Abortive Lytic Reactivation of KSHV in CBF1/CSL Deficient Human B Cell Lines

Abstract

Since Kaposi's sarcoma associated herpesvirus (KSHV) establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade.

Author Summary

Kaposi's sarcoma associated herpesvirus (KSHV) establishes a life-long persistent infection in B cells, which constitute the viral reservoir for reactivation and production of progeny virus. Viral reactivation is associated with multiple AIDS related malignancies including Kaposi's sarcoma, an endothelial tumor, and two B cell lymphoproliferative malignancies, the primary effusion lymphoma and the multicentric Castleman's disease. CBF1/CSL is a cellular DNA binding protein that can recruit transactivators or repressors to regulatory sites in the viral and cellular genome. The replication and transcription activator (RTA) plays an essential role in the switch between latency and lytic reactivation. RTA can either bind to DNA directly or is recruited to DNA via anchor proteins like CBF1/CSL and activates transcription. In this study we used a novel cell culture model to analyze the contribution of the CBF1/CSL protein to the process of viral reactivation in human B cells. Two isogenic CBF1/CSL proficient or deficient B cell lines were latently infected with recombinant KSHV. Lytic viral gene expression, viral replication and virus production were compared. Our results suggest that viral lytic gene expression is severely attenuated but not abolished at multiple stages before and after the onset of lytic replication while virus production is below detection levels in CBF1/CSL deficient B cells.