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. 2012 Jan;8(1):e1002479.
doi: 10.1371/journal.ppat.1002479. Epub 2012 Jan 12.

The RBP-Jκ binding sites within the RTA promoter regulate KSHV latent infection and cell proliferation

Jie Lu  1 , Subhash C Verma, Qiliang Cai, Abhik Saha, Richard Kuo Dzeng, Erle S Robertson
Affiliations

The RBP-Jκ binding sites within the RTA promoter regulate KSHV latent infection and cell proliferation

Jie Lu et al. PLoS Pathog. 2012 Jan.

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of two recombinant KSHV BACmids with deletion of RBP-Jκ sites in the RTA promoter.
(A) Schematic diagram showing generation of BAC RTA1st, a recombinant BAC36 with first RBP-Jκ site deletion in the RTA promoter (B) Schematic diagram showing generation of BAC RTAall, a recombinant BAC 36 with deletion of first, 2nd and 3rd RBP-Jκ site in the RTA promoter. (C) Ethidium bromide-stained gel and southern blots with BAC36 wt (lane 1) and the mutated BACmid, RTA1st (Unfloxed, lane 2) and RTA1st (floxed, lane 3), cleaved with XhoI. (D) PCR analysis for Bac36 wt and RTA1st recombinant virus at the junction of deletion of RBP-Jκ site within RTA promoter. (E) Ethidium bromide-stained gel and southern blots with BAC36 wt (lane 1) and the mutated BACmid, RTAall (Unfloxed, lane 2) and RTAall (floxed, lane 3), cleaved with XhoI. (F) PCR analysis for BAC36 wt and RTA1st recombinant virus at the junction of deletion of RBP-Jκ sites within RTA promoter. DNA fragments were sequenced and confirmed the mutation.
Figure 2
Figure 2. Transfection of 293 cells with BAC36 wt, BAC RTA1st and BAC RTAall DNAs.
(A) Cells were transfected with BAC36 wt, BAC RTA1st and BAC RTAall DNAs. GFP expression levels were monitored by fluorescent microscopy 2 days after transfection, and the transfected cells were split and selected with Hygromycin B. The hygromycin-resistant cells were pooled and passed three to four times. The homogenous population of GFP-positive cells harboring KSHV wild-type and mutant genomes were obtained. (B). Immunofluorescence analysis for LANA in BAC36-293 BAC RTA1st-293 and BAC RTAall-293 cells.
Figure 3
Figure 3. Comparisons of effect on deletion of RBP-Jκ sites within RTA promoter in KSHV lytic life cycle.
(A) Comparison of levels of viral gene expression of BAC36 wt, RTA1st and RTAall recombinant viruses in 293 cells. Western blot for RTA and LANA at 0, 24, 48, 72 hours lytic induction of BAC36 wt, RTA1st and RTAall stable 293 cell lines in the presence of Butyrate acid and TPA. The same blot was also reprobed with anti-GAPDH antibody to ensure equal loading of each sample. RD: relative density. (B) Detection of intracellular KSHV viral genome copies using TR primer in BAC36 wt-293, RTA1st-293 and RTAall-293 cells induced by Butyrate acid and TPA. (C) Detection of extracellular KSHV progeny virion production using TR primer in the supernatant of BAC36 wt-293, RTA1st-293 and RTAall-293 induced by Butyrate acid and TPA. *P<0.05; **P<0.01; ***P<0.001 by Student's t test.
Figure 4
Figure 4. Comparison of cell growth and proliferation for WT, RTA1st and RTAall -293 cells.
(A) BAC36 wt, RTA1st and RTAall transfected 293 cells selected for 3–4 weeks were assayed by FACS analysis based on GFP signals. (B) Relative GFP density in the BAC36 wt, BAC RTA1st and BAC RTAall transformed 293 cells. (C) BAC36 wt, RTA1st and RTAall-293 cells were starved in DMEM with 0.1% FBS for overnight. Next day, media were replaced with DMEM supplement with 5% FBS. Cells were cultured for 24 hrs and harvested for analysis by flow cytometer. (D) 293 cells transfected with BAC36 wt, BAC RTA1st and BAC RTAall viruses were selected with Hygromycin for a 4-week selection, cells were seed on the plates. Colony were monitored and photographed under fluorescence microscopy after growth for two weeks. (E) The plates were scanned by Typhoon 9200 based on GFP signals. (F) The colony number was quantized using odyssey V 3.0.
Figure 5
Figure 5. Comparisons of relative infectivity for BAC36 wt, RTA1st and RTAall recombinant viruses.
(A) PBMCs were infected by KSHV BAC36 wt, RTA1st and RTAall viruses with equally loading. GFP expression was monitored under a fluorescent microscope after 2dpi, 4dpi and 7dpi. (B) Intracellular KSHV viral genome copies were measured by a real-time PCR with primers to TR at 1 dpi, 2 dpi, 4 dpi and 7 dpi. (C) Extracellular KSHV progeny virion progenies were analyzed by a real-time PCR with primers to TR at 1 dpi, 2 dpi, 4 dpi and 7 dpi. Dpi, days post infection. *P<0.05; **P<0.01; ***P<0.001 by Student's t test.
Figure 6
Figure 6. Quantitative analysis of KSHV BAC36 wt, RTA1st and BAC RTAall recombinant viruses infected PBMCs at 1dpi, 2dpi, 4dpi and 7dpi.
(A) Immunofluorescence assay for PBMCs infected by KSHV BAC36 wt, RTA1st and RTAall recombinant viruses at 2 dpi, 4 dpi and 7 dpi. Uninfected and infected PBMCs at 2 dpi, 4 dpi and 7 dpi were stained for LANA protein expression. PBMCs expressed GFP, indicating the presence of viral genome. (B, C). Quantitative analysis for determination of latent and lytic infection in PMBC cells infected by KSHV BAC36 wt, RTA1st and RTAall recombinant viruses. Total RNAs were extracted, treated with DNase I, and reverse transcribed to cDNA after 1 dpi, 2 dpi, 4 dpi and 7dpi. Quantitative Real-time PCR analysis with the primers for LANA and RTA was performed using StepOnePlus Real-Time PCR System. Error bars indicate standard deviations from three separate experiments.
Figure 7
Figure 7. Quantitative analysis of ORF 49, ORF6, K8 and K9 expression in KSHV infected PBMCs at 1dpi, 2dpi, 4dpi and 7dpi.
Total RNAs were prepared and transcribed to cDNA and the qRT-PCR analysis with the primers for (A) ORF 49, (B) ORF6, (C) K8 and (D) K9 was performed using the Power SYBR green PCR master mix with GAPDH as control. Error bars indicate standard deviations from three separate experiments.
Figure 8
Figure 8. FACS analysis of T cells, B cells and GFP-positive cells in BAC36 wt, RTA1st and RTAall recombinant viruses infected PBMCs.
(A, B) KSHV BAC36 wt, RTA1st and RTAall viruses infected PBMCs were harvested at 1dpi, 2dpi, 4dpi and 7dpi. T cells and B cells were detected by using the APC–conjugated anti-CD3 and PercpCy 5.5 -conjugated anti-CD19 mAbs. GFP signals were monitored KSHV positive cells. Data were acquired on FACSCalibur equipped with CellQuest Pro software and analyzed using FlowJo software. (C) KSHV infected T cells (CD3+ GFP+). (D) KSHV infected B cells (CD19+ GFP+). (E) Total KSHV infected PBMC cells (GFP +). *P<0.05; **P<0.01; ***P<0.001 by Student's t test (N = 9).
Figure 9
Figure 9. Analysis of KSHV BAC36 wt, RTA1st and RTAall recombinant viruses infected TIVE cells at 1, 2 and 4 weeks post infection (wpi).
(A) TIVE cells were infected with concentrated KSHV BAC36 wt, RTA1st and RTAall recombinant viruses as described in materials and methods. GFP expression was used to monitor infection under fluorescence microscope at 1, 2 and 4 wpi. (B) Intracellular KSHV viral genome copies were measured by a real-time PCR with primers to TR at 1, 2 and 4 wpi. (C) Flow cytometer was performed for KSHV wt, RTA1st and RTAall recombinant viruses infected TIVE cells at 1, 2 and 4wpi. Wpi, week post-infection. *P<0.05; **P<0.01; ***P<0.001 by Student's t test.
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