Charting Latency Transcripts in Kaposi's Sarcoma-Associated Herpesvirus by Whole-Genome Real-Time Quantitative PCR

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

The latency-associated nuclear antigen of Kaposi sarcoma–associated herpesvirus induces B cell hyperplasia and lymphoma

Kaposi sarcoma–associated herpesvirus (KSHV) is a human lymphotropic herpesvirus. It is implicated in B cell neoplasias such as primary effusion lymphoma and multicentric Castleman disease in AIDS patients. The KSHV latency-associated nuclear antigen (LANA) is consistently expressed in all KSHV-associated tumor cells and was shown to bind the tumor suppressor proteins p53 and pRb. To test LANA’s contribution to lymphomagenesis in vivo we generated transgenic mice expressing LANA under the control of its own promoter, which is B cell specific. All of the transgenic mice developed splenic follicular hyperplasia due to an expansion of IgM(+)IgD(+) B cells and showed increased germinal center formation. We also observed lymphomas, implying that LANA can activate B cells and provide the first step toward lymphomagenesis

The Viral Latency-Associated Nuclear Antigen Augments the B-Cell Response to Antigen In Vivo▿

Gammaherpesviruses, including Kaposi sarcoma-associated herpesvirus (KSHV), establish latency in B cells. We hypothesized that the KSHV latency-associated nuclear antigen (LANA/orf73) provides a selective advantage to infected B cells by driving proliferation in response to antigen. To test this, we used LANA B-cell transgenic mice. Eight days after immunization with antigen without adjuvant, LANA mice had significantly more activated germinal center (GC) B cells (CD19+ PNA+ CD71+) than controls. This was dependent upon B-cell receptor since LANA did not restore the GC defect of CD19 knockout mice. However, LANA was able to restore the marginal zone defect in CD19 knockout mice

Fusion between hematopoietic and epithelial cells in adult human intestine.

Following transplantation of hematopoietic lineage cells, genetic markers unique to the transplanted cells have been detected in non-hematopoietic recipient cells of human liver, vascular endothelium, intestinal epithelium and brain. The underlying mechanisms by which this occurs are unclear. Evidence from mice suggests it is due in part to fusion between cells of hematopoietic and non-hematopoietic origins; however, direct evidence for this in humans is scant. Here, by quantitative and statistical analysis of X- and Y-chromosome numbers in epithelial and non-epithelial intestinal cells from gender-mismatched hematopoietic cell transplant patients, we provide evidence that transplanted cells of the hematopoietic lineage incorporate into human intestinal epithelium through cell fusion. This is the first definitive identification of cell fusion between hematopoietic cells and any epithelial cell type in humans, and provides the basis for further understanding the physiological and potential pathological consequences of cell fusion in humans

Long-Term-Infected Telomerase-Immortalized Endothelial Cells: a Model for Kaposi's Sarcoma-Associated Herpesvirus Latency In Vitro and In Vivo

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. Most KS tumor cells are latently infected with KSHV and are of endothelial origin. While PEL-derived cell lines maintain KSHV indefinitely, all KS tumor-derived cells to date have lost viral genomes upon ex vivo cultivation. To study KSHV latency and tumorigenesis in endothelial cells, we generated telomerase-immortalized human umbilical vein endothelial (TIVE) cells. TIVE cells express all KSHV latent genes 48 h postinfection, and productive lytic replication could be induced by RTA/Orf50. Similar to prior models, infected cultures gradually lost viral episomes. However, we also obtained, for the first time, two endothelial cell lines in which KSHV episomes were maintained indefinitely in the absence of selection. Long-term KSHV maintenance correlated with loss of reactivation in response to RTA/Orf50 and complete oncogenic transformation. Long-term-infected TIVE cells (LTC) grew in soft agar and proliferated under reduced-serum conditions. LTC, but not parental TIVE cells, formed tumors in nude mice. These tumors expressed high levels of the latency-associated nuclear antigen (LANA) and expressed lymphatic endothelial specific antigens as found in KS (LYVE-1). Furthermore, host genes, like those encoding interleukin 6, vascular endothelial growth factor, and basic fibroblast growth factor, known to be highly expressed in KS lesions were also induced in LTC-derived tumors. KSHV-infected LTCs represent the first xenograft model for KS and should be of use to study KS pathogenesis and for the validation of anti-KS drug candidates

Differentiation status of cells with abnormal sex-karyotypes.

<p><b>(</b>A) FABP2/IFABP expression in a control and (B) transplant patient sample with an example of an XXY cell. Brackets indicate differentiated (high Fabp2/Ifabp expression, black brackets) and undifferentiated (low Fabp2/Ifabp expression, gray brackets) regions of epithelium within each sample. (C) Enlarged view of boxed region from <i>B</i>, in an adjacent tissue section stained for Lamin B1.(D) Enlarged view of boxed region from panel C, showing X- (green) and Y- (red) chromosomes and Lamin B1 (white). Arrowhead indicates the same nucleus in panels C and D. Dashed lines indicate boundaries of epithelial and non-epithelial compartments.</p

Frequency of nuclei with 2 or 3 X-chromosomes.

<p>Percent of Y-chromosome-positive nuclei with two or three X-chromosomes, observed in epithelial (epi) and non-epithelial (non-epi) compartments of normal male and male-into-female gender mismatched bone marrow transplant patients (transplant). *, p = 0.0016, Fisher's exact test.</p

Epithelial compartmentalization and sex-karyotyping of intestinal cells.

<p>(A) Hematoxylin and Eosin stained intestinal biopsy; epithelial compartment is labeled. (B) Adjacent tissue section to that from panel A stained for X- (green) and Y- (red) chromosomes and Lamin B1 (white). Arrows indicate Y-chromosome-positive epithelial cells and the arrowhead points to a Y-chromosome-positive non-epithelial cell. Inset shows a sub-region stained for cytokeratin (blue); arrows and arrowhead serve as positional references. (C) Enlarged views of cells indicated in panel B by arrows and arrowhead; sex-karyotype is indicated for each. (D) Independent patient sample also stained for X- (green) and Y- (red) chromosomes and Lamin B1 (white). Arrows indicate Y-chromosome-positive epithelial cells. Dashed lines in all panels indicate boundaries of epithelial and non-epithelial compartments.</p