KSHV persists in KS and PEL tumor cells, which are of endothelial and B-cell lineage, respectively. In latently infected asymptomatic carriers, KSHV has been detected in circulating CD19 B cells (10
). Macrophages have also been shown to harbor KSHV genomes (2
). In ≥90% of infected tumor cells, KSHV is locked into the latent mode of infection and protein expression is restricted to only a few viral genes (21
). Most prominent among them is LANA, which is involved in transcriptional regulation (25
), required for episome maintenance (1
), and associated with viral oncogenesis (16
). LANA mRNA transcription is under the control of LANAp. LANAp is active in the absence of other viral gene products in transient assays and in latent PEL and KS tumor cells in vivo (11
). In contrast to all other KSHV transcription units, neither LANA mRNA nor LANAp is induced upon KSHV lytic reactivation in PEL cells (11
). This implies that LANAp activity is shielded from other transcriptional events on the viral genome but is subject to cellular regulation, which justifies an analysis of LANAp in isolation.
Here we present an in vivo analysis of LANAp’s tissue-specific activity in several independently derived lines of transgenic mice. While overall LANAp activity was low compared to those of murine housekeeping genes (apoB and TBP), it was clearly detectable by quantitative real-time RT-PCR analysis. In the spleen, LANAp-directed transgene expression was consistently observed in CD19-positive B lymphocytes but never in CD3-positive, mature T lymphocytes. In addition to B cells, we also detected a CD19- and CD3-double-negative population which supports LANAp activity. While one expects these to be macrophages or NK cells, we have been unable to obtain an unambiguous answer, since those cells have extremely high endogenous β-galactosidase activity. These data demonstrate that all cis elements, which are sufficient for authentic B-cell-specific LANA expression in vivo, are contained within the proximal 1,299 bp relative to the transcription start site (and the 5′ untranslated region). We cannot exclude the possibility that sequence elements further upstream modulate tissue specificity further. This conclusion could not have been drawn from tissue culture experiments, since cell lines only approximate the assembly of transcription factors, which determines authentic B-cell function, and it is currently not feasible to construct recombinant KSHV strains.
Of the major organs, only the kidney and, to a lesser degree, the liver showed appreciable transgene expression by whole-mount analysis. Other organs (brain and skin) either were consistently negative in every animal we investigated or showed scattered lacZ
-positive cells associated with the blood vessels (heart and muscle) upon closer inspection. In the latter cases, we cannot exclude the possibility that these stem from migrating cells or represent low-level activity in the target organ. Since circulating endothelial-cell precursors are recognized as a source of tissue vasculature (and potentially disseminate KS and KSHV), further experiments are needed to shed light on this issue. To exclude background effects from the analysis, we examined tie-2p-lacZ
transgenic and mock-transgenic mice side by side. In the kidney, LANAp activity localized to the distal and proximal collecting tubules rather than the vasculature, i.e., LANAp activity was observed in kidney epithelial cells. The extent of transgene expression varied, which is consistent with this organ's ontogeny: The convoluted tubes arise from individual nephrogenic mesenchymal stem cells (approximately 1,000 to 2,000), which are induced to vascularize by proximity to the ureteric epithelium and subsequently fuse with it to form individual nephrons. Hence, LANAp activity is subject to varying degrees of induction in kidney development. This result seems at first contradictory, since endothelial lineage cells after extravascularization, rather than mesenchymal cells, constitute the bulk of KS tumor cells. A conservative interpretation of the data would conclude either that the cis
elements required for the endothelial-cell activity of LANAp are not present in the transgene or that LANAp endothelial-cell specificity is mediated by trans
-acting factors which are not evolutionarily conserved. Alternatively, the kidney specificity of LANAp seen in our transgenic-mouse study suggests a new target cell population for KSHV latent persistence. The shedding of KSHV in saliva (33
) and prepubescent transmission in regions of endemicity (4
) argue that epithelial cells should not be excluded as a natural reservoir of this virus.
Based on sequence similarity, several Sp1 binding motifs have been identified in LANAp (11
), some of which bind purified Sp1 in vitro and are essential for LANAp activity (J. H. Jeong and D. P. Dittmer, unpublished data). OCT-1 and GATA sites have also been located in LANAp, and these could mediate promoter activity in B lymphocytes, though their functional significance has not been established. The transgenic mice developed in this study mimic an important aspect of the KSHV replication cycle. They establish the starting point and in vivo validation for in-depth mutagenesis studies, which will identify tissue-specific cellular transcription factors that regulate LANAp activity and thereby LANA, v-cyclin, and v-FLIP protein levels. Conversely, we are using LANAp to direct the expression of other viral oncogenes in an effort to investigate their transforming ability in the relevant tissue context and the appropriate expression level.
Why should KSHV promoter elements respond to murine trans
-activating factors? A great number of transcriptional regulators are conserved between mice and humans. One of the earliest transgenic models, that of the SV40 promoter/enhancer driving large T antigen, exhibited brain-specific expression, thus mirroring the observed tropisms of SV40 and human papovaviruses (BC virus and JC virus) for kidney and neuronal cells, respectively (5
). This provided a model for JC virus- and SV40-associated malignant mesotheliomas. In contrast, alpha- and betaherpesvirus regulatory elements have proven to have less tissue specificity in transgenic mice. The fundamental importance of tissue specificity for gammaherpesviruses was recognized early on, as these viruses were grouped together based on their tissue tropism prior to any sequence analysis.
Finally, the tissue specificity required for long-term LANAp activity is consistent with the observed loss of KSHV episomes from explanted KS tumor material (17
) or infected primary endothelial cell cultures. Our experiments show that LANAp activity is tissue specific rather than constitutive. As primary KS cells are selected for long-term growth in culture, they generally dedifferentiate. Presumably, LANAp activity cannot be maintained, LANA protein expression ceases, and the KSHV episome is lost during later cell division cycles. Consistent with this model, primary cells whose life is extended by human papillomavirus E6/E7 or ectopic telomerase expression maintain a more differentiated state and support extended LANAp activity and KSHV maintenance (31
). This suggests a scenario in which KSHV entry into cells is minimally restricted, but the tissue-specific activity of LANAp ensures latent KSHV persistence only in specialized cell lineages.