Array-based transcript profiling and limiting-dilution reverse transcription-PCR analysis identify additional latent genes in Kaposi's sarcoma-associated herpesvirus

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is a B-lymphotropic herpesvirus strongly linked to both lymphoproliferative diseases and Kaposi's sarcoma. The viral latency program of KSHV is central to persistent infection and plays important roles in the pathogenesis of KSHV-related tumors. Up to six polypeptides and 18 microRNAs are known to be expressed in latency, but it is unclear if all major latency genes have been identified. Here, we have employed array-based transcript profiling and limiting-dilution reverse transcription-PCR (RT-PCR) methodologies to explore this issue in several KSHV-infected cell lines. Our results show that RNAs encoding the K1 protein are found at low levels in most latently infected cell lines. The gene encoding v-IL-6 is also expressed as a latent transcript in some contexts. Both genes encode powerful signaling molecules with particular relevance to B cell biology: K1 mimics signaling through the B cell receptor, and v-IL-6 promotes B cell survival. These data resolve earlier controversies about K1 and v-IL-6 expression and indicate that, in addition to core latency genes, some transcripts can be expressed in KSHV latency in a context-dependent manner.

FIG. 1.

SLK.219 cells produce infectious virus. SLK.219 cells were either mock treated or induced to enter lytic replication with 1.2 mM valproic acid (VA) and AdRTA (adenovirus harboring the ORF50 cDNA). Reactivated cells express RFP. Extent of reactivation was measured using fluorescence-activated cell sorting (FACS) analysis. Only VA/AdRTA-treated SLK.219 cells display substantial reactivation. Virus in the supernatants of the mock- and VA/AdRTA-treated SLK.219 cells was concentrated by centrifugation, resuspended in TIME cell medium, and then used to infect TIME cells. The rKSHV.219 virus harbors a constitutively expressed GFP gene. Infected TIME cells are detected by measuring GFP fluorescence after receiving SLK.219-derived virus.

FIG. 2.

KSHV transcriptome analysis during latent infection. KSHV tiling microarray data (ordered by genome position) are displayed for 13,746 unique probes. In each group of arrays (A, B, C), the color bar indicates the fold change relative to the level for the appropriate mock-infected cells. (A) Viral gene expression analysis of uninfected BJAB and uninduced BCBL-1 cells. (B) Viral expression analysis of stably infected SLK.219 cells and the parental SLK cells. (C) Viral expression analysis of stably infected HFF.219 and the parental HFF cells. The asterisk indicates a series of neighboring probes that detect a transcript antisense to ORF K9 that is likely a spurious transcript (see text for details).

FIG. 3.

Detection of latent transcripts by use of serial cell dilution analysis. (A) Schematic of the method for evaluating latent viral gene expression. Uninduced, KSHV-infected cultures often harbor a small subpopulation of spontaneously reactivated cells. When such cultures are serially diluted, large dilutions are likely to generate some purely latently infected group of cells. qRT-PCR can be performed with each dilution to detect lytic and latent genes. Therefore, the ability to detect lytically expressed genes in a dilution series will end before the ability to detect latently expressed genes. (B) The 2-fold cell dilution series was generated on uninduced SLK.219 cells, ranging from dilution 1 (with approximately 1,500 cells) to dilution 10 (with approximately 3 cells). Six viral RNAs were analyzed by qRT-PCR with each dilution, and the results of the analysis are presented as 40 minus the C_T value. Three classic lytic genes are indicated by the blue curves. LANA, a known latently expressed gene, is indicated by the blue curve. The remaining two genes (K1 and v-IL-6) are likely expressed during latent infection, as they are detectable in cell dilutions in which lytic genes are not.

FIG. 4.

Northern analysis of K1 and v-IL-6. Poly(A)-enriched RNAs from indicated cells were resolved by formaldehyde-agarose gel electrophoresis, transferred to nylon membranes, and hybridized to radioactively labeled riboprobes that detect K1 (A) and v-IL-6 (B) transcripts.

FIG. 5.

K1 transcript structure. (A) The K1 transcript structure is based on RT-PCR and 3′ RACE analysis performed in this study and 5′ RACE analysis performed by Bowser et al. (3). (B) The nucleotide positions of important structural elements include the 5′ nucleotide of the transcript (nt 29), the splice donor site (nt 981), the splice acceptor site (nt 2625), the predicted poly(A) signal (nt 2937 to 2942), the site of polyadenylation (nt 2972), and the coding region (nt 105 to 971). All coordinates are based on the KSHV genome sequence (GenBank accession number U75698.1).