The RBP-Jκ Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is tightly linked to at least two lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). However, the development of KSHV-mediated lymphoproliferative disease is not fully understood. Here, we generated two recombinant KSHV viruses deleted for the first RBP-Jκ binding site (RTA _1st) and all three RBP-Jκ binding sites (RTA _all) within the RTA promoter. Our results showed that RTA _1st and RTA _all recombinant viruses possess increased viral latency and a decreased capability for lytic replication in HEK 293 cells, enhancing colony formation and proliferation of infected cells. Furthermore, recombinant RTA _1st and RTA _all viruses showed greater infectivity in human peripheral blood mononuclear cells (PBMCs) relative to wt KSHV. Interestingly, KSHV BAC36 wt, RTA _1st and RTA _all recombinant viruses infected both T and B cells and all three viruses efficiently infected T and B cells in a time-dependent manner early after infection. Also, the capability of both RTA _1st and RTA _all recombinant viruses to infect CD19+ B cells was significantly enhanced. Surprisingly, RTA _1st and RTA _all recombinant viruses showed greater infectivity for CD3+ T cells up to 7 days. Furthermore, studies in Telomerase-immortalized human umbilical vein endothelial (TIVE) cells infected with KSHV corroborated our data that RTA _1st and RTA _all recombinant viruses have enhanced ability to persist in latently infected cells with increased proliferation. These recombinant viruses now provide a model to explore early stages of primary infection in human PBMCs and development of KSHV-associated lymphoproliferative diseases.

Author Summary

Kaposi's sarcoma-associated herpesvirus (KSHV) is tightly linked to at least two lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). The life cycle of KSHV consists of latent and lytic phase. RTA is the master switch for viral lytic replication. In this study, we first show that recombinant viruses deleted for the RBP-Jκ sites within the RTA promoter have a decreased capability for lytic replication, and thus enhanced colony formation and proliferation of infected cells. Interestingly, the recombinant viruses show greater infectivity in human peripheral blood mononuclear cells (PBMCs). The recombinant viruses also infected CD19+ B cells and CD3+ T cells with increased efficiency in a time-dependent manner and now provide a model which can be used to explore the early stages of primary infection in human PBMCs, as well as the development of KSHV-associated lymphoproliferative diseases.