Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Sep;73(9):7334-42.
doi: 10.1128/JVI.73.9.7334-7342.1999.

Functional analysis of human herpesvirus 8-encoded viral interferon regulatory factor 1 and its association with cellular interferon regulatory factors and p300

L Burýsek  1 , W S Yeow, B Lubyová, M Kellum, S L Schafer, Y Q Huang, P M Pitha
Affiliations

Functional analysis of human herpesvirus 8-encoded viral interferon regulatory factor 1 and its association with cellular interferon regulatory factors and p300

L Burýsek et al. J Virol. 1999 Sep.

Abstract

Human herpesvirus 8/Kaposi sarcoma-associated virus (HHV-8/KSHV) contains, in addition to genes required for viral replication, a unique set of nonstructural genes which may be part of viral mimicry and contribute to viral replication and pathogenesis in vivo. Among these, HHV-8 encodes four open reading frames (ORFs) that showed homology to the transcription factors of the interferon regulatory factor (IRF) family. The ORF K9, viral IRF 1 (vIRF-1), has been cloned, and it was shown that, when overexpressed, it down modulates the interferon-mediated transcriptional activation of the interferon-stimulated gene 15 (ISG 15) promoter, and the role of vIRF-1 in viral mimicry was implied. However, the molecular mechanism of this effect has not been clarified. Here, we extend this observation and show that vIRF-1 also downregulates the transcriptional activity of IFNA gene promoter in infected cells by interfering with the transactivating activity of cellular IRFs, including IRF-1 and IRF-3. We further show that ectopic expression of vIRF-1 in NIH 3T3 cells confers resistance to tumor necrosis factor alpha-induced apoptosis. While vIRF-1 is unable to bind DNA with the same specificity as cellular IRFs, we demonstrate by in vitro binding assay that it can associate with the family of cellular IRFs, such as IRF-1 and the interferon consensus sequence binding protein. vIRF-1 interaction domain was localized between amino acids (aa) 152 and 243. While no binding between the full-size IRF-3 and vIRF-1 could be detected by the same assay, we show that vIRF-1 also targets the carboxy-terminal region (aa 1623 to 2414) of the transcriptional coactivator p300 which could also bind IRF-3 and IRF-1. These results demonstrate that vIRF-1 can modulate the transcription of the IFNA genes by direct heterodimerization with members of the IRF family, as well as by competitive binding with cellular transcription factors to the carboxy-terminal region of p300.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
vIRF-1 inhibited synergistic activation of IFNA4 promoter by NDV and IRF-1 or IRF-3. The IFNA4 CAT reporter plasmid (1 μg) was cotransfected into NIH 3T3 cells with 1 μg of either pcDNA vector (Con), IRF-1, or IRF-3 expression plasmids, together with increasing amount of vIRF-1 expression plasmid (1 and 4 μg). Cells were infected with NDV 24 h after transfection for 16 h and analyzed for CAT activity. Error bars show standard errors for triplicate experiments.
FIG. 2
FIG. 2
NIH 3T3 cells expressing vIRF-1 showed resistance to TNF-α-mediated apoptosis. (A) Expression of vIRF-1 mRNA in the clone (3T3/vIRF-1) selected for studies of apoptosis (Northern blot). The NIH 3T3 cell line expressing vIRF-1 was generated as described in Materials and Methods. (B) Comparison of TNF-α-induced cell killing in control NIH 3T3 cells and NIH 3T3/vIRF-1 cells. Cells were treated with the indicated concentrations of mouse TNF-α and actinomycin D (1 μg/ml) for 24 h, and the cell viability was determined by MTT assay. The data represent an average (± the standard error of the mean) of two separate experiments done in triplicate.
FIG. 3
FIG. 3
(A) Analysis of interactions between vIRF-1 and cellular IRF transcription factors by in vitro pull-down assay. Recombinant GST–vIRF-1 fusion protein was immobilized on glutathione-agarose beads and incubated with indicated 35S-labeled IRF proteins as described in Materials and Methods. In lanes 1, 5, 8, and 11, 20% of the respective in vitro-labeled input IRF was loaded onto the gel. Lanes 2, 6, 9, and 12 show binding to beads containing GST protein only. Lanes 3, 7, 10, and 13 show binding of IRF-1, IRF-2, IRF-3, and ICSBP, respectively. The binding in the presence of antibody (1 μg) against C-terminal peptide of IRF-1 is shown in lane 4. (B) Virus infection does not modulate IRF-1–GST–vIRF-1 interaction in cell lysates. NIH 3T3 cells were transfected with IRF-1 expression plasmid, and 24 h later cells were infected with NDV (multiplicity of infection of 5) for 16 h. Cell lysates from control (−) or infected cells (NDV) were then subjected to pull-down assay. GST–vIRF-1-bound proteins were resolved on SDS–10% PAGE and immunoblotted with anti-IRF-1 antibody (lane 3). Input (lane 1) represents 1% of total protein added into binding reaction. No detectable interaction was observed with control, GST-containing beads (lane 2). (C) Detection of vIRF-1 and vIRF-2 protein interactions. In vitro 35S-labeled vIRF-1 and vIRF-2 proteins were incubated with GST–vIRF-1 or GST bound to glutathione-agarose beads. Bound proteins were resolved by SDS–10% PAGE. Input vIRF-1 (lane 1) represents 20% of 35S-labeled vIRF-1 translation mixture added to beads.
FIG. 4
FIG. 4
Mapping of vIRF-1 interaction domain. (A) Positional scheme of vIRF-1 deletion mutants. Numbers correspond to amino acid boundaries of respective fragments. (B) Coomassie blue-stained gel illustrating the purity and size of the respective GST–vIRF-1 fusion fragments. (C) In vitro interaction between IRF-1 and different vIRF-1 fragments as detected by pull-down assay. 35S-labeled IRF-1 input (20%) and its binding to GST beads are shown in lanes 1 and 2, respectively. IRF-1 was pulled down by GST–vIRF-1C′ (lane 4) and not by GST–vIRF-1N′ (lane 3). Both GST–vIRF-1A (lane 5) and GST–vIRF-1B (lane 6) fragments actively bound IRF-1, while no IRF-1 binding was detected with GST–vIRF-1C fragment (lane 7).
FIG. 5
FIG. 5
(A) The C′-terminal peptides of vIRF-1 disrupt the specific activation of the IFNA4 promoter by NDV and IRF-1. The cotransfections were done as described in Fig. 1 by using 1 μg of each IRF-1, IFNA4 CAT, and the respective plasmid encoding the C′-terminal vIRF-1 peptides. The relative levels of vIRF-1 peptides in transfected cells were comparable (data not shown). Error bars show the standard errors for triplicate experiments. (B) vIRF-1 modulates the binding of IRF-1 to PRD-I oligonucleotide. Gel retardation assays were performed with purified recombinant GST–IRF-1 and His6–vIRF-1 proteins and 32P-labeled PRD-I probe. A total of 1 ng of GST–IRF-1 was preincubated alone (lane 1) or in the presence of 3, 5, or 7 ng of His6–vIRF-1 (lanes 2 to 4). The same reaction as in lane 2 was performed in the presence of 3 μl of vIRF-1 antiserum (AS, lane 5).
FIG. 6
FIG. 6
In vitro binding analysis of vIRF-1 and IRF-1 to p300. (A) Interaction of vIRF-1 and IRF-1 with the N′- and C′-terminal parts of p300. Binding of in vitro 35S-labeled N′-terminal half of p300 to immobilized GST–vIRF-1 and GST–IRF-1 is shown in lanes 3 and 4. The binding of the in vitro 35S-labeled C-terminal half of p300 to immobilized GST–vIRF-1 and GST–IRF-1 is shown in lanes 7 and 10. The binding of the C-terminal half of the p300 to N- and C-terminal parts of GST–vIRF-1 is shown in lanes 8 and 9, respectively. Lanes 2 and 6 show the binding to GST beads only, and lanes 1 and 5 show the input of the 35S-labeled N- or C-terminal part of p300 (20%). (B) Positional scheme of p300 deletion mutants. The numbers correspond to the amino acid boundaries of the respective fragments. (C) Mapping of p300 binding site for vIRF-1 and IRF-1 by in vitro pull-down assay. Recombinant GST–vIRF-1C′ and GST–IRF-1 fusion proteins were immobilized on glutathione-agarose beads and incubated with indicated 35S-labeled p300 peptides as shown in Fig. 6B. In lanes 1 to 3, 20% of the in vitro-labeled p300 fragments A, B, and C were analyzed on an SDS gel. Lanes 4 to 6 show the binding to beads containing GST protein only. Lanes 7 to 9 show the binding of the indicated p300 fragment to GST–vIRF-1C′ beads, and lanes 10 to 12 show the binding of the indicated p300 fragment to GST–IRF-1 beads.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Albrecht J-C, Nicholas J, Biller D, Cameron K R, Biesinger B, Newman C, Wittmann S, Craxton M A, Coleman H, Fleckenstein B, Hones R W. Primary structure of the herpesvirus saimiri genome. J Virol. 1992;66:5047–5058. - PMC - PubMed
    1. Au W-C, Moore P A, LaFleur D W, Tomball B, Pitha P M. Characterization of intereferon regulatory factor-7, and its potential role in the transcriptional activation of interferon A genes. J Biol Chem. 1998;273:29210–29217. - PubMed
    1. Au W-C, Moore P A, Lowther W, Juang Y-T, Pitha P M. Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc Natl Acad Sci USA. 1995;92:11657–11661. - PMC - PubMed
    1. Baer R, Bankier A T, Biggin M D, Deininger P L, Farrell P J, Gibson T J, Hatfull G, Hudson G S, Satchwell S C, Seguin C, et al. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984;310:207–211. - PubMed
    1. Balachandran S, Kim C N, Yeh W C, Mak T W, Bhalla K, Barber G N. Activation of the dsRNA-dependent protein kinase, PKR, induces apoptosis through FADD-mediated death signaling. EMBO J. 1998;17:6888–6902. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources