Kaposi's sarcoma-associated herpesvirus latent and lytic gene expression as revealed by DNA arrays

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is associated with three human tumors, Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. KSHV encodes a number of homologs of cellular proteins involved in the cell cycle, signal transduction, and modulation of the host immune response. Of the virus complement of over 85 open reading frames (ORFs), the expression of only a minority has been characterized individually. We have constructed a nylon membrane-based DNA array which allows the expression of almost every ORF of KSHV to be measured simultaneously. A PEL-derived cell line, BC-3, was used to study the expression of KSHV during latency and after the induction of lytic replication. Cluster analysis, which arranges genes according to their expression profile, revealed a correlation between expression and assigned gene function that is consistent with the known stages of the herpesvirus life cycle. Furthermore, latent and lytic genes thought to be functionally related cluster into groups. The correlation between gene expression and function also infers possible roles for KSHV genes yet to be characterized.

FIG. 1

(A) Quantitation of results from two independent uninduced samples. The data from the two arrays were normalized using a subset of the cellular genes. The signal variation between the two samples is shown by the error bar for each element which represents the mean value. KSHV genes are indicated by the open bar and are ordered colinearly with the genome from 5′ to 3′ as indicated. Cellular genes are indicated by a solid bar and are separated from viral genes by a vertical line. The signal from the majority of array elements forms a baseline, while the expression of a few genes is detected at levels significantly above this (labeled). MDC, Molecular Dynamics counts. (B) Quantitation of the results for duplicate samples taken at 24 h after TPA induction. The chart is in the same format as panel A. Array elements showing strong signals are labeled. (C) Scatter plot comparing the results from panels A and B. The values plotted represent the means from duplicate experiments. KSHV genes are indicated by open circles; cellular genes are indicated by filled circles. Identities of the points representing those labeled in panels A and B are shown.

FIG. 2

(A) Hierarchical clustering of gene expression data. Each row represents a separate amplicon on the array; each column represents the results from one array, hybridized with the sample detailed above. Columns 2 to 8 (−TPA) show untreated cells at successive time points shown in hours; lanes 9 to 21 (+TPA) show time points after the induction of lytic replication with TPA. Two independent experiments were conducted for the 24-, 34-, 48-, and 72-h time points for TPA induction. Column 1 (No KSHV) shows the array results from Ramos cell RNA, a KSHV-negative B-cell line. Levels of expression are relative to double the median level of expression for all genes averaged over all uninduced samples. The magnitude of this ratio is color coded according to the scale; shades of red signify detectable expression, and shades of green illustrate expression below the baseline level. The dendrogram on the left represents similarities of patterns of gene expression. The branch colored green is discussed in the text. (B) Expanded view of the uppermost cluster (red in panel A), which represents genes whose expression is detectable in uninduced cells. (C) Fold increase in expression at each time point (relative to time zero) after the induction of lytic replication with TPA. The genes form the cluster shown in panel B. The values are relative to the mean of the two time zero samples, with the values for 24, 34, 48, and 72 h being the averages of two experiments.

FIG. 3

Hierarchical clustering of genes and samples after the induction of lytic replication with TPA. The genes are ordered using a self-organizing map algorithm (18). The normalized log expression ratio is color coded according to the scale at the bottom. ORFs and corresponding gene names are listed on the right and color coded according to putative function shown by the key above. The dendrogram on the left represents relatedness of the patterns of gene expression. The three major branches are color coded according to the class of genes they represent and the times at which expression is first detected: primary lytic genes (0 to 10 h), secondary lytic genes (10 to 24 h), and tertiary lytic genes (48 to 72 h). Each column represents a sample taken at different times in hours after TPA induction (labeled above). The dendrogram at the top relates the samples according to the pattern of gene expression.

FIG. 4

(A) Mean patterns of expression of the genes contained in each major branch of the tree shown in Fig. 4. The data shown are identical to those used in the cluster analysis. Values above the line y = 0 are red in Fig. 4; values below are green. The values for 24, 34, 48, and 72 h represent the averages of two experiments. (B) Mean patterns of expression of genes grouped by putative function (Fig. 3). The initial time when significant expression is detected is extrapolated from the point where the lines cross y = 0 and thus become significantly above the baseline expression in uninduced cells. The values for 24, 34, 48, and 72 h represent the averages of two experiments.

FIG. 5

(A) Alignment of DNA binding domains of the human IRFs IRF-4 (GI:2497445) and ICSB-1 (GI:4504567) with those of vIRF-1 (GI:1718311), vIRF-2 (GI:3152729), K10.1, and a novel putative ORF, K10.7. The amino acid sequences for K10.1 and K10.7 were translated from the genomic sequence of KSHV (U75698). The sequence shown for each protein is bounded by the amino acid positions shown on either side. The alignment was performed with ClustalW (46). (B) RT-PCR products showing transcripts encoding vIRF-1 and the putative ORFs K10/10.1, K10.5/10.7, and K11/vIRF-2. The primers used are complementary to either end of the putative long ORFs (see Materials and Methods). RNA was extracted from BC-3 cells 24 h after the addition of TPA. RT-negative controls and positive controls from KSHV genomic DNA are included for each RT-PCR. The RT-PCR product sizes are predicted to be 1,350 (K9), 2,736 (K10/10.1), 1,701 (K10.5/10.7), and 2,043 (K11/vIRF-2) bp. (C) Positions (relative to genomic sequence U75698) and organization of the IRF-related genes of KSHV. The location of the novel putative ORF K10.7 is shown. Secondary lytic genes are shaded in black. The region of each ORF whose translated sequence is shown in panel A is indicated by the grey bars above. Sequenced transcripts shown in panel B are drawn below the corresponding ORFs. Each transcript encodes a predicted protein with full-length homology to known IRFs. The predicted size of the encoded protein is indicated below each transcript. Introns are located between 88343 and 88443 (K10/10.1), 90846 and 90939 (K10.5/10.7), and 93519 and 93639 (K11/vIRF-2). (D) Sequenced transcripts encoding K10/10.1 and K10. The positions of start and stop codons and introns are shown relative to U75698. RT-PCR products corresponding to the two transcripts are shown on the right. The primers used (see Materials and Methods) are labeled (small arrows). RNA templates was taken from BC-3 cells 0 (latent) and 24 (lytic) h after the addition of TPA. The PCR product from KSHV genomic DNA is shown for size comparison.

FIG. 5

(A) Alignment of DNA binding domains of the human IRFs IRF-4 (GI:2497445) and ICSB-1 (GI:4504567) with those of vIRF-1 (GI:1718311), vIRF-2 (GI:3152729), K10.1, and a novel putative ORF, K10.7. The amino acid sequences for K10.1 and K10.7 were translated from the genomic sequence of KSHV (U75698). The sequence shown for each protein is bounded by the amino acid positions shown on either side. The alignment was performed with ClustalW (46). (B) RT-PCR products showing transcripts encoding vIRF-1 and the putative ORFs K10/10.1, K10.5/10.7, and K11/vIRF-2. The primers used are complementary to either end of the putative long ORFs (see Materials and Methods). RNA was extracted from BC-3 cells 24 h after the addition of TPA. RT-negative controls and positive controls from KSHV genomic DNA are included for each RT-PCR. The RT-PCR product sizes are predicted to be 1,350 (K9), 2,736 (K10/10.1), 1,701 (K10.5/10.7), and 2,043 (K11/vIRF-2) bp. (C) Positions (relative to genomic sequence U75698) and organization of the IRF-related genes of KSHV. The location of the novel putative ORF K10.7 is shown. Secondary lytic genes are shaded in black. The region of each ORF whose translated sequence is shown in panel A is indicated by the grey bars above. Sequenced transcripts shown in panel B are drawn below the corresponding ORFs. Each transcript encodes a predicted protein with full-length homology to known IRFs. The predicted size of the encoded protein is indicated below each transcript. Introns are located between 88343 and 88443 (K10/10.1), 90846 and 90939 (K10.5/10.7), and 93519 and 93639 (K11/vIRF-2). (D) Sequenced transcripts encoding K10/10.1 and K10. The positions of start and stop codons and introns are shown relative to U75698. RT-PCR products corresponding to the two transcripts are shown on the right. The primers used (see Materials and Methods) are labeled (small arrows). RNA templates was taken from BC-3 cells 0 (latent) and 24 (lytic) h after the addition of TPA. The PCR product from KSHV genomic DNA is shown for size comparison.

FIG. 6

Map of the KSHV genome (updated from reference 35). Each ORF is color coded according to its expression pattern: latent (class I), latent/lytic (induced by TPA), primary lytic genes, secondary lytic genes, and tertiary lytic genes. ORFs 35, 50, 60, 67.5, and 68 and K10.5 were not analyzed due to cross-hybridization or missing probes.