| Objective: To evaluate the effect of human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) on the lytic cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) and identify the signaling pathway(s) involved in this process.Methods: 1. Recombinant lentivirus containing HIV-1 Vpr gene was packaged and identified, and simultaneously the viral titer was checked. The mRNA transcription and protein expression of Vpr gene in BCBL-1 (body cavity-based lymphoma, BCBL, or primary effusion lymphoma, PEL) cells infected with the recombinant lentivirus were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. 2. The mRNA transcription and protein expression of lytic phase genes of KSHV in PEL cells infected with the lentivirus were detected by real-time quantitative PCR (Real-time PCR) and Western blot, respectively. Then the supernatants of infected BCBL-1 were collected to measure KSHV viral particles as well as to infect vero cells. Subsequently the mRNA transcription and protein expression of lytic phase genes of KSHV in vero cells were also detected by RT-PCR and Western blot, respectively. Furthermore, the luciferase reporter assay was performed after PEL cells were infected with recombinant lentivirus carrying Vpr gene and further transfected with luciferase reporter plasmid carrying KSHV ORF50 or ORF73 gene promoter. 3. The human signal transduction pathwayfinder PCR array was performed using the RNA of BCBL-1 cells infected with recombinant lentivirus carrying Vpr gene and the results were verified by Western blot. Finally, the lytic protein expression of KSHV was further determined by Western blot after the addition of the specific inhibitor or overexpression of mutant plasmid.Results: 1. The recombinant lentivirus carrying Vpr gene was packaged successfully with the viral titer of 4×107 Tu/ml. And the mRNA transcription and protein expression of Vpr gene could be detected in BCBL-1 infected with the recombinant lentivirus. 2. The overexpression of Vpr protein remarkably decreased not only the lytic mRNA transcription of ORF50 (switch gene of KSHV cycle replication) and ORF26 (encoding KSHV minor capsid protein), but also the lytic proteins of viral interleukin-6 (vIL-6) and replication and transcription activator (Rta). Meanwhile, the overexpression of Vpr in BCBL-1 cells significantly downregulated the copies of KSHV viral particles as well as the mRNA transcription of ORF73/LANA (latent-associated nuclear antigen), ORF26 and the protein expression of vIL-6 in vero cells. In addition, the Vpr protein inhibited the activation of KSHV ORF50 and ORF73 promoters directly. 3. Overexpression of Vpr protein decreased the phosphorylation of glycogen synthase kinase 3β(GSK-3β) in BCBL-1 cells. Addition of LiCl, the specific inhibitor of GSK-3β, or transfection of GSK-3β-S9A construct, the overexpression plasmid of GSK-3β, reinforced or partially blocked the effect of Vpr-downregulated expression of KSHV vIL-6. Vpr protein also decreased the phosphorylation of PTEN and the transfection of overexpression plasmid of PTEN also reinforced the effect of Vpr-downregulated expression of KSHV vIL-6. Finally, Vpr protein also inhibited the phosphorylation of c-Raf,MEK1/2 and ERK1/2 of MAKP signaling pathway .Conclusions: 1. Recombinant lentivirus containing Vpr gene could efficiently infect BCBL-1 cells, and Vpr protein expressed in BCBL-1 cells. 2. The Vpr protein significantly downregulated the lytic cycle replication of KSHV by inhibiting the activation of ORF50 and ORF73 promoters. 3. Vpr repressed KSHV replication by inhibiting the activation of GSK-3βand PTEN signaling pathways. Ras/c-Raf/MEK/ERK MAPK pathway might also play a role in modulation of Vpr-inhibited KSHV replication. These data suggest that in AIDS-related KS (AIDS-KS) patients, Vpr protein may help KSHV to escape from the immune clearance by inhibiting KSHV lytic replication, which facilitate the establishment of latent infection and tumor formation.
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