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The human immunodeficiency virus (HIV) genome not only contains structural genes, but also a series of accessory genes. The vpu gene is a unique accessory gene to HIV-1 and it encodes a small transmembrane protein [7]. Some studies have shown that Vpu is very important for the maturation and release of HIV-1 [7, 22]. The Vpu-deleted HIV-1 (HIV-1ΔVpu) virions accumulated in the intracellular regions and at the cell surfaces of the HIV-1 infected cells [16]. So the Vpu protein counteracts the effect of inhibitors on HIV-1 virion release. The accelerated effect of HIV-1 release by the protein Vpu is achieved through recruiting cullin-ring finger ubiquitin ligases to eliminate host cell proteins that impede replication and release [13]. In the HIV-1 life cycle, the Vpu protein reduced the cell surface expression of CD4 and recruited cullin1 to efficiently facilitate the release of HIV-1 virons from infected cells [13].
The effect of Vpu is counteracted by the tetherin transmembrane protein [16, 24]. Tetherin is an interferon-induced protein [23, 24], also termed HM1.24 [3], bone marrow stromal antigen 2 (BST2) [6] and cluster of differentiation 317 (CD317) [4]. Tetherin is a raft-associated membrane protein, with a N-terminus cytoplasmic tail, a membrane spanning domain, an extracellular domain, and a C-terminus glycosylpho sphatidylinositol (GPI) membrane anchor [9, 16, 23]. The C-terminal GPI structure enables tetherin localization within cholesterol enriched lipid rafts which is important for HIV-1 release at the plasma membrane [23]. HIV-1 selectively buds from the glycolipid-enriched membrane lipid rafts and HIV-1 virion particles containing the GPI-linked proteins [17]. So it is presumed that tetherin inhibits the HIV-1 release in this way.
Because of the counteractive relationship between Vpu and tetherin, inhibition of Vpu function and mobilization of tetherin's antiviral activity is a potential therapeutic strategy in HIV/AIDS [16]. In this study, Vpu-targeted small interfering RNA (siRNA) that effectively knocks down the expression of the HIV-1 Vpu protein was designed [13, 15]. As a host factor, tetherin that is counteracted by HIV-1 Vpu can also be over-expressed to enhance the antiviral ability of an infected cell [2, 5]. Replication and release of HIV-1 may be inhibited effectively by the combination of Vpu-targeted siRNA and a tetherin over-expressed retrovirus. In this study, both pSIREN-RetroQ-vpuRNAi and pMSCV-tetherin retroviral vector plasmids were packaged into retroviruses in the same packaging cell line. Mixed plasmids were then transfected into the PT67 cells at different ratios, and the mixed retroviruses were harvested. The inhibition efficiency of mixed retroviruses was detected in the TZM-bl cells pre-infected with HIV-1 NL4.3. Then the combined inhibition of vpu RNAi and tetherin over-expression was evaluated based on the individual inhibition effect. Finally, we identified the optimum ratio of mixed retrovirus that achieved the best inhibitory effect on HIV-1 replication.
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The HEK293T cell line, derived from transformation of HEK293 cells with the SV40 large T gene [19], was used for the expression of recombinant genes. The PT67 cell line (Cat. No. 631510, Clontech) derived from NIH-3T3 cell contains the moloney murine leukemia virus (MoMuLV) gag, pol, and env (10A1-derived) genes [14] and was used for production of replication-incompetent retrovirus after transfecting with pMSCVneo. The TZM-bl cell line (also called JC.53bl-13) is a HeLa cell derivative that was engineered by amphotropic retroviral transduction and was used to express CD4 and CCR5 and integrated copies of the luciferase and -galactosidase genes under control of the HIV-1 promoter [8, 18]. All of the above cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 0.1% penicillin, and 0.1% streptomycin at 37 ℃ with 5 % CO2.
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The plasmid pSIREN-RetroQ was double enzyme digested by Bam H Ⅰ and Eco R Ⅰ. The 6.4 kb fragment was gel purified by a gel extraction kit (DC3511, BIOMIGA). Sequences of Vpu-targeted siRNA were obtained from the online siRNA design tool (http://sivirus.rnai.jp/HIV/) designed by Naito et al. [15]. Target sequences were synthesized as oligonucleotides by Sangon Biotech (Shanghai) and are shown in Table 1. Each oligonucleotide was resuspended in Tris-EDTA (TE) buffer to a concentration of 100 μmol/L and the top strand and the bottom strand of oligonucleotides were mixed at a 1 : 1 ratio up to 30 μL. The mixture was then annealed at 95 ℃ 30 s / 72 ℃ 2 min/ 37 ℃ 2 min/ 25 ℃ 2 min for two cycles. The annealed product was ligated into the purified RNAi-Ready pSIREN-RetroQ vector. Detailed of the protocols are available in the BD™ Knockout RNAi Systems User Manual (PT3739-1).
Table 1. Oligonucleotides used to produce shRNA expression vectors and plasmids construction
The pMSCVneo plasmid was double enzyme digested by Xho Ⅰ and Bgl Ⅱ. The 6.5 kb fragment was gel purified by a gel extraction kit (DC3511, BIOMIGA). The tetherin gene was obtained from the pflag-BST2 plasmid, which is a flag-BST2 fusion protein expressing plasmid, by polymerase chain reaction (PCR). Primers used to amplify tetherin are shown in Table 1. The two enzymes sites Xho Ⅰ and Bgl Ⅱ were added upstream and downstream of the tetherin gene. The tetherin gene was purified with a PCR purification kit (DC3514, BIOMIGA) and ligated into the pMSCVneo vector.
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In order to get a broad-spectrum inhibition effect on the vpu gene silencing, the vpu gene siRNA target sites should be well conserved. Sequences of Vpu-targeted siRNA were obtained from the online siRNA design tool (http://sivirus.rnai.jp/HIV/) [15]. Five siRNA target sequences were found from 495 HIV-1 vpu sequences which included subtype A, B, C, D, F, G, H, J, K, and are summarized in Table 1. All of these five sequences were annealed and ligated into the purified RNAi-Ready pSIREN-RetroQ vector as described above. To demonstrate whether the siRNA can repress the vpu expression successfully, a vpu-EGFP fusion transcript reporter system was constructed. Primers used to amplify the vpu gene are shown in Table 1. The vpu gene was ligated into the pEGFP-N1 plasmid at the enzyme digestion sites of Eco R Ⅰ and Bam H Ⅰ. The pVpu-EGFP plasmid and pSIREN-RetroQ-vpuRNAi-1~5 plasmids were co-transfected in HEK293T cells (5×105 cells/well on 6 well plates). pSIREN-RetroQ plasmid was used as a negative control and co-transfected with the pVpu-EGFP plasmid. After 48 h, HEK293T cells were observed by fluorescence microscope and images were taken. The RNAi inhibition efficiency was denoted by fluorescence intensity.
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Tetherin expression of pMSCV-tetherin plasmid was checked by Western blot analysis. The pMSCV-tetherin was transfected into HEK293T cells for the high expression of the recombinant gene. HEK293T cells were seeded in 6 well plates (5×105 cells in each well). 2 μg pMSCV-tetherin plasmid was transfected into cells, and the pMSCVneo plasmid was also transfected as a negative control. After 48 h, cells were harvested for Western blotting assay. Cells were washed twice by 2 mL phosphate buffer solution (PBS) per well, and then cells were detached with 2 mL PBS. The mixture was transferred to a 1.5 mL microtube, centrifuged at 5, 000 rpm for 2 min, and supernatant were discarded. The same volumes of 2x loading buffer (0.1mol/L Tris-HCl pH=6.8, 4% sodium dedecyl sulphate, 20% glycerol, 1% β-Mercaptoethanol, 0.1% bromphenolblue) and 5μL β-Mercaptoethanol were added. Samples were boiled for 15~25min until the solution become clear, centrifuged at 12000 rpm for 5min and supernatants were collected. Supernatants were run on a 10% SDS-polyacrylamide gel along with pre-stained Marker (#SM0671, Fermentas). Blotting, blocking and antibody incubation were performed as previously described in Liu et al. [11, 12], using the mouse polyclonal to BST2 (ab88523, Abcam) at a ratio of 1:1000. Horseradish peroxidase-conjugated goat anti-mouse antibody (AB503, SinoBio) diluted to a ratio of 1:1000 was used as secondary antibody. In order to ensure equal protein loading, a polyclonal anti-β-actin antibody (AB101, SinoBio) was diluted to a ratio of 1:1000 as the primary antibody. Finally, the membrane was treated with western lightning substrates (NEL103001EA, PerKinElmer) using a ChemiDoc XRS imaging system (Bio-Rad).
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Retroviral particles were packaged in cell line PT67 by transfection of corresponding plasmids. Plasmids pSIREN-RetroQ-vpuRNAi and pMSCV-tetherin were transfected into the PT67 cells (1.2×107 cells in 15 cm dish) using polyethylenimine (PEI). Total plasmids and PEI were mixed for 10 min in the serum-free DMEM with a ratio of 16 μg : 80 μg. Fresh medium was changed at 8 h post transfection. Then 48 h after transfection, the supernatant was harvested and concentrated with ultracentrifugation at 4℃ 32000 rpm for 1.5 h. The supernatant was discarded gently after ultracentrifugation, and 200 µL of serum-free DMEM medium was added into the tube. The pellet was allowed to resuspend at 4℃overnight [20]. Next day, the virus was dispensed and stored at -80℃ for further study.
HIV NL4.3 virus was packaged in cell line HEK293T by transfection of the infectious clone pNL4.3 plasmid [7, 8]. HEK293T cells were transfected with 16 μg pNL4.3 plasmid, supernatant was collected at 60h post transfection. The 50% tissue culture infective dose (TCID50) of virus stock was determined by infecting TZM-bl cells with fourfold serial dilutions of virus [8]. A multiplicity of infection (MOI) was the ratio of TCID50 to cell number [2, 8]. An equal MOI was needed during virus infection in triplicate experiments.
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Two retrovirus plasmids expressing tetherin and vpu-RNAi were mixed in varying ratios, as shown in Table 2, and then co-transfected into PT67 packaging cells in 15 cm dishes. The detailed process of harvesting virus followed the virus packaging protocol. The harvested mixture of the two retroviruses was then used to infect an indicator cell line TZM-bl in the presence of HIV-1 NL4.3. Inhibition effect was checked in the 96 well plates. The TZM-bl cells were seeded in 96 well plates (4×103 cells in each well) and were infected with HIV-1 NL4.3 with a multiplicity of infection (MOI) of 0.5. After 8 h, the mixed retroviruses at varying ratios were added with a MOI of 0.01. The interference retroviruses mixture was added every 24 h, a total of 4 times, and at 16 h after the last treatment, cells were harvested and luciferase activities were detected to measure the replication of HIV-1. Luciferase assay was according to the rapid protocol for the Steady-Glo assay system (E2520, Promega). The value of relative luciferase activities (RLA) were used to evaluate the effect of the interference retroviruses on HIV-1 NL4.3 replication.
Table 2. Ratios of the two kinds of plasmids used in the virus packaging