CRISPR/RfxCas13d guide RNAs used in this study.

KSHV has not been found to transform human B cells alone. We show now it infects both peripheral and tonsillar B cells inefficiently and cooperates with EBV to co-transform a similar, small fraction of cells from both sources. These cells yield immortalized progeny, one hallmark of transformation, and depend on both viruses for their continued growth. The cells secrete multiple cytokines that support their paracrine growth and express viral genes that mediate expression of these cytokines. The co-transformed cells grow preferentially as tumors in the peritoneal cavities of NSG mice outgrowing B cells transformed only by EBV. The levels of EBV genes expressed in co-transformed cells decrease during growth in vitro and more so during tumor growth in vivo while those of KSHV increase, mirroring that found In Primary Effusion Lymphoma (PEL) derived cell lines. The expression of cellular genes changes too, to reflect that of PEL biopsies. Both KSHV and EBV oncogenes are expressed in the co-transformed cells that regulate the gene signature of PELs, making these co-transformed cells a tractable model with which to understand this unique lymphoma associated with these two tumor viruses. Studies of this model will also illuminate the individual contributions of each virus to the many cancers they cause.