Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor

Abstract

Interferons (IFNs) are a family of multifunctional cytokines with antiviral activities. The K9 open reading frame of Kaposi's sarcoma-associated herpesvirus (KSHV) exhibits significant homology with cellular IFN regulatory factors (IRFs). We have investigated the functional consequence of K9 expression in IFN-mediated signal transduction. Expression of K9 dramatically repressed transcriptional activation induced by IFN-alpha, -beta, and -gamma. Further, it induced transformation of NIH 3T3 cells, resulting in morphologic changes, focus formation, and growth in reduced-serum conditions. The expression of antisense K9 in KSHV-infected BCBL-1 cells consistently increased IFN-mediated transcriptional activation but drastically decreased the expression of certain KSHV genes. Thus, the K9 gene of KSHV encodes the first virus-encoded IRF (v-IRF) which functions as a repressor for cellular IFN-mediated signal transduction. In addition, v-IRF likely plays an important role in regulating KSHV gene expression. These results suggest that KSHV employs an unique mechanism to antagonize IFN-mediated antiviral activity by harboring a functional v-IRF.

FIG. 1

(A) Identification of KSHV K9 protein. Lysates from BJAB cells (lane 1), BC-1 cells (lane 2), BCBL-1 cells (lane 3), 293 cells transfected with the pFJ vector (lane 4), and 293 cells transfected with pFJ-K9 (lane 5) were used for immunoblot assay with anti-K9 antibody. (B) Construction of the NIH 3T3-K9 cell line. After selection with puromycin, lysates of NIH 3T3-babe cells (lane 1) and NIH 3T3-K9 cells (lane 2) were immunoblotted with anti-K9 antibody. Arrows indicate the K9 protein. Sizes are indicated in kilodaltons.

FIG. 2

Expression of K9 represses IFN-mediated induction of GAS, GBP, and ISG15 promoter activity. NIH 3T3-babe (▧) and NIH 3T3-K9 (░⃞) cells were cotransfected with GBP-ISRE-luc (A), GAS-luc or GAS_mt-luc (B), or ISG15-ISRE-luc (C) together with pGKβgal, which expresses β-galactosidase from a phosphoglucokinase promoter. After transfection, cells were cultured in the presence or absence of mouse IFN-α/β or IFN-γ for 48 h. Assays for luciferase or β-galactosidase activity were performed as described in Materials and Methods. Values represent the average of three independent experiments.

FIG. 3

Transformation of NIH 3T3 cells by the KSHV K9 gene. Puromycin-resistant cells were obtained after transfection with the retroviral vector pBabe-puro or pBabe-K1. Puromycin-resistant cells were plated at 10⁶ cells per 100-mm-diameter tissue culture dish. Preconfluent puromycin-resistant cells were photographed at a magnification of ×100 (A). After 14 days incubation, cells were photographed to show morphologic transformation at ×40 (B). For serum dependence (C), 10⁶ cells were placed in 100-mm-diameter tissue culture dish, maintained in 0.1% serum for 14 days, and photographed at ×40.

FIG. 4

Decreased K9 expression in BCBL-1/anti-K9 cells. Cell lysates were used for immunoblot assay with the anti-K9 antibody. Lane 1, control BJAB cells; lane 2, BCBL-1 cells; lane 3, BCBL-1/anti-K9 cells with tetracycline; lane 4, BCBL-1/anti-K9 cells without tetracycline. The arrow indicates K9 protein. Sizes are indicated in kilodaltons.

FIG. 5

Increased level of IFN-mediated transcriptional activity by decreased K9 expression. BCBL-1 (▧) and BCBL-1/anti-K9 (░⃞) cells were electroporated with GAS-luc (A) or ISG15-ISRE-luc (B) together with pGKβgal. After electroporation, cells were incubated with or without human IFN-β or IFN-γ for 48 h. Assays for luciferase or β-galactosidase activity were performed as described in Materials and Methods. Values represent the average of three independent experiments.

FIG. 6

Downregulation of KSHV gene expressions in BCBL-1/anti-K9 cells. (A) Immunoblot assay of KSHV proteins. Lysates from BCBL-1 cells (lane 1) and BCBL-1/anti-K9 cells (lane 2) were immunoblotted with KS-negative normal human serum (a), KS-positive human serum 19 (b), KS-positive human serum 20 (c), or rabbit anti-vIL-6 antibody (d). Arrows indicate locations of the specific proteins. Sizes are indicated in kilodaltons. (B) Northern blot analysis of KSHV genes. Total RNA from BCBL-1 cells (lane 1) and BCBL-1/anti-K9 cells (lane 2) was separated on a 1% agarose gel, transferred to nitrocellulose, and hybridized with ³²P-labeled PAN/T1.1, vIL-6, K8, sVCA, or cellular actin. Arrows indicate the location of the specific transcripts. (C) Southern blot analysis of KSHV genes. Purified genomic DNA of BCBL-1 (lane 1) or BCBL-1/anti-K9 cells (lane 2) was digested with restriction enzyme PstI. Labeled vIL-6 or orf73 probe was used for the hybridization. The vIL-6-positive band is expected to be a 6.3-kb DNA fragment, and the orf73-positive band is expected to be a 1.5-kb DNA fragment. Arrows indicate locations of the vIL-6- and orf73-specific bands. Sizes are indicated in kilobases.

FIG. 7

Reduced vIL-6 expression in BCBL-1 cells containing the vIL-6 antisense sequence. (A) Genetic organization of LXSG and LXSG-anti-vIL-6. LTR, long terminal repeat; SV40, simian virus 40. (B) FACS analysis of BCBL-1 cells electroporated with either LXSG or LXSG-anti-vIL-6. a, BCBL-1 (M1 < 0.5%); b, BCBL/LXSG (M1 = 15%); c, BCBL-1/LXSG-anti-vIL-6 (M1 = 35%). M1 indicates the gated cells for the green fluorescence. (C) Immunoblot assays. The same amounts of proteins from the sorted cells were used for immunoblot assay with anti-vIL-6 antibody (a), anti-K9 antibody (b), and human KS serum (c). Lane 1, BCBL-1/LXSG; lane 2, BCBL-1/LXSG-anti-vIL-6. Sizes are indicated in kilodaltons.