Murine gamma-herpesvirus immortalization of fetal liver-derived B cells requires both the viral cyclin D homolog and latency-associated nuclear antigen

Abstract

Human gammaherpesviruses are associated with the development of lymphoproliferative diseases and B cell lymphomas, particularly in immunosuppressed hosts. Understanding the molecular mechanisms by which human gammaherpesviruses cause disease is hampered by the lack of convenient small animal models to study them. However, infection of laboratory strains of mice with the rodent virus murine gammaherpesvirus 68 (MHV68) has been useful in gaining insights into how gammaherpesviruses contribute to the genesis and progression of lymphoproliferative lesions. In this report we make the novel observation that MHV68 infection of murine day 15 fetal liver cells results in their immortalization and differentiation into B plasmablasts that can be propagated indefinitely in vitro, and can establish metastasizing lymphomas in mice lacking normal immune competence. The phenotype of the MHV68 immortalized B cell lines is similar to that observed in lymphomas caused by KSHV and resembles the favored phenotype observed during MHV68 infection in vivo. All established cell lines maintained the MHV68 genome, with limited viral gene expression and little or no detectable virus production - although virus reactivation could be induced upon crosslinking surface Ig. Notably, transcription of the genes encoding the MHV68 viral cyclin D homolog (v-cyclin) and the homolog of the KSHV latency-associated nuclear antigen (LANA), both of which are conserved among characterized γ2-herpesviruses, could consistently be detected in the established B cell lines. Furthermore, we show that the v-cyclin and LANA homologs are required for MHV68 immortalization of murine B cells. In contrast the M2 gene, which is unique to MHV68 and plays a role in latency and virus reactivation in vivo, was dispensable for B cell immortalization. This new model of gammaherpesvirus-driven B cell immortalization and differentiation in a small animal model establishes an experimental system for detailed investigation of the role of gammaherpesvirus gene products and host responses in the genesis and progression of gammaherpesvirus-associated lymphomas, and presents a convenient system to evaluate therapeutic modalities.

Figure 1. Generation of MHV68 immortalized fetal liver-derived B cell lines.

(A) Two stages in vitro culture system for establishing MHV68 infected fetal liver-derived B cell lines. (B) Purification of MHV68-YFP infected fetal liver cells by flow cytometry. Shown are representative pre- and post-sort analyses of YFP-expression. (C) Morphology of a representative MHV68-YFP infected culture at 2.5 weeks post-infection, showing extensive clumping of cells and lymphoblastoid phenotype.

Figure 2. Surface phenotyping MHV68 infected cell lines derived from infection of day 15 fetal liver cells.

Cell surface expression was analyzed for individual cell lines derived from single cell cloning by flow cytometry. Day 15 fetal liver cells (FLC d15) and splenocytes harvested from naive C57BL/6 mice (BL6 splenocytes) were used as staining controls.

Figure 3. Analyses of viral genome frequency, viral gene expression and induction of virus reactivation from MHV68 immortalized B cell lines.

(A) The MHV68 genome is stably maintained in the established fetal-liver derived B cell lines. Shown was limiting dilution PCR detection of the MHV68 genome. The intersection of the horizontal and vertical dashed lines depicts the predicted point, based on a Poisson distribution, where every cell harbors viral genome. (B) Semi-quantitative RT-PCR of a panel of viral genes known to be associated with either virus replication and/or latency. cDNA synthesized from total RNA isolated from individual cell lines was serially diluted 1∶1, 1∶5, or 1∶25 prior to PCR amplification. (C) Immunoblot analyses of viral lytic gene expression after treating cells with candidate reactivation stimuli. The cells were stimulated with the indicated reagents for 72 hr, followed by immunoblot detection with either a rabbit anti-MHV68 antiserum, a chicken anti-ORF59 antibody, a rabbit anti-vCyclin antibody or a mouse anti-actin antibody.

Figure 4. The MHV68 cyclin D homolog is required for immortalization of fetal liver-derived B cells.

Fetal liver (FL) cells, isolated from embryos at day 15 of gestation, were infected with wild-type (WT) MHV68-YFP or v-Cyclin.Stop-YFP viruses at an MOI of 10 by spinoculation. (A) Colonies were readily apparent in WT infected FL cell cultures by 2.5 weeks post-infection, but not in v-Cyclin.Stop-infected FL cell cultures. (B and C) The colonies derived from WT-infected FL cells grew rapidly and contained numerous YFP bright cells. In contrast, there was little evidence of cellular proliferation or colony formation in the v-Cyclin.Stop infected FL cell cultures. The images were taken at magnification 200X (A) or 100X (B and C), and both brightfield (top panels) and YFP-fluorescence images (bottom panels) are shown.

Figure 5. Induction of lymphomas by MHV68 immortalized FL cell lines in athymic nude and Rag 2-deficient mice.

(A) Subcutaneous tumors at the site of injection on the rear flank of Nu/J mice (upper panel). Also shown in a representative H&E stained tumor section (lower panel). (B) Representative H&E stained tumor sections of either mock treated or Nu/J mice inoculated intraperitoneally with the SL-1 MHV68 transformed B cell line. Lymphomas were apparent in both the spleen and lymph node in Nu/J mice injected with MHV68 immortalized FL cell lines. (C) Summary of lymphoma induction using the indiciated MHV68 immortalized B cell lines inoculated into athymic nude, Rag 2-deficient, C57Bl/6 or MuMT (B cell-deficient) mice.