Winship Cancer Institute, Emory University, 1365-B Clifton Rd NE, Atlanta, GA 30322, USA
Correspondence
Margaret K. Offermann
mofferm{at}emory.edu
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ABSTRACT |
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Present address: Georgia Cancer Specialists, Atlanta, GA, USA.
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INTRODUCTION |
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Interferons (IFNs) are cytokines that play a central role in host defence against viral infection. Type I IFNs (IFN- and IFN-
) are produced and secreted by a wide variety of virally infected cells, whereas Type II IFN (IFN-
) is produced primarily by natural killer cells, CD4+ T helper (Th1) cells and CD8+ cytotoxic T cells (Biron, 1999
; Biron et al., 1999
). The two types of IFNs signal through distinct receptors, but there is some overlap in the Jak-STAT signalling molecules involved in transducing responses (Doly et al., 1998
; Schindler, 1999
, 2002
; Schindler & Brutsaert, 1999
). While both types of IFNs induce expression of antiviral proteins, divergent responses to the two types of IFN have been observed in patients with KS as well as in HHV-8-infected cells in culture. Patients with KS who have adequate CD4 counts and who lack constitutional symptoms often respond to IFN-
with regression of KS lesions (Fischl et al., 1996
; Krown, 1991
; Krown et al., 1992
; Sawyer et al., 1990
), whereas clinical trials testing the efficacy of IFN-
for the treatment of KS were halted when several patients experienced dramatic worsening of lesions (Aboulafavia et al., 1989
; Ganser et al., 1986
; Krigel et al., 1985
). IFN-
prevents HHV-8-infected BCBL-1 cells from entering the lytic phase of virus replication in response to incubation with TPA, thereby preventing production of infectious virus (Chang et al., 2000
; Monini et al., 1999
; Pozharskaya et al., 2004
). This occurs in part because entry into the lytic phase of virus replication sensitizes BCBL-1 cells to the pro-apoptotic effects of IFN-
(Pozharskaya et al., 2004
). In contrast, IFN-
induces a several fold increase in the percentage of cells that express lytic viral proteins (Blackbourn et al., 2000
; Chang et al., 2000
; Mercader et al., 2000
). IFN-
is often expressed at elevated levels in KS lesions (Fiorelli et al., 1998
). No studies have directly examined changes in infectious virus resulting from IFN-
, but the increase in lytic viral gene expression led to the hypothesis that the expression of IFN-
in KS lesions stimulates infectious virus production (Blackbourn et al., 2000
; Chang et al., 2000
). Alternatively, the expression of IFN-
in KS lesions might not increase viral load and might merely reflect the host response to the viral infection.
Some IFN-induced changes directly affect cellular responses to viral infection, whereas other responses to IFN are of an immunological nature. Proteins induced by IFNs that directly contribute to cellular antiviral responses include the double-stranded RNA activated protein kinase (PKR), 2'5'-oligoadenylate synthetase (OAS) and Mx (Barber, 2001; Ronni et al., 1995
; Samuel, 2001
; Williams, 1999
; Yaffe et al., 1996
). IFNs also induce the transcription factor IRF-1 that regulates expression of mRNA for immunomodulatory and growth regulatory proteins (Kimura et al., 1994
; Kirchhoff et al., 1995
; Taniguchi et al., 2001
). Both types of IFN can also augment acquired immunity by inducing higher levels of expression of major histocompatibility complex (MHC) class I, a protein that is important in the presentation of viral antigens to cytotoxic T lymphocytes (Kessler et al., 2002
). IFN-
affects the host response by favouring Th1 differentiation over Th2 (Lohoff et al., 1997
; Novelli et al., 1997
), thereby favouring cell-mediated immune responses.
Despite studies exploring responses to IFN- in vitro and in vivo, it is not known whether IFN-
affects the production of infectious HHV-8. In this report, we demonstrate that both IFN-
and IFN-
induce expression of multiple IFN-responsive genes in the primary effusion lymphoma cell line, BCBL-1, but the patterns of induction are distinct for the two types of IFN. Neither type of IFN induces a detectable increase in the production of infectious virus, and both types function as antiviral cytokines to decrease the amount of infectious virus that results from TPA-induced entry into the lytic phase.
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METHODS |
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A reporter cell line for quantification analysis of infectious HHV-8, T1H6 (Inoue et al., 2003), was maintained in Dulbecco's modification of Eagle's medium (Cellgro) supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 200 U penicillin ml1, 10 U streptomycin ml1 and 50 µg hygromycin B ml1 (Invitrogen), and split 1 : 10 every 34 days.
Northern blot analysis.
Total cellular RNA was isolated from BCBL-1 cells using Trizol (GibcoBRL) and was size-fractionated on a 1 % agarose formaldehyde gel in the presence of 1 µg ethidium bromide ml1. RNA was transferred to nitrocellulose and covalently linked by ultraviolet irradiation using a Stratalinker (Stratagene) and by baking in vacuo for 2 h at 80 °C. DNA probes included a 1·2 kb EcoRI fragment from a cDNA of 2'5'-OAS (Benech et al., 1985), a 1·2 kb cDNA insert containing IRF-1 from pCOIRF-1 (Whiteside et al., 1994
) and a 1·2 kb EcoRI cDNA fragment of GAPDH from clone HHVMC32 (Adams et al., 1992
). The MHC I probe was a 6 kb EcoRI fragment of the human genomic HLA-B7 gene. The probe for PKR was a 1043 nt PCR fragment from the cDNA (Meurs et al., 1990
). The probes for vIRF-1 (Zimring et al., 1998
) and vIL-6 (Yu et al., 1999
) consisted of the entire coding regions. All probes were labelled with [32P]dCTP using an oligolabelling kit (Stratagene) in accordance with the manufacturer's recommendations. Hybridization was performed at 42 °C in 5x SSC, 1 % SDS, 5x Denhart's, 100 µg denatured salmon sperm DNA ml1, 50 % formamide and 10 % dextran sulfate. Nitrocellulose was washed with a final stringency of 0·2x SSC in 0·1 % SDS at 55 °C. Blots were serially probed for the indicated sequences, and the nitrocellulose was stripped using boiling water prior to rehybridization with other probes. Densitometry was used to quantitate relative differences in mRNA levels.
Western blot analysis.
BCBL-1 cells were pelleted by centrifugation and lysed in cell lysis buffer (Cell Signalling Technology) to which protease inhibitor cocktail (BD PharMingen) was added. Total protein was size fractionated by electrophoresis using 12·5 % acrylamide SDS-PAGE. Proteins were transferred from the gel to PVDF membranes. Membranes were blocked in 5 % dry non-fat milk and probed with antibodies against vIRF-1 at 1 : 1000 dilution, LANA (ORF73, clone LN53; Advanced Biotechnologies) at 1 : 500 dilution, PKR (B-10; Santa Cruz Biotechnology) at 1 : 1000 dilution and actin (I-19; Santa Cruz Biotechnology) at 1 : 1000 dilution. After the secondary antibody reaction, the membranes were washed in TBS-Tween (0·02 M Tris, pH 7·6, 0·1 M sodium chloride, 0·05 % Tween-20) and visualized using enhanced chemiluminescence reaction (Amersham Pharmacia).
Assay for extracellular infectious HHV-8 production.
The T1H6 reporter cell line contains the lacZ gene under control of the PAN promoter and responds to infection with HHV-8 in a sensitive and quantitative manner that accurately assesses the amount of infectious HHV-8 present (Inoue et al., 2003; Krug et al., 2004
; Pozharskaya et al., 2004
). Briefly, 8x104 T1H6 cells per well were seeded in 48-well plates in triplicate. The next day BCBL-1 cell medium, filtered through 0·45 µm filter, was added to T1H6 cells in the presence of 8 µg polybrene (Sigma) ml1. Plates with T1H6 cells were centrifuged at 400 g for 30 min at room temperature and incubated at 37 °C for 1·5 h. Then the medium was changed, and cells were incubated for 2 days at 37 °C. After three freeze-thaw cycles with 50 µl PBS, cell lysates were harvested, and their
-galactosidase activities were measured by luminescent
-galactosidase assay (Clontech Lab) using LUMIstar Galaxy luminometer (BMG LabTechnologies). For the standard curve, dilution series of infectious virus were used as well as serial dilutions of the
-galactosidase.
Cell viability.
Viable BCBL-1 cells were detected by direct counting with trypan blue exclusion using a haemocytometer. Mean and standard deviations (SD) from replicates were determined.
Flow cytometry.
BCBL-1 cells (1x106) were washed in staining buffer (Dulbecco's PBS with 2 % fetal bovine serum without Mg2+ or Ca 2+, pH 7·4) and incubated with anti-IFN- receptor
-rabbit polyclonal antibody (1 : 50, C-20; Santa Cruz Biotechnology) or anti-IFN-
/
receptor rabbit polyclonal antibody (1 : 50, C-18; Santa Cruz Biotechnology) for 30 min at 4 °C. After cells were washed twice in staining buffer, secondary goat anti-rabbit antibody conjugated to Alexa Fluor 488 (1 : 500; Molecular Probes) was applied for 30 min at 4 °C. BCBL-1 cells were washed again in staining buffer and fixed in Cytofix/Cytoperm (BD PharMingen) solution for 20 min at 4 °C. As a negative control, BCBL-1 cells were also incubated with secondary antibody without primary antibody. For each sample, 10 000 events were acquired on a FACScan flow cytometer (Becton Dickinson).
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RESULTS |
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A lack of a measurable increase in production of infectious virus might occur if the increase was below the level of detection by the assay. Alternatively, the increase in PKR, 2'5'-OAS and other antiviral genes induced by IFN- might have restricted the ability of HHV-8 to complete its life cycle, thereby preventing production of infectious virus. To examine these possibilities, we induced entry of HHV-8 into the lytic phase using TPA and examined the ability of IFN-
and IFN-
to modulate the production of infectious virus resulting from the TPA. Combined treatment of the cells with IFN-
and TPA reduced the amount of infectious virus that resulted from incubation with TPA by approximately 60 % when examined on days 3, 4 and 5 (Fig. 5a
). The reduction was less than the complete disruption of production of infectious virus that resulted when IFN-
was present during incubation with TPA, and the combination of IFN-
and IFN-
was comparable to incubation with IFN-
alone. We hypothesized that the reduction in TPA-stimulated production of infectious virus that resulted from incubation with IFN-
or IFN-
was due to an increase in cell death prior to completion of virus replication. When IFN-
was present during incubation with TPA, the number of viable cells was significantly reduced on days 4 and 5, by 43 and 50 %, respectively, compared with TPA treatment alone (Fig. 5b
). When IFN-
was present during incubation with TPA, cell death occurred at early time points, leading to a progressive reduction in the number of cells compared with the number present at time 0. This early cell death was accompanied by a complete disruption of TPA-induced production of infectious virus.
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DISCUSSION |
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Incubation with TPA had a more profound impact on the number of viable BCBL-1 cells than incubation with either IFN- or IFN-
. Entry into the lytic phase is accompanied by exit from the cell cycle as cellular resources are shifted from cellular replication to virus replication (Pozharskaya et al., 2004
; Wang et al., 2003
; Wu et al., 2002
). Considerable apoptosis also occurs in response to TPA-induced lytic replication of HHV-8 in BCBL-1 cells, but this does not prevent release of infectious virus (Pozharskaya et al., 2004
). Multiple viral proteins are expressed during lytic replication that block apoptosis and responses to IFNs (Gao et al., 1997
; Seo et al., 2002
; Zimring et al., 1998
), but they are not expressed at sufficient levels or for sufficient duration to prevent many changes induced by IFN-
and IFN-
(D'Agostino et al., 1999
; Pozharskaya et al., 2004
). We demonstrated that enhanced cell death and disruption of the production of infectious HHV-8 accompanied the enhanced expression of antiviral proteins that resulted from incubation with IFN-
and IFN-
. The host response to viral infection is generally greater during lytic replication when multiple viral genes are present that can trigger an innate antiviral response. BCBL-1 cells supporting lytic replication of HHV-8 are more sensitive to IFN-
-induced apoptosis than latently infected cells (D'Agostino et al., 1999
; Pozharskaya et al., 2004
). The heightened expression of PKR and 2'5'-OAS that resulted from incubation with IFN-
and IFN-
probably contributed to the disruption of viral production by decreasing expression of viral proteins and by enhancing apoptosis. Both 2'5'-OAS and PKR contain double-stranded binding domains that regulate enzymic activation (Clemens & Elia, 1997
; Justesen et al., 2000
; Tan & Katze, 1999
). Activation generally occurs when sufficient double-stranded RNA is expressed during viral infection to activate these enzymes. Entry into the lytic cascade of HHV-8 occurs in response to expression of Rta, the product of ORF 50 (Lukac et al., 1998
, 1999
; Sun et al., 1998
). Expression of Rta is accompanied by co-expression of complementary transcripts (Lukac et al., 1999
), indicating the presence of viral double-stranded RNA that might activate PKR and 2'5'-OAS. Activated 2'5'-OAS catalyses the formation of oligoadenylates in 2'5' linkage that activate a latent ribonuclease, RNase L (Castelli et al., 1998
; Hartmann et al., 1998
; Marie et al., 1999
; Sarkar & Sen, 1998
). PKR catalyses the phosphorylation of eIF2
, a translation factor whose phosphorylation blocks the initiation of protein synthesis (Clemens & Elia, 1997
; Samuel et al., 1997
). Together, activated 2'5'-OAS and PKR decrease translation of both viral and cellular proteins and contribute to apoptosis of virally infected cells (Balachandran et al., 2000
; Barber, 2001
; Castelli et al., 1998
; Der et al., 1997
).
Incubation of non-TPA stimulated BCBL-1 cells with IFN- and IFN-
had very little effect on cell number until late time points when there was also an increase in expression of lytic-associated mRNAs. This suggests that IFN-induced cellular antiviral proteins were not fully activated in latently infected cells, and spontaneous entry into the lytic phase of virus replication during prolonged incubation with either IFN-
or IFN-
activated innate defences, thereby triggering cell death. The studies reported here were done in BCBL-1 cells, a primary effusion lymphoma cell line that is persistently infected with HHV-8. IFN-
also functions as an antiviral cytokine in HHV-8-infected transformed human microvascular endothelial cells to suppress the expression of HHV-8 lytic transcripts without affecting the percentage of cells that support latent replication (Milligan et al., 2004
).
These studies indicate that both IFN- and IFN-
induced the expression of cellular antiviral genes in HHV-8-infected BCBL-1, enhanced cell death and decreased the production of infectious virus. The ability of IFN-
and IFN-
to block production of infectious HHV-8 was not dependent on changes in the immune system since no immune effector cells were present during these studies. Thus, IFN-
more effectively disrupted production of infectious HHV-8 than IFN-
by modifying the function of innate cellular antiviral pathways.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Adams, M. D., Dubnick, M., Kerlavage, A. R., Moreno, R., Kelley, J. M., Utterback, T. R., Nagle, J. W., Fields, C. & Venter, J. C. (1992). Sequence identification of 2,375 human brain genes. Nature 355, 632634.[CrossRef][Medline]
Balachandran, S., Roberts, P. C., Brown, L. E., Truong, H., Pattnaik, A. K., Archer, D. R. & Barber, G. N. (2000). Essential role for the dsRNA-dependent protein kinase PKR in innate immunity to viral infection. Immunity 13, 129141.[Medline]
Barber, G. N. (2001). Host defense, viruses and apoptosis. Cell Death Differ 8, 113126.[CrossRef][Medline]
Benech, P., Mory, Y., Revel, M. & Chebath, J. (1985). Structure of two forms of the interferon-induced (2'5')-oligo A synthetase of human cells based on cDNAs and gene sequences. EMBO J 4, 22492256.[Abstract]
Biron, C. A. (1999). Initial and innate responses to viral infections - pattern setting in immunity or disease. Curr Opin Microbiol 2, 374381.[CrossRef][Medline]
Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P. & Salazar-Mather, T. P. (1999). Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol 17, 189220.[CrossRef][Medline]
Blackbourn, D. J., Fujimura, S., Kutzkey, T. & Levy, J. A. (2000). Induction of human herpesvirus-8 gene expression by recombinant interferon gamma. AIDS 14, 9899.[CrossRef][Medline]
Castelli, J. C., Hassel, B. A., Maran, A., Paranjape, J., Hewitt, J. A., Li, X. L., Hsu, Y. T., Silverman, R. H. & Youle, R. J. (1998). The role of 2'-5' oligoadenylate-activated ribonuclease L in apoptosis. Cell Death Differ 5, 313320.[CrossRef][Medline]
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]
Chang, Y., Ziegler, J., Wabinga, H. & 8 other authors (1996). Kaposi's sarcoma-associated herpesvirus and Kaposi's sarcoma in Africa. Uganda Kaposi's Sarcoma Study Group. Arch Intern Med 156, 202204.[Abstract]
Chang, J., Renne, R., Dittmer, D. & Ganem, D. (2000). Inflammatory cytokines and the reactivation of Kaposi's sarcoma-associated herpesvirus lytic replication. Virology 266, 1725.[CrossRef][Medline]
Clemens, M. J. & Elia, A. (1997). The double-stranded RNA-dependent protein kinase PKR: structure and function. J Interferon Cytokine Res 17, 503524.[Medline]
D'Agostino, G., Arico, E., Santodonato, L. & 9 other authors (1999). Type I consensus IFN (IFN-con1) gene transfer into KSHV/HHV-8-infected BCBL-1 cells causes inhibition of viral lytic cycle activation via induction of apoptosis and abrogates tumorigenicity in SCID mice. J Interferon Cytokine Res 19, 13051316.[CrossRef][Medline]
Der, S. D., Yang, Y. L., Weissmann, C. & Williams, B. R. (1997). A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis. Proc Natl Acad Sci U S A 94, 32793283.
Dictor, M., Rambech, E., Way, D., Witte, M. & Bendsoe, N. (1996). Human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) DNA in Kaposi's sarcoma lesions, AIDS Kaposi's sarcoma cell lines, endothelial Kaposi's sarcoma simulators, and the skin of immunosuppressed patients. Am J Pathol 148, 20092016.[Abstract]
Doly, J., Civas, A., Navarro, S. & Uze, G. (1998). Type I interferons: expression and signalization. Cell Mol Life Sci 54, 11091121.[CrossRef][Medline]
Fiorelli, V., Gendelman, R., Sirianni, M. C. & 8 other authors (1998). -Interferon produced by CD8+ T cells infiltrating Kaposi's sarcoma induces spindle cells with angiogenic phenotype and synergy with human immunodeficiency virus-1 Tat protein: an immune response to human herpesvirus-8 infection? Blood 91, 956967.
Fischl, M. A., Finkelstein, D. M., He, W., Powderly, W. G., Triozzi, P. L. & Steigbigel, R. T. (1996). A phase II study of recombinant human interferon-alpha 2a and zidovudine in patients with AIDS-related Kaposi's sarcoma. AIDS Clinical Trials Group. J Acquir Immune Defic Syndr Hum Retrovirol 11, 379384.[Medline]
Ganser, A., Brucher, W., Brodt, H. R., Busch, W., Brandhorst, I., Helm, E. B. & Hoelzer, D. (1986). Treatment of AIDS-related Kaposi's sarcoma with recombinant gamma-interferon. Onkologie 9, 163166.[Medline]
Gao, S. J., Boshoff, C., Jayachandra, S., Weiss, R. A., Chang, Y. & Moore, P. S. (1997). KSHV ORF K9 (vIRF) is an oncogene which inhibits the interferon signaling pathway. Oncogene 15, 19791985.[CrossRef][Medline]
Grundhoff, A. & Ganem, D. (2003). The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus permits replication of terminal repeat-containing plasmids. J Virol 77, 27792783.
Hartmann, R., Norby, P. L., Martensen, P. M., Jorgensen, P., James, M. C., Jacobsen, C., Moestrup, S. K., Clemens, M. J. & Justesen, J. (1998). Activation of 2'-5' oligoadenylate synthetase by single-stranded and double-stranded RNA aptamers. J Biol Chem 273, 32363246.
Hu, J., Garber, A. C. & Renne, R. (2002). The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus supports latent DNA replication in dividing cells. J Virol 76, 1167711687.
Inoue, N., Winter, J., Lal, R. B., Offermann, M. K. & Koyano, S. (2003). Characterization of entry mechanisms of human herpesvirus 8 by using an Rta-dependent reporter cell line. J Virol 77, 81478152.
Justesen, J., Hartmann, R. & Kjeldgaard, N. O. (2000). Gene structure and function of the 2'-5'-oligoadenylate synthetase family. Cell Mol Life Sci 57, 15931612.[Medline]
Kessler, B. M., Glas, R. & Ploegh, H. L. (2002). MHC class I antigen processing regulated by cytosolic proteolysis-short cuts that alter peptide generation. Mol Immunol 39, 171179.[CrossRef][Medline]
Kimura, T., Nakayama, K., Penninger, J. & 8 other authors (1994). Involvement of the IRF-1 transcription factor in antiviral responses to interferons. Science 264, 19211924.[Medline]
Kirchhoff, S., Koromilas, A. E., Schaper, F., Grashoff, M., Sonenberg, N. & Hauser, H. (1995). IRF-1 induced cell growth inhibition and interferon induction requires the activity of the protein kinase PKR. Oncogene 11, 439445.[Medline]
Krigel, R. L., Odajnyk, C. M., Laubenstein, L. J., Ostreicher, R., Wernz, J., Vilcek, J., Rubinstein, P. & Friedman-Kien, A. E. (1985). Therapeutic trial of interferon-gamma in patients with epidemic Kaposi's sarcoma. J Biol Response Mod 4, 358364.[Medline]
Krown, S. E. (1991). Interferon and other biologic agents for the treatment of Kaposi's sarcoma. Hematol Oncol Clin North Am 5, 311322.[Medline]
Krown, S. E., Paredes, J., Bundow, D., Polsky, B., Gold, J. W. & Flomenberg, N. (1992). Interferon-alpha, zidovudine, and granulocyte-macrophage colony-stimulating factor: a phase I AIDS Clinical Trials Group study in patients with Kaposi's sarcoma associated with AIDS. J Clin Oncol 10, 13441351.[Abstract]
Krug, L. T., Pozharskaya, V. P., Yu, Y., Inoue, N. & Offermann, M. K. (2004). Inhibition of infection and replication of human herpesvirus 8 in microvascular endothelial cells by alpha interferon and phosphonoformic acid. J Virol 78, 83598371.
Lohoff, M., Ferrick, D., Mittrucker, H. W., Duncan, G. S., Bischof, S., Rollinghoff, M. & Mak, T. W. (1997). Interferon regulatory factor-1 is required for a T helper 1 immune response in vivo. Immunity 6, 681689.[Medline]
Lukac, D. M., Renne, R., Kirshner, J. R. & Ganem, D. (1998). Reactivation of Kaposi's sarcoma-associated herpesvirus infection from latency by expression of the ORF 50 transactivator, a homolog of the EBV R protein. Virology 252, 304312.[CrossRef][Medline]
Lukac, D. M., Kirshner, J. R. & Ganem, D. (1999). Transcriptional activation by the product of open reading frame 50 of Kaposi's sarcoma-associated herpesvirus is required for lytic viral reactivation in B cells. J Virol 73, 93489361.
Marie, I., Rebouillat, D. & Hovanessian, A. G. (1999). The expression of both domains of the 69/71 kDa 2',5' oligoadenylate synthetase generates a catalytically active enzyme and mediates an anti-viral response. Eur J Biochem 262, 155165.
Mercader, M., Taddeo, B., Panella, J. R., Chandran, B., Nickoloff, B. J. & Foreman, K. E. (2000). Induction of HHV-8 lytic cycle replication by inflammatory cytokines produced by HIV-1-infected T cells. Am J Pathol 156, 19611971.
Meurs, E., Chong, K., Galabru, J., Thomas, N. S., Kerr, I. M., Williams, B. R. & Hovanessian, A. G. (1990). Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell 62, 379390.[Medline]
Milligan, S., Robinson, M., O'Donnell, E. & Blackbourn, D. (2004). Inflammatory cytokines inhibit Kaposi's sarcoma-associated herpesvirus lytic gene transcription in in vitro-infected endothelial cells. J Virol 78, 25912596.
Monini, P., Carlini, F., Sturzl, M. & 19 other authors (1999). Alpha interferon inhibits human herpesvirus 8 (HHV-8) reactivation in primary effusion lymphoma cells and reduces HHV-8 load in cultured peripheral blood mononuclear cells. J Virol 73, 40294041.
Moore, P. S. & Chang, Y. (2001). Kaposi's sarcoma-associated herpesvirus. In Fields Virology, 4th edn, pp. 28032833. Edited by B. Fields, D. Knipe & P. Howley. Philadelphia: Lippincott-Raven Publishers.
Novelli, F., D'Elios, M. M., Bernabei, P., Ozmen, L., Rigamonti, L., Almerigogna, F., Forni, G. & Del Prete, G. (1997). Expression and role in apoptosis of the alpha- and beta-chains of the IFN-gamma receptor on human Th1 and Th2 clones. J Immunol 159, 206213.[Abstract]
Pozharskaya, V. P., Weakland, L. L., Zimring, J. C., Krug, L. T., Unger, E. R., Neisch, A., Joshi, H., Inoue, N. & Offermann, M. K. (2004). Short duration of elevated vIRF-1 expression during lytic replication of human herpesvirus 8 limits its ability to block antiviral responses induced by alpha interferon in BCBL-1 cells. J Virol 78, 66216635.
Rainbow, L., Platt, G. M., Simpson, G. R., Sarid, R., Gao, S. J., Stoiber, H., Herrington, C. S., Moore, P. S. & Schulz, T. F. (1997). The 222- to 234-kilodalton latent nuclear protein (LNA) of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) is encoded by orf73 and is a component of the latency-associated nuclear antigen. J Virol 71, 59155921.[Abstract]
Roizman, B. & Philip, P. E. (2001). The family herpesviridae: a brief introduction. In Fields Virology, 4th edn, pp. 23812397. Edited by B. Fields, D. Knipe & P. Howley. Philadelphia: Lippincott-Raven Publishers.
Ronni, T., Sareneva, T., Pirhonen, J. & Julkunen, I. (1995). Activation of IFN-alpha, IFN-gamma, MxA, and IFN regulatory factor 1 genes in influenza A virus-infected human peripheral blood mononuclear cells. J Immunol 154, 27642774.
Samuel, C. E. (2001). Antiviral actions of interferons. Clin Microbiol Rev 14, 778809.
Samuel, C. E., Kuhen, K. L., George, C. X., Ortega, L. G., Rende-Fournier, R. & Tanaka, H. (1997). The PKR protein kinase - an interferon-inducible regulator of cell growth and differentiation. Int J Hematol 65, 227237.[CrossRef][Medline]
Sarid, R., Flore, O., Bohenzky, R., Chang, Y. & Moore, P. S. (1998). Transcription mapping of the Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) genome in a body cavity-based lymphoma cell line (BC-1). J Virol 72, 10051012.
Sarkar, S. N. & Sen, G. C. (1998). Production, purification, and characterization of recombinant 2', 5'-oligoadenylate synthetases. Methods 15, 233242.[CrossRef][Medline]
Sawyer, L. A., Metcalf, J. A., Zoon, K. C., Boone, E. J., Kovacs, J. A., Lane, H. C. & Quinnan, G. V. Jr. (1990). Effects of interferon-alpha in patients with AIDS-associated Kaposi's sarcoma are related to blood interferon levels and dose. Cytokine 2, 247252.[CrossRef][Medline]
Schindler, C. (1999). Cytokines and JAK-STAT signaling. Exp Cell Res 253, 714.[CrossRef][Medline]
Schindler, C. W. (2002). Series introduction. JAK-STAT signaling in human disease. J Clin Invest 109, 11331137.
Schindler, C. & Brutsaert, S. (1999). Interferons as a paradigm for cytokine signal transduction. Cell Mol Life Sci 55, 15091522.[CrossRef][Medline]
Seo, T., Lee, D., Shim, Y. S., Angell, J. E., Chidambaram, N. V., Kalvakolanu, D. V. & Choe, J. (2002). Viral interferon regulatory factor 1 of Kaposi's sarcoma-associated herpesvirus interacts with a cell death regulator, GRIM19, and inhibits interferon/retinoic acid-induced cell death. J Virol 76, 87978807.
Sun, R., Lin, S. F., Gradoville, L., Yuan, Y., Zhu, F. & Miller, G. (1998). A viral gene that activates lytic cycle expression of Kaposi's sarcoma-associated herpesvirus. Proc Natl Acad Sci U S A 95, 1086610871.
Tan, S. L. & Katze, M. G. (1999). The emerging role of the interferon-induced PKR protein kinase as an apoptotic effector: a new face of death? J Interferon Cytokine Res 19, 543554.[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]
Wang, S. E., Wu, F. Y., Fujimuro, M., Zong, J., Hayward, S. D. & Hayward, G. S. (2003). Role of CCAAT/enhancer-binding protein alpha (C/EBP) in activation of the Kaposi's sarcoma-associated herpesvirus (KSHV) lytic-cycle replication-associated protein (RAP) promoter in cooperation with the KSHV replication and transcription activator (RTA) and RAP. J Virol 77, 600623.[CrossRef][Medline]
Whiteside, S. T., King, P. & Goodbourn, S. (1994). A truncated form of the IRF-2 transcription factor has the properties of a postinduction repressor of interferon-beta gene expression. J Biol Chem 269, 2705927065.
Williams, B. R. (1999). PKR; a sentinel kinase for cellular stress. Oncogene 18, 61126120.[CrossRef][Medline]
Wu, F. Y., Tang, Q. Q., Chen, H., ApRhys, C., Farrell, C., Chen, J., Fujimuro, M., Lane, M. D. & Hayward, G. S. (2002). Lytic replication-associated protein (RAP) encoded by Kaposi sarcoma-associated herpesvirus causes p21CIP-1-mediated G1 cell cycle arrest through CCAAT/enhancer-binding protein-. Proc Natl Acad Sci U S A 99, 1068310688.
Yaffe, A., Schwarz, Y., Hacohen, D., Kinar, Y., Nir, U. & Salzberg, S. (1996). Inhibition of 2-5A synthetase expression by antisense RNA interferes with interferon-mediated antiviral and antiproliferative effects and induces anchorage-independent cell growth. Cell Growth Differ 7, 969978.[Abstract]
Yu, Y., Black, J. B., Goldsmith, C. S., Browning, P. J., Bhalla, K. & Offermann, M. K. (1999). Induction of human herpesvirus-8 DNA replication and transcription by butyrate and TPA in BCBL-1 cells. J Gen Virol 80, 8390.[Abstract]
Zimring, J. C., Goodbourn, S. & Offermann, M. K. (1998). Human herpesvirus 8 encodes an interferon regulatory factor (IRF) homologue that represses IRF-1 mediated transcription. J Virol 72, 701707.
Received 22 April 2004;
accepted 1 July 2004.