, S. B., Bablaman, R. & Esteban, M. (1996). Regulated expression of the interferon-induced protein kinase p68 (PKR) by vaccinia virus recombinants inhibits the replication of vesicular stomatitis virus but not that of poliovirus. J Interferon Cytokine Res 16, 10731078.[Medline]
Li, M., Lee, H., Guo, J., Niepel, F., Fleckenstein, B., Ozato, K. & Jung, J. U. (1998). Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor. J Virol 72, 54335440.
Lloyd, R. M. & Shatkin, A. J. (1993). Translational stimulation by reovirus polypeptide sigma substitution for VAI RNA and inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2. J Virol 66, 68786884.
Lu, Y., Wambach, M., Katze, M. G. & Krug, R. M. (1995). Binding of the influenza virus NSI protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the eIF2 translation initiation factor. Virology 214, 222228.[CrossRef][Medline]
Lubyova, B. & Pitha, P. M. (2000). Characterization of a novel human herpesvirus 8-encoded protein vIRF3, that shows homology to viral and cellular interferon regulatory factors. J Virol 74, 81948201.
Mamane, Y., Heylbroeck, C., Genin, P. & 7 other authors (1999). Interferon regulatory factors: the next generation. Gene 237, 114.[CrossRef][Medline]
Moore, P. S., Gao, S. J., Dominguez, G. & 7 other authors (1996). Primary characterization of a herpesvirus agent associated with Kaposi's sarcoma. J Virol 70, 549558.[Abstract]
Nguyen, H., Hiscott, J. & Pitha, P. M. (1997). The growing family of interferon regulatory factors. Cytokine Growth Factor Rev 8, 293312.[CrossRef][Medline]
Petryshyn, R., Chen, J. J. & London, I. M. (1984). Growth-related expression of a double-stranded RNA-dependent protein kinase in 3T3 cells. J Biol Chem 259, 1473614742.
Ploegh, H. L. (1998). Viral strategies of immune evasions. Science 280, 248253.
Rivas, C., Thlick, A. E., Parravicini, C., Moore, P. S. & Chang, Y. (2001). Kaposi's sarcoma-associated herpesvirus LANA2 is a B-cell-specific latent viral protein that inhibits p53. J Virol 75, 429438.
Rowlands, A. G., Panniers, R. & Henshaw, E. C. (1988). The catalytic mechanism of guanine nucleotide exchange factor action and competitive inhibition by phosphorylated eukaryotic initiation factor 2. J Biol Chem 263, 55265533.
Samuel, C. E. (1991). Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology 183, 111.[Medline]
Sarid, R., Olsen, S. J. & Moore, P. S. (1999). Kaposi's sarcoma-associated herpesvirus: epidemiology, virology, and molecular biology. Adv Virus Res 52, 139232.[Medline]
Soulier, J., Grollet, L., Oksenhendler, E. & 7 other authors (1995). Kaposi's sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman's disease. Blood 86, 12751280.
Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H. & Schreiber, R. D. (1998). How cells respond to interferons. Annu Rev Biochem 67, 227264.[CrossRef][Medline]
Taniguchi, T., Ogasawara, K., Takaoka, A. & Tanaka, N. (2001). IRF family of transcription factors as regulators of host defense. Annu Rev Immunol 19, 623655.[CrossRef][Medline]
Taylor, D. R., Shi, S. T., Romano, P. R., Barber, G. N. & Lai, M. M. (1999). Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 285, 107110.
Thornberry, N. A. & Lazebnik, Y. (1998). Caspases: enemies within. Science 281, 13121316.
Vazquez, M. I. & Esteban, M. (1999). Identification of the functional domains in the 14-kilodalton envelope protein (A27L) of vaccinia virus. J Virol 73, 90989109.
Vilcek, J. & Sen, G. (1996). Interferons and other cytokines. In Fields Virology, 3rd edn, vol. 1, pp. 375399. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Zhou, A., Paranjape, J., Brown, T. L. & 8 other authors (1997). Interferon action and apoptosis are defective in mice devoid of 2',5'-oligoadenylate-dependent RNase L. EMBO J 16, 63556363.
Zimring, J. C., Goodbourn, S. & Offermann, M. K. (1998). Human herpesvirus 8 encodes an interferon regulatory factor (IRF) homolog that represses IRF-1 mediated transcription. J Virol 72, 701707.
Received 27 November 2002;
accepted 3 February 2003.