Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV associated malignancies become manifested in immunocompromised patients with previous history of KSHV infection. KSHV is classified as a gamma-2 herpesvirus and displays a tightly regulated gene expression similar to the other Herpesviridae. KSHV lytic genes are expressed during viral progeny production and destructive release from the host cell. Latent genes, on the other hand, are expressed in the absence of viral reactivation and are commonly the only genes expressed during KSHV induced neoplastic transformation. We hypothesized that KSHV latent genes are a unique cluster of genes that are regulated differently in the KSHV genome and that we would be able to detect their expression by developing a real-time quantitative PCR array. The data confirmed that latency associated nuclear antigen (LANA/ORF73), v-cyclin, v-FLIP, and LANA-2/vIRF-3 are a unique latency cluster in KSHV infected primary effusion cells (PEL) in culture (chapter I). Among KSHV latent genes LANA is the only one that is expressed as a multi-functional protein under the control of its own promoter and that is B cell specific. LANA is the most studied latent protein due to its essential role in viral episome maintenance and its consistent expression in all KSHV-associated tumor cells. In vitro studies confirmed that LANA is able to bind p53 and pRb tumor suppressor proteins as well as glycogen synthase kinase 3 b; however, no animal studies had confirmed the in vivo effects of LANA. We hypothesized that LANA contributes to the transforming potential of KSHV and that a transgenic mouse model for LANA would contribute significantly to understanding the in vivo mechanism of B cell transformation to malignant lymphomas. LANA transgenic mice were developed by pronuclear injection of KSHV LANA DNA under the control of the LANA promoter and the offspring were backcrossed for 6 generations to generate LANA transgenic mice in a C57BL/6 background. LANA transgenic mice were viable and had no reproductive abnormalities. LANA mRNA and protein were detected specifically in the bone marrow and the splenic B cells, which confirmed previous reports of B cell specificity of the LANA promoter. We developed a multicolor flowcytometry assay to look at the mature B cell populations in the spleen and found that LANA induced hyperplasia of mature follicular B cells (IgM super(+)IgD super(+)) and no other B cell populations including MZ, plasma cells, and memory cells were affected. These data were statistically significant to p,0.05. Using immunohistochemistry, we further analyzed the surface characteristics of these hyperplastic IgM super(+)IgD super( +) follicular cells using specific germinal center markers and confirmed that these cells were PNA super(+) activated early germinal center cells that localized to large patches and extra-follicular regions of the spleen. The preneoplastic B cell hyperplasia phenotype in the mice was age-dependent and was more pronounced in older mice. At a low frequency of 11% the LANA transgenic mice (older than 300 days) developed monoclonal splenic B cell tumors. Albeit low, this frequency is about twice the frequency reported for the C57BL/6 strain. These data suggested that LANA contributed to B cell transformation in vivo by inducing hyperplasia, which is a preneoplastic condition that would prime mature B cells to complete neoplastic transformation upon acquiring mutations (Chapter 11). These findings suggested a number of mechanisms for LANA induced B cell hyperplasia: (1) LANA alone could behave like an oncogene that would transform a single B cell to proliferate and cause tumor development, (2) LANA can effect the normal B cell activation and proliferation in the spleen by lowering the threshold of activation and causing follicular B cells to produce an augmented antigenic response to normal environmental antigens, priming a larger number of B cells to be activated (PNA super(+)) and therefore increasing the likelihood of a second site mutation, which would lead to tumor development. If hypothesis (1) is true we would expect to find a monoclonal population of activated proliferating PNA super(+) B cells in the spleen of each transgenic mouse, if hypothesis (2) is true we would expect to find a polyclonal population of PNA super(+) B cells that would have the same surface phenotype as activated PNA super(+) germinal center cells that develop following antigenic stimulation. The data confirmed that monoclonal B cell proliferation was only evident in 11% of the mice that developed splenic tumors at older ages; however, the preneoplastic PNA super(+) activated B cells in the spleen of the LANA transgenic mice were polyclonal. We developed a multicolor flowcytometry assay to look at activated, and germinal center B cells based on their surface phenotype, and found that the LANA transgenic PNA super(+) B cells had the same surface phenotype as the PNA super(+) activated germinal center B cells following primary immunization with a T-cell dependent antigen (NP-KLH). The data also suggested that a percentage of the PNA super(+) early GC B cells in the LANA mice had the potential to complete the germinal center reaction and express late germinal center surface markers (Chapter III). These findings confirmed that LANA is not an oncogene in the traditional sense, however, it can prime mature splenic B cells to produce an augmented response to normal environmental antigens and develop PNA super(+) early germinal center cells in the absence of immunization. Based on these findings we hypothesized that LANA induces B cell proliferation by modulating cellular pathways that are normally only activated following antigen-dependent immune response. The BCR signaling pathway is the most important cellular pathway associated with antigen-induced B cell proliferation and CD19 is the most crucial element of this pathway. We hypothesized that (1) LANA modulates BCR signaling at the transcriptional level distal to CD19, or (2) alternatively augments antigen-dependent BCR signaling and requires CD19 expression. We developed CD19 knock out mice that express LANA, by crossing LANA transgenic mice with available CD19 knock out mice. We compared F2 and eliminated background effects by using F2 CD19 knock out mice that are LANA negative as age-matched littermate controls. We found that LANA did not produce follicular B cell hyperplasia or germinal center development in the CD19 knock out mice, which suggests that LANA indeed modulates the BCR signaling pathway and requires CD19 to induce B cell proliferation (Chapter IV). We also found that LANA partially rescues the MZ B cell defect of the CD19 knock out mice and allowed MZ development in the absence of CD19 expression. This phenotype was very similar to Bcl-2-induced MZ development in CD19 knock out mice that express Bcl-2 ectopically. These findings suggest that LANA activates MZ precursor cells in the CD19-/- mice sufficiently to up-regulate cell survival factors that allow MZ differentiation and colonization. These findings were expected based on the in vitro findings that had previously shown LANA's association with survival genes p53 and pRb, as well as the data in LANA transgenic mice that suggest the absence of PARP from PNA super(+ ) GC foci in a number of LANA transgenic mice.
Oncogenes; Herpesviridae; Germinal centers; Statistical analysis; Kaposi's sarcoma-associated herpesvirus; Differentiation; Cell proliferation; Transcription; Human herpesvirus 8; Mutation; Cell activation; Gene expression; Transformation; Lymphocytes B; Promoters; Infection; Transgenic mice; Lymphocytes T; Animal models; Data processing; Lymphoma; Hyperplasia; Spleen; Tumors; Signal transduction; p53 protein; Cell culture; Immunization; Cell survival; Plasma cells; mRNA; Progeny; CD19 antigen