Efficient Infection of a Human B Cell Line with Cell-Free Kaposi's Sarcoma-Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is causatively linked to two B cell lymphoproliferative disorders, multicentric Castleman's disease and primary effusion lymphoma. Latently infected B cells are a major KSHV reservoir, and virus activation from tonsillar B cells can result in salivary shedding and virus transmission. Paradoxically, human B cells (primary and continuous) are notoriously refractory to infection, thus posing a major obstacle to the study of KSHV in this cell type. By performing a strategic search of human B cell lymphoma lines, we found that MC116 cells were efficiently infected by cell-free KSHV. Upon exposure to recombinant KSHV. 219, enhanced green fluorescent protein reporter expression was detected in 17 to 20% of MC116 cells. Latent-phase transcription and protein synthesis were detected by reverse transcription-PCR and detection of latency- associated nuclear antigen expression, respectively, in cell lysates and individual cells. Selection based on the puromycin resistance gene in KSHV. 219 yielded cultures with all cells infected. After repeated passaging of the selected KSHV-infected cells without puromycin, latent KSHV was maintained in a small fraction of cells. Infected MC116 cells could be induced into lytic phase with histone deacetylase inhibitors, as is known for latently infected non-B cell lines, and also selectively by the B cell-specific pathway involving B cell receptor cross-linking. Lytic-phase transition was documented by red fluorescent protein reporter expression, late structural glycoprotein (K8.1A, gH) detection, and infectious KSHV production. MC116 cells were CD27(-)/CD10(+), characteristic of transitional B cells. These findings represent an important step in the establishment of an efficient continuous B cell line model to study the biologically relevant steps of KSHV infection.