Regulation And Mechanism Of Viral Replicative Cycle Of Kaposi's Sarcoma-associated Herpesvirus (KSHV) By Regulator Of Virion Protein Expression Of Human Immunodeficiency Virus Type 1 (HIV-1)

Background:Human immunodeficiency virus type 1 (HIV-1) infection significantly increases the risk of Kaposi's sarcoma (KS) occurrence in individuals infected with Kaposi'ssarcoma-associated herpesvirus (KSHV). KSHV infection appeared to be necessary but some cofactors also played important roles in KS pathogenesis. Previous studies have demonstrated that HIV-1 particles, inflammatory cytokines and soluble proteins released by infected cells could modulate the lytic cycle replication of KSHV. Our previous study also demonstrated that HIV-1 trans-activative transcription protein (Tat) was one of the cofactors that activated lytic cycle replication of KSHV. Regulator of virion protein expression (Rev) was another significant HIV-1 regulator as well as Tat, and these two genes overlapped in most of the nucleotide sequences.Objective:To study on the effect of HIV-1 Rev protein on the replicative cycle of KSHV in primary effusion lymphoma (PEL) cell lines including BCBL-1 cells.Methods : The fragment of Rev gene was cloned into the lentivirus vector pHAGE-CMV-MCS-IZsGreen, then the recombinant plasmid named pLenti-Rev, packaging vector psPAX2 and envelope vector pMD2.G were cotransfected into the 293T cells. Culture media were harvested and filtered through a 0.45μm filter to obtain the recombinant lentivirus and the viral titer was checked by fluorescence microscopy. After infected with the recombinant lentivirus, the mRNA transcription and protein expression of Rev gene in 293T and BCBL-1 cells were detected by fluorescence microscopy,RT-PCR and Western blot. To determine whether the level of expression of Rev protein modulates the replicative cycle of KSHV, Rev-Flag, replication and transcription activator (Rta) and viral interleukin-6 (vIL-6) protein of KSHV from BCBL-1 infected with the recombinant lentivirus and transfected with pCI-neo-Rev were detected by Western blot, respectively. Furthermore, mRNA transcription of Rta in BCBL-1 transfected with pCI-neo-Rev, which represented the high-level expression of Rev protein, was detected quantitatively by Real-time quantitive PCR. To examine whether the the low-level expressed Rev protein represented by the lentivirus infection influences the replicative cycle of KSHV, after addition of TPA to BCBL-1 cells infected with the recombinant lentivirus, RT-PCR and Western blot were utilized to detect KSHV mRNAs and viral proteins in BCBL-1 cells, which were infected by lentivirus with various multiplicity of infection (MOI) or with different time-course after infection. To detect the effect of Rev protein on the release of the infectious virion of KSHV, the supernatants from BCBL-1 cells infected with the recombinant lentivirus after induction of TPA at different time points were harvested and used to infect the Vero cells. Then, PCR, RT-PCR and Western blot were performed to analysize KSHV gene sequence, mRNAs and viral proteins. To further explore the molecular mechanisms by which Rev modulates KSHV replication, luciferase reporter assay was employed to measure the Rta and ORF73 promoter activity induced by Rev protein in PEL cells infected by lentivirus. And Western blot was used to detect whether MAPK and PI3K/GSK-3βsignal pathways play a role in Rev-induced KSHV lytic replication.Results:The recombinant lentivirus vector containing Rev gene was constructed successfully with the viral titer of 2×107 TU/ml. BCBL-1 cells could be efficiently infected by the recombinant lentivirus and Rev protein was expressed correctly in these cells. The results of Western blot showed that the expression of KSHV Rta and vIL-6 were significantly upregulated when expression level of Rev protein was low, and vice versa. Real-time quantitive PCR indicated that the mRNA transcriptional level of KSHV Rta in Rev overexpressed-cells was significantly decreased compared with the corresponding control. RT-PCR and Western blot was applied to detect the effect of Rev protein on KSHV replication in BCBL-1 infected by lentivirus which representing of the low-level expression of Rev protein, and the results showed that both mRNA transcriptional levels of latency-associated nuclear antigen (LANA), ORF26 (encoding KSHV minor capsid protein), ORF57, vIL-6 and protein expression levels of Rta and vIL-6 were upregulated in BCBL-1 cells infected by the Rev lentivirus. In the virus releasing assay, the KSHV virus copies in Rev virus-infected BCBL-1 cells were higher. In addition, the mRNA transcriptional levels of viral flice-inhibitory protein (vFLIP), ORF26, T1.1 and vIL-6 of KSHV in Vero cells infected with the supernatants of Rev virus-infected BCBL-1 cells were significantly upregulated at some degree at different time points. Finally, luciferase reporter assay indicated that low-level expressed Rev could inhibit KSHV Rta promoter activity but induce ORF73 promoter activity, which suggested that Rev might induce the replication of KSHV in an indirect manner. Western blot results showed that ERK and GSK-3βphosphorylation levels were upregulated in varying degrees in Rev-lentivirus groups, suggesting that Rev protein might participate in the regulation of ERK MAPK and PI3K/GSK-3βpathways to induce KSHV lytic replication.Conclusion:HIV-1 Rev protein could be expressed effectively in BCBL-1 infected with the recombinant lentivirus. Forthermore, low-level expression of Rev protein could elevate the KSHV replication and high-level expression of Rev protein could inhibit its replication. Rev protein induced the lytic replication of KSHV via an indirect way instead of directly binding to Rta promoter. Moreover, regulation of ERK MAPK and PI3K/GSK-3βpathways might contribute to Rev-induced KSHV replication, indicating that these pathways might be of therapeutic value in the treatment of AIDS-KS.