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

Jie Lu,Richard Kuo Dzeng,Qiliang Cai,Erle S Robertson,Subhash C Verma,Abhik Saha,Blossom Damania

doi:10.1371/journal.ppat.1002479

Jie Lu, Richard Kuo Dzeng + Show 5 more

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https://doi.org/10.1371/journal.ppat.1002479

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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 (RTA1st) and all three RBP-Jκ binding sites (RTAall) within the RTA promoter. Our results showed that RTA1st and RTAall 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 RTA1st and RTAall viruses showed greater infectivity in human peripheral blood mononuclear cells (PBMCs) relative to wt KSHV. Interestingly, KSHV BAC36 wt, RTA1st and RTAall 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 RTA1st and RTAall recombinant viruses to infect CD19+ B cells was significantly enhanced. Surprisingly, RTA1st and RTAall 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 RTA1st and RTAall 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.

Highlights

Kaposi sarcoma-associated herpesvirus (KSHV, known as human herpesvirus 8 [HHV8]) infection is pivotal to the development of Kaposi sarcoma (KS)
The recombinant viruses infected CD19+ B cells and CD3+ T cells with increased efficiency in a time-dependent manner and provide a model which can be used to explore the early stages of primary infection in human peripheral blood mononuclear cells (PBMCs), as well as the development of Kaposi’s sarcoma-associated herpesvirus (KSHV)-associated lymphoproliferative diseases
The activity of the replication and transcription activator (RTA) promoter was reduced about 40% in the truncations of first RBPJk and all three recombination signal binding protein Jk (RBP-Jk) binding site within RTA promoter compared to the wt promoter [30]

Summary

Introduction

Kaposi sarcoma-associated herpesvirus (KSHV, known as human herpesvirus 8 [HHV8]) infection is pivotal to the development of Kaposi sarcoma (KS). In comparison to lytic cycle replication, fewer genes are expressed in latent infection and a number of these genes are involved in disruption of the cell cycle, and in maintenance of the viral genome. One of those latent genes is Latency-associated nuclear antigen (LANA), encoded by KSHV open reading frame 73 (ORF73), which is critical for persistence of the viral episome and maintenance of latent infection in KSHV infected cells [3]. The replication and transcription activator (RTA) is encoded by KSHV ORF50 and plays an essential role in the control of the lytic replication cycle. MTA), ORF59 (polymerase processivity factor, PF-8), ORF 74 (vGPCR), K2 (vIL-6), K5 (MIR-2), K6 (vMIP-1), K8 (k-bZIP), K9 (vIRF), K12

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