Transcription program of human herpesvirus 8 (kaposi's sarcoma-associated herpesvirus)

Abstract

Human herpesvirus 8 (HHV-8), a gammaherpesvirus implicated in Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease, encodes several pathogenically important cellular homologs. To define the HHV-8 transcription program, RNA obtained from latently infected body cavity-based lymphoma 1 cells induced to undergo lytic replication was used to query a custom HHV-8 DNA microarray containing nearly every known viral open reading frame. The patterns of viral gene expression offer insights into the replication and pathogenic strategies of HHV-8.

FIG. 1

Microarray images from BCBL-1 cells induced with TPA. Poly(A)⁺ RNA was isolated from TPA-induced and uninduced BCBL-1 cells at 0, 3, 8, 10, 12, 24, 36, 48, 72, and 96 h after induction. Poly(A)⁺ RNA was reverse transcribed into fluorescently labeled cDNA in the presence of Cy3-dUTP (uninduced; pseudocolored green) or Cy5-dUTP (induced; pseudocolored red). The labeled cDNAs were hybridized to an HHV-8 array containing viral ORFs and expressed messages (V regions), as well as a subset of cellular genes (C regions). The arrays were scanned, and pseudocolor images were constructed. The intensities of gene expression are depicted on the color scale below the image. Down-regulated spots appear dark green, greenish-yellow spots indicate no change in expression between untreated and treated samples, and spots corresponding to genes overexpressed during HHV-8 replication appear red. White spots indicate maximal gene expression intensity (saturation) in the Cy5 channel (635 nm), which indicates very high expression of that particular viral gene.

FIG. 2

Hierarchical clustering of HHV-8 gene expression data. Calibrated expression ratios for each gene were cataloged based on the hierarchical clustering program (average-linkage algorithm) in which the temporal expression ratios of genes were compared pairwise and grouped according to their similarity (Pearson's correlation coefficient). Columns indicate separate time points, and every row displays the expression profile of a single ORF. The normalized expression ratios across all the time points are color coded, with green boxes indicating expression ratios lower than the mean and red boxes indicating gene expression greater than the mean. Black boxes indicate an intermediate level of expression. The magnitude of up-regulation from the mean is shown by differing intensities of red, with deep red illustrating lower expression and bright red showing the highest levels of expression. The dendrogram at the left clusters the ORFs based on the relatedness of their gene expression patterns. DHFR, dihydrofolate reductase; BHV4, bovine herpesvirus 4; ssDNA, single-stranded DNA; EBV, Epstein-Barr virus.

FIG. 3

Northern analyses for HHV-8 genes. HHV-8 genes, ORF 17, ORF 57, ORF 59, and ORF K4.2, were selected for Northern analysis to confirm the results obtained using microarrays. The same PCR-amplified fragments used to construct the arrays were labeled via random priming and hybridized to blots made from the same RNA used in the cDNA synthesis step for microarray assays. Data were collected with a PhosphorImager. The expression patterns obtained using the Northern blots are those expected for the genes, with expression for the immediate-early genes ORF K4.2 and ORF 57 peaking approximately 24 to 48 hpi, that for the early lytic gene ORF 59 peaking at 36 hpi, and that for the late lytic gene ORF 17 peaking at 48 hpi. The data were normalized to account for basal gene expression levels by using uninduced BCBL-1 cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 4

HHV-8 map. The expression patterns for HHV-8 genes were placed onto a physical map of the virus (32). ORFs are color coded by function. Graphs are shaded by the time at which a doubling in calibrated ratio is seen compared to time 0 hpi (baseline). The expression levels plotted in the graphs are normalized to the maximum expression (set to equal 100) for each gene.