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. 2003 Apr;77(7):4205-20.
doi: 10.1128/jvi.77.7.4205-4220.2003.

Global changes in Kaposi's sarcoma-associated virus gene expression patterns following expression of a tetracycline-inducible Rta transactivator

Hiroyuki Nakamura  1 , Michael Lu, Yousang Gwack, John Souvlis, Steven L Zeichner, Jae U Jung
Affiliations

Global changes in Kaposi's sarcoma-associated virus gene expression patterns following expression of a tetracycline-inducible Rta transactivator

Hiroyuki Nakamura et al. J Virol. 2003 Apr.

Abstract

An important step in the herpesvirus life cycle is the switch from latency to lytic reactivation. In order to study the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), we developed a gene expression system in KSHV-infected primary effusion lymphoma cells. This system uses Flp-mediated efficient recombination and tetracycline-inducible expression. The Rta transcriptional activator, which acts as a molecular switch for lytic reactivation of KSHV, was efficiently integrated downstream of the Flp recombination target site, and its expression was tightly controlled by tetracycline. Like stimulation with tetradecanoyl phorbol acetate (TPA), the ectopic expression of Rta efficiently induced a complete cycle of viral replication, including a well-ordered program of KSHV gene expression and production of infectious viral progeny. A striking feature of Rta-mediated lytic gene expression was that Rta induced KSHV gene expression in a more powerful and efficient manner than TPA stimulation, indicating that Rta plays a central, leading role in KSHV lytic gene expression. Thus, our streamlined gene expression system provides a novel means not only to study the effects of viral gene products on overall KSHV gene expression and replication, but also to understand the natural viral reactivation process.

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Figures

FIG. 1.
FIG. 1.
Summary of Flp-In/TetR system. (A) Schematic representation of plasmid constructs and positions of primers used for establishment of TREx BCBL1-Rta cells. P1 to P7 are primers, as detailed in Table 1. pFRT/lacZeo contains the FRT site for site-specific recombination downstream of the initiation codon (ATG) and expresses lacZ-Zeocin fusion protein (lacZ-Zeo) driven by the SV40 early promoter. pcDNA6/TR constitutively expresses TetR driven by the CMV promoter and also expresses the blasticidin resistance gene (Blast) driven by the SV40 promoter. pOG44 contains Flp recombinase (FLP), which mediates the FRT site-specific recombination. pcDNA5/FRT/TO-Rta contains the FRT site followed by the hygromycin resistance gene (Hygro), which carries neither its promoter nor its start codon. This vector also contains myc/His-tagged Rta cDNA (Rta) downstream of the CMV promoter and tetracycline operator sequences (CMVp/2xTetO2). (B) Schematic diagram of the establishment of TREx BCBL1-Rta cells. Plasmid vectors and drugs used to establish the system are depicted with blue and red letters, respectively. R, resistant; S, sensitive.
FIG. 2.
FIG. 2.
Survey of FRT site-specific recombination. (A) PCR analysis. Genomic DNAs from BCBL-1 (lanes 1), Flp-In BCBL1 (lanes 2), TREx BCBL1 (lanes 3), TREx BCBL1-Rta (lanes 4), TREx BCBL1-vector (lanes 5), BJAB (lanes 6), and H2O (lanes 7) were used for PCR amplification. PCR primers P1 to P9 are shown in Fig. 1 and Table 1. The arrows indicate appropriate PCR products. M, molecular size marker. (B) β-Galactosidase assay of BCBL-1 (bar 1), Flp-In BCBL1 (bar 2), TREx BCBL1 (bar 3), TREx BCBL1-Rta (bar 4), and TREx BCBL1-vector (bar 5). The values represent averages of three individual experiments, with the error bars showing standard deviations.
FIG. 3.
FIG. 3.
KSHV protein expression in TPA-induced BCBL-1 cells (A) or Dox-induced TREx BCBL1-Rta cells (B). At the indicated times after stimulation with TPA (20 ng/ml) or Dox (1 μg/ml), equal amounts of total proteins were analyzed by immunoblotting with KSHV-specific antibodies. Anti-actin antibody was used to monitor protein amounts.
FIG. 4.
FIG. 4.
Multiprobe RPA of KSHV genes. Total RNAs (30 μg) extracted from TPA-induced BCBL-1 cells and Dox-induced TREx BCBL1-Rta cells at the indicated time points were subjected to RPA. The protected RNA fragments were visualized with a phosphorimager.
FIG. 5.
FIG. 5.
Hierarchical clustering of KSHV gene expression induced by Rta. (A) Color-coded expression scale. Expression of KSHV genes was strongly induced by Rta up to 48-fold by 24 h. (B) Color scale representing the degree of expression based on a calibrated ratio of Dox-stimulated Cy3-dUTP-labeled viral cDNA signal intensity over empty vector control Cy5-dUTP-labeled cDNA signal intensity. A calibrated ratio of 1, represented by black, indicates no change in expression. A calibrated ratio much greater than 1, represented by red, indicates a very strong increase in expression. Note the early increase in expression after 2 h of Rta induction (ORF 50), indicated by the arrowhead. While all genes were strongly overexpressed following Rta induction, it is apparent that certain genes show increases in expression earlier than others. The top scale is log 2 (CalRatio).
FIG. 6.
FIG. 6.
Expression rate profiling of KSHV gene expression induced by Rta. The colored lines represent the kinetic classes defined by the rate or change in expression over change in time (see the text for more detail). The bars represent the percentages of maximal expression, set at 100% (tallest bar), normalized to time zero.
FIG. 7.
FIG. 7.
SOM analysis of KSHV gene expression. SOM was used to analyze KSHV gene expression upon Rta expression. A 2-by-2 SOM grid with 250,000 iterations was used to group the KSHV expression patterns into four groups by similarity (Pearson's correlation). The graphs represent each SOM grouping of gene patterns normalized to the percentage of maximum expression.
FIG. 8.
FIG. 8.
Assays for release of infectious viral progeny. (A) Electron microscopy to detect virion particles. TPA-treated BCBL-1 cells (top) and Dox-treated TREx BCBL1-Rta cells (bottom) were subjected to transmission electron microscopy. Bars, 500 (top) and 200 (bottom) nm. (B) ORF29-specific RT-PCR analysis to examine KSHV infectivity. cDNA synthesis reactions were performed with (+) or without (−) RT and amplified with ORF29 primers. Lanes: M, molecular size markers; 1 and 2, BCBL-1 cells; 3 and 4, 293 cells with mock infection; 5 and 6, 293 cells with the supernatant from uninduced TREx BCBL1-Rta cells; 7 and 8, 293 cells with the supernatant from Dox-induced TREx BCBL1-Rta cells. The arrow indicates the ORF29 cDNA fragment.
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