Charting latency transcripts in Kaposi's sarcoma-associated herpesvirus by whole-genome real-time quantitative PCR

Abstract

The division into a latent or lytic life cycle is fundamental to all herpesviridae. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8), latent genes have been implicated in cell autonomous transformation, while certain lytic genes procure a tumor friendly milieu through paracrine mechanism. To query KSHV transcription, we devised and validated a high-throughput, high-specificity, high-sensitivity, real-time quantitative reverse transcription-PCR array. This novel methodology is applicable to many human pathogens. Its first use demonstrated that the mRNA levels for KSHV LANA, v-cyclin, and v-FLIP do not increase at any time after viral reactivation. The mRNA for LANA-2/vIRF-3 is similarly resistant to viral reactivation. In contrast, every other latent or lytic message was induced. Hence, LANA, v-FLIP, v-cyclin, and LANA-2 constitute a group of uniquely regulated transcripts in the KSHV genome.

FIG. 1.

Ethidium bromide-stained 2% agarose gel of the PCR products for each KSHV ORF after 40 cycles. The template was reverse transcribed poly(A) mRNA from BCBL-1 cells 48 h after TPA induction. Most amplicons are of the same size; exceptions are noted by name.

FIG. 2.

Melting curve profile for selected KSHV ORFs after 40 cycles (amplification products for orf26, K9spl, LANA-2spl, actin, KbZIP, orf40spl, K9, LANA-2unspl, v-cyc, orf40unspl, orf57spl, orf57unspl). The template was reverse transcribed poly(A) mRNA from BCBL-1 cells 48 h after TPA induction. The temperature is shown on the horizontal axis. The vertical axis shows the value of the first derivative of the change in fluorescence intensity at each temperature. Thus, the peak identifies the phase transition.

FIG. 3.

(A) C_T values for each primer pair in the KSHV array. Closed circles indicate the C_T values if poly(A) mRNA without RT was used (RT^neg); open circles indicate the C_T values if the mRNA was subjected to RT (RT^pos). The vertical axis indicates the C_T value, and the horizontal axis indicates the percentile of primers. (B) Frequency histogram of the dC_T difference between RT^pos and RT^neg reactions for each primer pair. Superimposed is the expected normal distribution. The relative frequency is shown on the vertical, and the dC_T is shown on the horizontal axis. (C) SD (on the horizontal scale) and mean (n = 3) C_T values for a group of amplicons. The dotted line indicates the mean of means.

FIG. 4.

dC_T values, which were normalized to C_Tgapdh for each primer pair in the KSHV array. The template was poly(A) mRNA from untreated BCBL-1 cells held in culture for 24, 48, and 72 h. The vertical axis indicates the dC_T value, and the horizontal axis indicates the percentile of primers.

FIG. 5.

(A) Plot of the dC_T values (normalized to gapdh) for each primer pair in the KSHV array (circles). The vertical axis indicates the dC_T value from mock-treated BCBL-1 cells, and the horizontal axis indicates the dC_T value from 48-h-TPA-treated BCBL-1 cells. Also shown is the regression line, equation, and R² value through the values for latency type one mRNAs (solid dots). The dotted line traces the values for orfK8.1 under either condition. The “LANA” primer pairs are located either within the LANA ORF or within the LANA 5′UTR Similarly, for LANA-2/vIRF-3 we designed two primer pairs, one per exon. (B) Plot of the difference for each primer pair in the KSHV array between mock- and TPA-treated BCBL-1 cells at 48 h after induction. C_T values were normalized to GAPDH to yield dC_T, which were then compared to yield ddC_T. The vertical axis indicates the ddC_T value, the horizontal axis the percentile of the total distribution. Note that the ddC_T values represent a log transformation of the relative abundance of the target mRNA. A value of ddC_T = 0 indicates identical abundance of the target mRNA under each condition. A positive ddC_T value indicates induction, and a negative ddC_T value indicates suppression. Arrows indicate the values of latency type one genes.

FIG. 6.

Plot of dC_T (normalized to GAPDH) for each primer pair in the KSHV array for TPA-treated BCBL-1 cells at 24, 48, and 72 h after induction as well as for mock-treated cells at 24 h. The vertical axis indicates the dC_T value, and the horizontal axis indicates the percentile of the total distribution. Note that the ddC_T values represent a log transformation of the relative abundance of the target mRNA. The arrows indicate primer pairs for which all three time points, as well as mock-treated cells, exhibit similar dC_T values, i.e., the mRNA did not change upon TPA treatment. Primer pairs which measured mRNAs that did not respond to TPA in a consistent manner are indicated with asterisks.

FIG. 7.

Cluster analysis with dC_T (normalized to GAPDH) values of each primer pair in the KSHV array for TPA-treated BCBL-1 cells at 24, 48, and 72 h after induction, as well for as mock-treated cells at 24 h. Data were median centered by gene and time point. Hence, mRNAs that are underrepresented are in shades of green, and those that are overrepresented are in red.