Characterization of a novel human herpesvirus 8-encoded protein, vIRF-3, that shows homology to viral and cellular interferon regulatory factors

Abstract

The genome of the human herpesvirus 8 (HHV-8) contains a cluster of open reading frames (ORFs) encoding proteins with homology to the cellular transcription factors of the interferon regulatory factor (IRF) family. Two of these homologues, vIRF-1 and vIRF-2, were previously identified and functionally analyzed. In this study, we have characterized a novel gene, designated vIRF-3, encoded within the previously predicted ORF K10.5 and our newly identified ORF K10. 6. Northern blotting of RNA extracted from BCBL-1 cells with a vIRF-3-specific probe and reverse transcription-PCR analyses revealed a single transcript of 2.2 kb with a splice present in the coding region. The vIRF-3 mRNA levels in BCBL-1 cells were increased upon 12-O-tetradecanoylphorbol-13-acetate treatment, with kinetics of expression similar to those of the early immediate genes. The vIRF-3 ORF encodes a 73-kDa protein with homology to cellular IRF-4 and HHV-8-encoded vIRF-2 and K11. In transient transfection assays with the IFNACAT reporter, vIRF-3 functioned as a dominant-negative mutant of both IRF-3 and IRF-7 and inhibited virus-mediated transcriptional activity of the IFNA promoter. Similarly, the overexpression of vIRF-3 in mouse L929 cells resulted in inhibition of virus-mediated synthesis of biologically active interferons. These results suggest that by targeting IRF-3 and IRF-7, which play a critical role in the activation of alpha/beta interferon (IFN) genes, HHV-8 has 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.

FIG. 1

Genomic organization of HHV-8-encoded vIRF-3. (A) Schematic diagram of the 83- to 95-kb region of the HHV-8 genome (GenBank accession no. U93872) showing the cluster of ORFs with homology to cellular IRFs. Diagram of vIRF-3 ORF is shown below. (B) Map of the vIRF-3 ORF. A putative CCAAT box, TATA box, AP-1 binding sites, and poly(A) signals are boxed and in boldface. The nucleotide sequence corresponding to the intron is boxed; the splice donor (GT…) and splice acceptor (…AG) sites are in boldface. The primers (V3A, V3B, V3C, V3D, V3E, V3F, V3G, and V3H) used for RT-PCR analyses are underlined. The arrows indicate the orientations of the primers.

FIG. 2

Expression of the vIRF-3 gene in BCBL-1 cells. (A) Northern blot analysis of total RNA isolated from BCBL-1 cells treated with TPA (50 ng/ml) for 0, 2, 8, and 24 h. The vIRF-3-specific transcript was detected by using K10.5 ORF cDNA as a probe. The levels of β-actin mRNA at different times postinduction are shown for comparison. (B) Expression of the sense strand of vIRF-3 ORF. Total RNA was isolated from BCBL-1 cells treated with TPA for 24 h and was reverse transcribed (RT+). The primer (V3B) used for cDNA synthesis was complementary to the sense strand at the 3′ end of the vIRF-3 ORF. The cDNA was amplified by PCR using the primers V3A and V3B. The RT-PCR reaction in the absence of reverse transcriptase (RT−) and PCR amplification of genomic vIRF-3 ORF (Genomic DNA) were used as controls. (C) RT-PCR was carried out with RNA extracted from uninduced or TPA-induced (24 h) BCBL-1 cells; extracted DNA served as a control against detection of genomic DNA. Amplification of HHV-8 genomic DNA yielded a 440-nucleotide fragment (lane 5). However, a fragment of only 346-nucleotides was amplified by RT-PCR from RNA extracted from TPA-treated and untreated BCBL-1 cells (lanes 1 and 3). The RT-PCR reactions in the absence of reverse transcriptase (RT−) were used as controls (lanes 2 and 4). Schematic representation of the primers used in the assay is shown in the lower panel. (D) RT-PCR analysis of RNA extracted from BCBL-1 cells after 24 h of TPA treatment. Total RNA was reverse transcribed by using oligo(dT)_12–18 primers and PCR amplified with primers (V3E and V3D, and V3C and V3F) located in the 5′ and 3′ untranslated regions. PCR amplification of BCBL-1 DNA served as a control.

FIG. 3

Quantitative RT-PCR analysis of the vIRF-3 ORF. Total RNA isolated from BCBL-1 cells at different time points of TPA treatment (0, 2, 4, 8, 16, and 24 h) was reverse transcribed by using oligo(dT)_12–18 primers and was subsequently PCR amplified with primers complementary to the 5′ and 3′ regions of the vIRF-3 ORF. Products were visualized by agarose gel electrophoresis (upper panel). The middle panel represents the Southern blot of RT-PCR products diluted 25 times and probed with vIRF-3 cDNA. The levels of GAPDH expression are shown for comparison (lower panel). Kinetics of vIRF-3 transcript expression is summarized in the graph.

FIG. 4

(A) Multiple alignment of HHV-8-encoded IRF homologues. The alignment was constructed of vIRF-3 (accession no. AF157602), vIRF-2 (accession no. AF045550), and K11 (accession no. U93872) amino acid sequences by using CLUSTAL W software. Identical and homologous residues are shaded in black and gray, respectively. (B) Alignment of homologous regions between vIRF-3 and IRF-4 (accession no. U52682). The asterisks below the sequence indicate the residues conserved in the IRF association domain of most cellular IRFs.

FIG. 5

Analysis of vIRF-3 protein synthesized in vivo and in vitro. (A) Schematic diagram of vIRF-3 deletion constructs. (B) Western blot analysis of cell lysates obtained from HeLa cells transfected with FLAG-tagged vIRF-3 expression constructs. To generate the vIRF-3-FLAG, vIRF-3-N′-FLAG, and vIRF-3-C′-FLAG constructs, the vIRF-3 ORF was amplified by RT-PCR from RNA of TPA-induced (24 h) BCBL-1 cells using primers V3A and V3B, V3A and V3H, and V3G and V3B, respectively (see Fig. 1B for position of primers). The primers V3A and V3G contained an EcoRI restriction site, and primers V3B and V3H contained a BamHi restriction site and a FLAG epitope (DYKDDDDK). The amplified products were digested and inserted into pcDNA3.1(+) (Invitrogen). To construct vIRF-3 genomic expression vectors, the viral DNA from HHV-8-harboring BCBL-1 cells was used as a template for PCR amplifications, with primers V3A and V3B containing a FLAG epitope on either the 5′ or 3′ end. The amplified products were digested with EcoRI and BamHI and inserted into pcDNA3.1(+) vector. Transfection of vIRF-3 cDNA and vIRF-3 genomic constructs containing the intron sequence (Genom-5′-FLAG and Genom-3′-FLAG) yielded proteins of the same size of approximately 73 kDa (lanes 1, 4, and 5). The sizes of N- and C-terminal parts of vIRF-3 were 35 and 38 kDa, respectively. (C) The vIRF-3, vIRF-3-N′, and vIRF-3-C′ proteins were synthesized and labeled with [³⁵S]methionine in vitro, by using the coupled transcription-translation system (Promega, Madison, Wis.). The vIRF-3 protein migrated at approximately 73 kDa. The sizes of in vitro-translated N- and C-terminal parts of vIRF-3 were similar to those expressed in vivo.

FIG. 6

Functional analysis of vIRF-3. (A) vIRF-3 protein inhibits IRF-3- and IRF-7-mediated activation of the IFNA4 promoter. The IFNA4CAT reporter plasmid (1 μg) was cotransfected with either empty vector or vectors expressing cellular IRFs (IRF-3 or IRF-7) (1 μg) and vIRF-3 (3 μg). The β-galactosidase-expressing plasmid (0.1 μg) was included as an internal standard. When indicated, 24 h after transfection, cells were infected with Sendai virus (SV) (multiplicity of infection = 5) for 16 h. The cells were harvested for CAT assay 40 h after transfection. (B) The synergistic activation of the IFNA4 promoter by Sendai virus and cellular IRF-3 or IRF-7 was inhibited by both the C-terminal and N-terminal portions of vIRF-3 protein. Transfection of genomic vIRF-3 construct (Genom-5′-FLAG) had the same inhibitory effect as vIRF-3 cDNA. Con represents cells transfected with the IFNA4CAT construct in the absence of virus infection. Error bars show standard errors for triplicate experiments.