Induction of Kshv Ie Gene Expression

Theoretically, herpesvirus IE genes can be selectively induced in the presence of inhibition of protein synthesis. Since protein synthesis inhibitors, such as cycloheximide, are in general toxic to host cells and cause apoptosis in these cells, a range of cycloheximide concentrations has to be determined in which protein synthesis is completely inhibited but cell viability rate remains high at least for 8-12 h.

1. BC-1 cells are grown in RPMI-1640 medium supplemented with 15% FBS.

2. Cycloheximide is added to BC-1 cells in T75 flasks to the following concentrations: 0, 10, 50, and 100 pg/mL. After 8 or 12 h, the cell viability of each treatment is assessed by trypan blue (0.4%) staining followed by microscopic examination (see Note 1).

3. Protein synthesis inhibition by cycloheximide is determined by [35S]methionine incorporation.

a. BC-1 cells are collected, washed with 1X phosphate-buffered saline (PBS), suspended in methionine-free growth medium (107/mL), and seeded in 24-well plates (5 x 106/well).

b. Cells are incubated with cycloheximide at various concentration (0, 10, 50, and 100 |g/mL) for 30 min at 37°C prior to the addition of [35S]methionine (50 |Ci/well).

c. Protein synthesis inhibition is determined after various periods by comparing the percentage of trichloroacetic acid-insoluble [35S]methionine from cyclo-heximide-treated cells with that from untreated cells (see Note 2).

4. Once the window of cycloheximide dosage that ensures a complete blockade of protein synthesis and maximal cell survival is determined, BC-1 cells are induced for KSHV reactivation in the presence of cycloheximide within the window.

a. Exponentially growing BC-1 cells (106/mL) are induced by treatment with 3 mM sodium butyrate in the presence of 50 |g/mL cycloheximide for 4 h.

b. To ensure a maximal blockade of protein synthesis upon virus reactivation, cells are induced with sodium butyrate 1-4 h after cycloheximide addition.

3.2. Mapping of the Regions That Are Transcriptionally Active in the IE Stage

1. Total RNAs are purified using TRIzol reagent (Gibco-BRL) from 108 BC-1 cells that have been treated with 3 mM sodium butyrate for 4 h in the presence of 50 | g/mL cycloheximide, as well as from the same number of cells that are not treated with sodium butyrate but incubated with cycloheximide for 8 h. Poly(A+) RNAs are purified using the PolyAtract mRNA isolation system (Promega).

2. The double-stranded cDNAs are synthesized using the Universal riboclone cDNA synthesis system (Promega).

3. These cDNAs are labeled using a random priming method with [a-32P]dCTP (Amersham, Arlington Heights, IL).

4. Six overlapping KSHV cosmid DNAs, which represent the whole KSHV genome, are digested with £coRI, BamHI, or both and separated in 0.8% agarose gels (Fig. 1). DNAs are transferred onto a Nytran membrane and probed with 32P-labeled cDNA prepared from sodium butyrate-induced and -uninduced BC-1 cells in the presence of cycloheximide. As illustrated in Fig. 1, a number of bands that are only present in the blot hybridized with the cDNA pool from the induced cells (Fig. 1D) is observed. Examples include the 2.2-kb EcoRI/BamHI fragment in cosmid 1 (22,409-24,637); the 2.6-kb EcoRI/BamHI fragments in cosmids 2 and 3 (66,444-69,094); the 3.1-kb £coRI/BamHI fragment in cosmid 2 (47,518-50,637), and the 3.8-kb £coRI/BamHI fragment in cosmid 3 (69,095-72,888) (Fig. 1D). The transcripts originating from these DNA fragments may correspond to KSHV genes induced by sodium butyrate in the presence of cycloheximide and thus are candidates for the IE mRNAs of KSHV (see Note 3).

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