Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 88 (4), 2183-94

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Targets Expression of Cellular IRF4 and the Myc Gene to Facilitate Lytic Replication

Affiliations

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Targets Expression of Cellular IRF4 and the Myc Gene to Facilitate Lytic Replication

Hye-Ra Lee et al. J Virol.

Abstract

Besides an essential transcriptional factor for B cell development and function, cellular interferon regulatory factor 4 (c-IRF4) directly regulates expression of the c-Myc gene, which is not only associated with various B cell lymphomas but also required for herpesvirus latency and pathogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma and primary effusion lymphoma, has developed a unique mechanism to deregulate host antiviral innate immunity and growth control by incorporating four viral homologs (vIRF1 to -4) of cellular IRFs into its genome. Previous studies have shown that several KSHV latent proteins, including vIRF3, vFLIP, and LANA, target the expression, function, and stability of c-Myc to establish and maintain viral latency. Here we report that the KSHV vIRF4 lytic protein robustly suppresses expression of c-IRF4 and c-Myc, reshaping host gene expression profiles to facilitate viral lytic replication. Genomewide gene expression analysis revealed that KSHV vIRF4 grossly affects host gene expression by upregulating and downregulating 118 genes and 166 genes, respectively, by at least 2-fold. Remarkably, vIRF4 suppressed c-Myc expression by 11-fold, which was directed primarily by the deregulation of c-IRF4 expression. Real-time quantitative PCR (RT-qPCR), single-molecule in situ hybridization, and chromatin immunoprecipitation assays showed that vIRF4 not only reduces c-IRF4 expression but also competes with c-IRF4 for binding to the specific promoter region of the c-Myc gene, resulting in drastic suppression of c-Myc expression. Consequently, the loss of vIRF4 function in the suppression of c-IRF4 and c-Myc expression ultimately led to a reduction of KSHV lytic replication capacity. These results indicate that the KSHV vIRF4 lytic protein comprehensively targets the expression and function of c-IRF4 to downregulate c-Myc expression, generating a favorable environment for viral lytic replication. Finally, this study further reinforces the important role of the c-Myc gene in KSHV lytic replication and latency.

Figures

FIG 1
FIG 1
vIRF4 robustly downregulates c-Myc gene expression in KSHV-infected PEL cells. (A) Genomewide analysis of vIRF4 effects on host gene expression. The heat map represents expression profiles of cellular mRNAs upon vIRF4 expression and represents averages for two independent experiments. The values were normalized, clustered on the basis of a Euclidean metric, and represented on a log2 scale. (B) Validation of microarray data by RT-PCR. The blots show the results of RT-PCR assays of c-Myc, Survivin, SIAH, β-catenin, and GAPDH expression in TRExBCBL-1 pcDNA or TRExBCBL-1 vIRF4/Au cells upon Doxy (1 μg/ml) treatment. (C) vIRF4-mediated suppression of c-Myc expression in a time-dependent manner. TRExBCBL-1 vIRF4/Au cells were treated with Doxy (1 μg/ml) for the indicated times, and the c-Myc, Survivn, and β-catenin mRNA levels were analyzed by RT-qPCR. (D) c-Myc expression in Doxy-induced TRExBCBL-1 vIRF4/Au cells. Upon Doxy (1 μg/ml) stimulation, equal amounts of total proteins were analyzed by immunoblotting (IB) with an anti-Myc antibody or anti-β-catenin antibody. Anti-tubulin and anti-Au antibodies were used to monitor protein amounts and vIRF4 expression, respectively.
FIG 2
FIG 2
vIRF4 downregulates c-Myc gene expression in a c-IRF4-dependent manner. (A) Effect of vIRF4 on expression of c-Myc and c-IRF4 in H929 cells. H929 cells were transduced by a lentivirus carrying either GFP (control) or vIRF4/Au. At 3 days postinfection, cell lysates were used for either IB with anti-Au antibody or RT-qPCR analyses with c-Myc-, c-IRF4-, and 18S specific primers. (B and C) Effects of vIRF4 on expression of c-Myc and c-IRF4 in BJAB (B) and 293T (C) cells. Twenty-four hours after treatment of TRExBJAB pcDNA and TRExBJAB vIRF4/Au cells (B) or TREx293T pcDNA and TREx293T vIRF4/Au cells (C) with Doxy, equal amounts of cell lysates were used for RT-qPCR and IB, as indicated. (D) c-Myc and c-IRF4 expression kinetics upon vIRF4 expression. (Left) TRExBCBL-1 vIRF4/Au cells were induced with Doxy treatment for the indicated times, and equal amounts of total RNAs were analyzed by RT-qPCR. (Right) TRExBCBL-1 vIRF4/Au cells were transduced by a lentivirus carrying c-IRF4/Flag and incubated for 48 h, followed by Doxy stimulation. Cells were harvested at the indicated times and used for RT-qPCR analysis.
FIG 3
FIG 3
vIRF4-mediated reduction of c-IRF4 and c-Myc mRNAs at the single-cell level. (A) c-Myc, c-IRF4, and vIRF4 mRNAs were individually codetected in TRExBCBL-1 pcDNA and TRExBCBL-1 vIRF4/Au cells with or without Doxy treatment. Cell boundaries are indicated by dashed lines. (B) Average numbers of c-Myc, c-IRF4, and vIRF4 transcripts (± standard deviations [SD]) in TRExBCBL-1 pcDNA and TRExBCBL-1 vIRF4/Au cells with or without Doxy treatment. (C) Scatter plots of numbers of c-Myc versus vIRF4 and c-IRF4 versus vIRF4 transcripts in individual TRExBCBL-1 vIRF4 cells upon Doxy treatment (N, sample size; ρ, Spearman's correlation coefficient). (D) Pairwise Spearman's correlation coefficients for c-Myc versus c-IRF4 in TRExBCBL-1 pcDNA and TRExBCBL-1 vIRF4/Au cells with or without Doxy treatment.
FIG 4
FIG 4
vIRF4 DNA-binding domain is necessary to suppress c-Myc gene expression. (A) vIRF4ΔDBD mutant fails to suppress c-Myc expression. Twenty-four hours after treatment with Doxy (1 μg/ml), TRExBCBL-1 pcDNA, TRExBCBL-1 vIRF4/Au, and TRExBCBL-1 vIRF4ΔDBD/Au cell lysates were used for RT-qPCR (left) and IB (right). (B) vIRF4ΔDBD mutant fails to suppress c-IRF4 expression. TRExBCBL-1 vIRF4/Au and TRExBCBL-1 vIRF4ΔDBD/Au cells were stimulated with Doxy (1 μg/ml) for 24 h and then used for RT-qPCR analysis.
FIG 5
FIG 5
vIRF4 competes with c-IRF4 for binding to the c-Myc promoter region. TRExBCBL-1 vIRF4/Au and TRExBCBL-1 vIRF4ΔDBD/Au cells were mock treated or treated with Doxy for 24 h. Au, c-IRF4, and histone 3 (H3) antibodies were used for ChIP assay, and ChIP DNAs were subjected to RT-qPCRs using primers for the c-Myc promoter regions.
FIG 6
FIG 6
The vIRF4-mediated downregulation of c-Myc expression contributes to efficient KSHV lytic replication. (Left) TRExBCBL-1 pcDNA, TRExBCBL-1 vIRF4/Au, and TRExBCBL-1 vIRF4ΔDBD/Au cells were treated with Doxy (1 μg/ml) for the indicated times, and cell lysates were used for RT-qPCR using specific primers for KSHV genes. (Right) Cell lysates were used for IB with anti-Au, anti-Myc, and anti-tubulin. vec, TRExBCBL-1 pcDNA cell lysates; wt, TRExBCBL-1 vIRF4/Au cell lysates; mut, TRExBCBL-1 vIRF4ΔDBD/Au cell lysates.
FIG 7
FIG 7
vIRF4ΔDBD KSHV mutant shows reduced lytic replication. (A and B) Construction of vIRF4ΔDBD KSHV BAC16. (A) Schematic of vIRF4ΔDBD KSHV BAC16. (B) NheI restriction enzyme digestion of WT, vIRF4ΔDBD, and R-vIRF4ΔDBD BAC16 clones. Lanes M and N indicate the midrange PFGF DNA marker and the 1-kb DNA marker, respectively. (C) WT vIRF4 or vIRF4ΔDBD mutant expression. iSLKWTBAC16, iSLKvIRF4ΔDBDBAC16, and iSLKR-R-vIRF4ΔDBDBAC16 cells were treated with Doxy (1 μg/ml) and 1 mM NaB. At 24 h and 48 h poststimulation, cell extracts were used for either RT-PCRs with four different vIRF4-specific primer pairs (P1 to P4) or IB with anti-vIRF4 antibody. (D) RT-qPCR analysis of KSHV viral gene expression. Twenty-four and 48 h after stimulation of iSLKWTBAC16, iSLKvIRF4ΔDBDBAC16, and iSLKR-R-vIRF4ΔDBDBAC16 cells with Doxy (1 μg/ml) and 1 mM NaB, KSHV gene expression was determined by RT-qPCR. The induction levels of each viral gene were calculated by comparing Doxy- and NaB-treated cells with mock-treated cells. *, P < 0.05; **, P < 0.001. (E) Viral genomic DNA copy numbers. Genomic DNAs were prepared from iSLKWTBAC16, iSLKvIRF4ΔDBDBAC16, and iSLKR-R-vIRF4ΔDBDBAC16 cells either mock treated (0 h; latency) or treated with 1 mM NaB (24 and 48 h). The levels of viral genomic DNA were determined by RT-qPCR and were calculated relative to those of the mock-treated samples (0 h; latency).

Similar articles

See all similar articles

Cited by 13 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback