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Dipeptidase

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W., Takaoka A., Honda K., Taniguchi T. binds SAMHD1 and recruits it to the CRL4DCAF1 E3 ubiquitin ligase via its interaction with DCAF1 to facilitate SAMHD1 ubiquitination and subsequent degradation (27, 28, 33, 34). Vpx is not essential for virus replication in tissue culture, but it is important for viral replication and disease progression in animal models (35,C37). The effect of Vpx is possibly linked to its ability to enhance virus replication in dendritic cells and macrophages in tissue culture (21, 23, 24, 26, 38). Myeloid cells are believed to be critical targets for HIV gene at the 3 end of the gene in pEGFP-C1(Clontech). The IL12p40 promoter luciferase reporter plasmid was a kind gift from Dr. Murphy. The lentiviral vector (pFLRu-MCS-YFP) was a gift from Dr. Thomson. HIV-2 (GH-1) with an N-terminal FLAG-Myc-HA tag was cloned into EcoRI and AgeI sites to yield pLenti-Vpx, and verified by DNA sequencing. pCMV-HA-MyD88 was a gift of Dr. Beutler (Addgene plasmid 12287). pcDNA3-IKK? was from Dr. Maniatis (Addgene plasmid 26201). pEF-Bos huTBK1 FLAG-His was a gift from Dr. Fitzgerald (Addgene plasmid 27241). The pLKO.Puro plasmid expressing shRNA 5 CCTTAACAAGAGCCGGGACTT or TBLR1 5 TGATAGTATCCGGCTACAGAT was used to deplete IRF5. Infectious lentiviral particles encoding HIV-2 Vpx were generated by transfecting 293T cells using TransIT (Mirus) with 4 parts transfer vector (pLenti-vpx), 3 parts packaging plasmid (pGag-Pol), and 1 part vesicular stomatitis virus (VSV) glycoprotein (VSVg) expression plasmid. Seventy two hours after transfection, the virus was collected and concentrated by ultracentrifugation through a 20% sucrose cushion. Human macrophage cell line, THP-1, was transduced with the lentivirus expressing HIV-2 Vpx or an empty vector for 2 days and selected for puromycin resistance for 2C3 more days. Transfection, Immunoprecipitation, and Western Blot Analysis For all transient assays, plasmids were transfected into either 293T or HT1080 cells using TransIT (Mirus) according to the manufacturer’s instructions. For immunoprecipitation, 293T cells were transfected with IRF5 and Vpx expression plasmids, and cell lysates were collected 48C72 h post-transfection in PBS, 0.5% Nonidet P-40, and protease inhibitors, and cell debris was removed by centrifugation in a microcentrifuge at 14,000 rpm for 5 min. Cell lysates were diluted with PBS so that the final concentration of Nonidet P-40 was 0.2% and incubated with a specific antibody at 4 C overnight. Then 30 l of protein A/G-agarose beads were mixed with the lysate for 1 h at 4 C and washed with PBS, 0.1% Nonidet P-40 three times, 10 min each time. Finally the beads were resuspended in sample buffer and boiled, before subjecting to SDS-PAGE. Co-precipitated proteins were identified by Western blotting using a femto Supersignal detection kit (Thermo Scientific). Immunofluorescence Microscopy Raw 267.4 cells grown on coverslips were transfected with pFMH-Vpx and pGFP-IRF5. THP-1 cells were transduced with the lentiviral vector encoding for 72 h and then treated with 100 nm PMA. After 24 h, cells were treated with 10 m R848 for 6 h and fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and stained with monoclonal anti-FLAG antibody and then with Alexa Fluor 954-conjugated goat anti-mouse IgG (Molecular Probes). Nuclei were visualized using 0.5 g/ml Hoechst 33258 pentahydrate (Molecular Probes). Cells were visualized with a Tis epifluorescence microscope (Nikon) equipped with Metamorph software (objective 40). Luciferase Assays 293T or HT1080 cells in 12-well plates were.Biol. and resting CD4+ T cells (27,C29). SAMHD1 is a deoxynucleotide triphosphohydrolase that blocks HIV-1 reverse transcription by depleting the intracellular pool of deoxynucleoside triphosphates (30,C32). Vpx neutralizes the antiviral activity of SAMHD1 by promoting its proteasome-dependent degradation. Vpx binds SAMHD1 and recruits it to the CRL4DCAF1 E3 ubiquitin ligase via its interaction with DCAF1 to facilitate SAMHD1 ubiquitination and subsequent degradation (27, 28, 33, 34). Vpx is not essential for virus replication in tissue culture, but it is important for viral replication and disease progression in animal models (35,C37). The effect of Vpx is possibly linked to its ability to enhance virus replication in dendritic cells and macrophages in tissue culture (21, 23, 24, 26, 38). Myeloid cells are believed to be critical targets for HIV gene at the 3 end of the gene in pEGFP-C1(Clontech). The IL12p40 promoter luciferase reporter plasmid was a kind gift from Dr. Murphy. The lentiviral vector (pFLRu-MCS-YFP) was a gift from Dr. Thomson. HIV-2 (GH-1) with an N-terminal FLAG-Myc-HA tag was cloned into EcoRI and AgeI sites to yield pLenti-Vpx, and verified by DNA sequencing. pCMV-HA-MyD88 was a gift of Dr. Beutler (Addgene plasmid 12287). pcDNA3-IKK? was from Dr. Maniatis (Addgene plasmid 26201). pEF-Bos huTBK1 FLAG-His was a gift from Dr. Fitzgerald (Addgene plasmid 27241). The pLKO.Puro plasmid expressing shRNA 5 CCTTAACAAGAGCCGGGACTT or 5 TGATAGTATCCGGCTACAGAT was used to deplete IRF5. Infectious lentiviral particles encoding HIV-2 Vpx were generated by transfecting 293T cells using TransIT (Mirus) with 4 parts transfer vector (pLenti-vpx), 3 parts packaging plasmid (pGag-Pol), and 1 part vesicular stomatitis virus (VSV) glycoprotein (VSVg) expression plasmid. Seventy two hours after transfection, the virus was collected and concentrated by ultracentrifugation through a 20% sucrose cushion. Human macrophage cell line, THP-1, was transduced with the lentivirus expressing HIV-2 Vpx or an empty vector for 2 days and selected for puromycin resistance for 2C3 more days. Transfection, Immunoprecipitation, and Western Blot Analysis For all transient assays, plasmids were transfected into either 293T or HT1080 cells using TransIT (Mirus) according to the manufacturer’s instructions. For immunoprecipitation, 293T cells were transfected with IRF5 and Vpx expression plasmids, and cell lysates were collected 48C72 h post-transfection in PBS, 0.5% Nonidet P-40, and protease inhibitors, and cell debris was removed by centrifugation in a microcentrifuge at 14,000 rpm for 5 min. Cell lysates were diluted with PBS so that the final concentration of Nonidet P-40 was 0.2% and incubated with a specific antibody at 4 C overnight. Then 30 l of protein A/G-agarose beads were mixed with the lysate for 1 h at 4 C and washed with PBS, 0.1% Nonidet P-40 three times, 10 min each time. Finally the beads were resuspended in sample buffer and boiled, before subjecting to SDS-PAGE. Co-precipitated proteins were identified by Western blotting using a femto Supersignal detection kit (Thermo Scientific). Immunofluorescence Microscopy Raw 267.4 cells grown on coverslips were transfected with pFMH-Vpx and pGFP-IRF5. THP-1 cells were transduced with the lentiviral vector encoding for 72 h and then treated with 100 nm PMA. After 24 h, cells were treated with 10 m R848 for 6 h and fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and stained with monoclonal anti-FLAG antibody and then with Alexa Fluor 954-conjugated goat anti-mouse Clobetasol propionate IgG (Molecular Probes). Nuclei were visualized using 0.5 g/ml Hoechst 33258 pentahydrate (Molecular Probes). Cells were visualized with a Tis epifluorescence microscope (Nikon) equipped with Metamorph software (objective 40). Luciferase Assays 293T or HT1080 cells in 12-well plates were transiently transfected with an IL12p40 promoter-firefly luciferase reporter plasmid (500 ng) and IRF5 and/or a Vpx expression plasmid. A pRL-TK (luciferase) reporter plasmid was used as an internal control for transfection efficiency using TransIT (Mirus) according to the instructions of the manufacturer. The total amount of DNA was kept constant by supplementation with an empty vector (pcDNA3.1). At 24 h post-transfection, the luciferase activity was measured by Dual-Luciferase assay. RNA Isolation and qRT-PCR Total RNA was extracted from THP-1 cells transduced with either an empty lentiviral vector or the vector encoding HIV-2 Vpx or was infected with HIV-2 viruses using the RNeasy mini kit (Qiagen). Real time qRT-PCR was performed using the one-step RT-PCR kit (Bio-Rad) to measure the mRNA level.K., Manel N., Florens L., Washburn M. dendritic cells (24, 26). SAMHD1 was recently identified as a potent restriction factor of HIV-1 in myeloid cells and resting CD4+ T cells (27,C29). SAMHD1 is a deoxynucleotide triphosphohydrolase that blocks HIV-1 reverse transcription by depleting the intracellular pool of deoxynucleoside triphosphates (30,C32). Vpx neutralizes the antiviral activity of SAMHD1 by promoting its proteasome-dependent degradation. Vpx binds SAMHD1 and recruits it to the CRL4DCAF1 E3 ubiquitin ligase via its Clobetasol propionate interaction with DCAF1 to facilitate SAMHD1 ubiquitination and subsequent degradation (27, 28, 33, 34). Vpx is not essential for virus replication in tissue culture, but it is important for viral replication and disease progression in animal models (35,C37). The effect of Vpx is possibly linked to its capability to improve trojan replication in dendritic cells and macrophages in tissues lifestyle (21, 23, 24, 26, 38). Myeloid cells are thought to be vital focuses on for HIV gene on the 3 end from the gene in pEGFP-C1(Clontech). The IL12p40 promoter luciferase reporter plasmid was a sort present from Dr. Murphy. The lentiviral vector (pFLRu-MCS-YFP) was something special from Dr. Thomson. HIV-2 (GH-1) with an N-terminal FLAG-Myc-HA label was cloned into EcoRI and AgeI sites to produce pLenti-Vpx, and confirmed by DNA sequencing. pCMV-HA-MyD88 was something special of Dr. Beutler (Addgene plasmid 12287). pcDNA3-IKK? was from Dr. Maniatis (Addgene plasmid 26201). pEF-Bos huTBK1 FLAG-His was something special from Dr. Fitzgerald (Addgene plasmid 27241). The pLKO.Puro plasmid expressing shRNA 5 CCTTAACAAGAGCCGGGACTT or 5 TGATAGTATCCGGCTACAGAT was utilized to deplete IRF5. Infectious lentiviral contaminants encoding HIV-2 Vpx had been generated by transfecting 293T cells using TransIT (Mirus) with 4 parts transfer vector (pLenti-vpx), 3 parts product packaging plasmid (pGag-Pol), and 1 component vesicular stomatitis trojan (VSV) glycoprotein (VSVg) appearance plasmid. Seventy two hours after transfection, the trojan was gathered and focused by ultracentrifugation through a 20% sucrose pillow. Individual macrophage cell series, THP-1, was transduced using the lentivirus expressing HIV-2 Vpx or a clear vector for 2 times and chosen for puromycin level of resistance for 2C3 even more times. Transfection, Immunoprecipitation, and Traditional western Blot Analysis For any transient assays, plasmids had been transfected into either 293T or HT1080 cells using TransIT (Mirus) based on the manufacturer’s guidelines. For immunoprecipitation, 293T cells had been transfected with IRF5 and Vpx appearance plasmids, and cell lysates had been gathered 48C72 h post-transfection in PBS, 0.5% Nonidet P-40, and protease inhibitors, and cell particles was removed by centrifugation within a microcentrifuge at 14,000 rpm for 5 min. Cell lysates had been diluted with PBS so the final focus of Nonidet P-40 was 0.2% and incubated with a particular antibody at 4 C overnight. After that 30 l of proteins A/G-agarose beads had been blended with the lysate for 1 h at 4 C and cleaned with PBS, 0.1% Nonidet P-40 3 x, 10 min every time. Finally the beads had been resuspended in test buffer and boiled, before subjecting to SDS-PAGE. Co-precipitated protein had been identified by Traditional western blotting utilizing a femto Supersignal recognition package (Thermo Scientific). Immunofluorescence Microscopy Fresh 267.4 cells harvested on coverslips were transfected with pFMH-Vpx and pGFP-IRF5. THP-1 cells had been transduced using the lentiviral vector Clobetasol propionate encoding for 72 h and treated with 100 nm PMA. After 24 h, cells had been treated with 10 m R848 for 6 h and set with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and stained with monoclonal anti-FLAG antibody and with Alexa Fluor 954-conjugated goat anti-mouse IgG (Molecular Probes). Nuclei had been visualized using 0.5 g/ml Hoechst 33258 pentahydrate (Molecular Probes). Cells had been visualized using a Tis epifluorescence microscope (Nikon) built with Metamorph software program (objective 40). Luciferase Assays 293T or HT1080 cells in 12-well plates had been transiently transfected with an IL12p40 promoter-firefly luciferase reporter plasmid (500 ng) and IRF5 and/or a Vpx appearance plasmid. A pRL-TK (luciferase) reporter plasmid was utilized as an interior control for transfection performance using TransIT (Mirus) based on the guidelines of the maker. The quantity of DNA was held continuous by supplementation with a clear vector (pcDNA3.1). At 24 h post-transfection, the luciferase activity was assessed by Dual-Luciferase.T. recruits it towards the CRL4DCAF1 E3 ubiquitin ligase via its connections with DCAF1 to facilitate SAMHD1 ubiquitination and following degradation (27, 28, 33, 34). Vpx isn’t essential for trojan replication in tissues culture, nonetheless it is very important to viral replication and disease development in animal versions (35,C37). The result of Vpx is normally possibly associated with its capability to improve trojan replication in dendritic cells and macrophages in tissues lifestyle (21, 23, 24, 26, 38). Myeloid cells are thought to be vital focuses on for HIV gene on the 3 end from the gene in pEGFP-C1(Clontech). The IL12p40 promoter luciferase reporter plasmid was a sort present from Dr. Murphy. The lentiviral vector (pFLRu-MCS-YFP) was something special from Dr. Thomson. HIV-2 (GH-1) with an N-terminal FLAG-Myc-HA label was cloned into EcoRI and AgeI sites to produce pLenti-Vpx, and confirmed by DNA sequencing. pCMV-HA-MyD88 was something special of Dr. Beutler (Addgene plasmid 12287). pcDNA3-IKK? was from Dr. Maniatis (Addgene plasmid 26201). pEF-Bos huTBK1 FLAG-His was something special from Dr. Fitzgerald (Addgene plasmid 27241). The pLKO.Puro plasmid expressing shRNA 5 CCTTAACAAGAGCCGGGACTT or 5 TGATAGTATCCGGCTACAGAT was utilized to deplete IRF5. Infectious lentiviral contaminants encoding HIV-2 Vpx had been generated by transfecting 293T cells using TransIT (Mirus) with 4 parts transfer vector (pLenti-vpx), 3 parts product packaging plasmid (pGag-Pol), and 1 component vesicular stomatitis trojan (VSV) glycoprotein (VSVg) appearance plasmid. Seventy two hours after transfection, the trojan was gathered and focused by ultracentrifugation through a 20% sucrose pillow. Individual macrophage cell series, THP-1, was transduced using the lentivirus expressing HIV-2 Vpx or a clear vector for 2 times and chosen for puromycin level of resistance for 2C3 even more times. Transfection, Immunoprecipitation, and Traditional western Blot Analysis For any transient assays, plasmids had been transfected into either 293T or HT1080 cells using TransIT (Mirus) based on the manufacturer’s instructions. For immunoprecipitation, 293T cells were transfected with IRF5 and Vpx expression plasmids, and cell lysates were collected 48C72 h post-transfection in PBS, 0.5% Nonidet P-40, and protease inhibitors, and cell debris was removed by centrifugation in a microcentrifuge at 14,000 rpm for 5 min. Cell lysates were diluted with PBS so that the final concentration of Nonidet P-40 was 0.2% and incubated with a specific antibody at 4 C overnight. Then 30 l of protein A/G-agarose beads were mixed with the lysate for 1 h at 4 C and washed with PBS, 0.1% Nonidet P-40 three times, 10 min each time. Finally the beads were resuspended in sample buffer and boiled, before subjecting to SDS-PAGE. Co-precipitated proteins were identified by Western Clobetasol propionate blotting using a femto Supersignal detection kit (Thermo Scientific). Immunofluorescence Microscopy Natural 267.4 cells produced on coverslips were transfected with pFMH-Vpx and pGFP-IRF5. THP-1 cells were transduced with the lentiviral vector encoding for 72 h and then treated with 100 nm PMA. After 24 h, cells were treated with 10 m R848 for 6 h and fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and stained with monoclonal anti-FLAG antibody and then with Alexa Fluor 954-conjugated goat anti-mouse IgG (Molecular Probes). Nuclei were visualized using 0.5 g/ml Hoechst 33258 pentahydrate (Molecular Probes). Cells were visualized with a Tis epifluorescence microscope (Nikon) equipped with Metamorph software (objective 40). Luciferase Assays 293T or HT1080 cells in 12-well plates were transiently transfected with an IL12p40 promoter-firefly luciferase reporter plasmid (500 ng) and IRF5 and/or a Vpx expression plasmid. A pRL-TK (luciferase) reporter plasmid was used as an internal control for transfection efficiency using TransIT (Mirus) according to the instructions of the manufacturer. The total amount of DNA was kept.As shown in Fig. pool of deoxynucleoside triphosphates (30,C32). Vpx neutralizes the antiviral activity of SAMHD1 by promoting its proteasome-dependent degradation. Vpx binds SAMHD1 and recruits it to the CRL4DCAF1 E3 ubiquitin ligase via its conversation with DCAF1 to facilitate SAMHD1 ubiquitination and subsequent degradation (27, 28, 33, 34). Vpx is not essential for computer virus replication in tissue culture, but it is important for viral replication and disease progression in animal models (35,C37). The effect of Vpx is usually possibly linked to its ability to enhance computer virus replication in dendritic cells and macrophages in tissue culture (21, 23, 24, 26, 38). Myeloid cells are believed to be crucial targets for HIV gene at the 3 end of the gene in pEGFP-C1(Clontech). The IL12p40 promoter luciferase reporter plasmid was a kind gift from Dr. Murphy. The lentiviral vector (pFLRu-MCS-YFP) was a gift from Dr. Thomson. HIV-2 (GH-1) with an N-terminal FLAG-Myc-HA tag was cloned into EcoRI and AgeI sites to yield pLenti-Vpx, and verified by DNA sequencing. pCMV-HA-MyD88 was a gift of Dr. Beutler (Addgene plasmid 12287). pcDNA3-IKK? was from Dr. Maniatis (Addgene plasmid 26201). pEF-Bos huTBK1 FLAG-His was a gift from Dr. Fitzgerald (Addgene plasmid 27241). The pLKO.Puro plasmid expressing shRNA 5 CCTTAACAAGAGCCGGGACTT or 5 TGATAGTATCCGGCTACAGAT was used to deplete IRF5. Infectious lentiviral particles encoding HIV-2 Vpx were generated by transfecting 293T cells using TransIT (Mirus) Clobetasol propionate with 4 parts transfer vector (pLenti-vpx), 3 parts packaging plasmid (pGag-Pol), and 1 part vesicular stomatitis computer virus (VSV) glycoprotein (VSVg) expression plasmid. Seventy two hours after transfection, the computer virus was collected and concentrated by ultracentrifugation through a 20% sucrose cushion. Human macrophage cell collection, THP-1, was transduced with the lentivirus expressing HIV-2 Vpx or an empty vector for 2 days and selected for puromycin resistance for 2C3 more days. Transfection, Immunoprecipitation, and Western Blot Analysis For all those transient assays, plasmids were transfected into either 293T or HT1080 cells using TransIT (Mirus) according to the manufacturer’s instructions. For immunoprecipitation, 293T cells were transfected with IRF5 and Vpx expression plasmids, and cell lysates were collected 48C72 h post-transfection in PBS, 0.5% Nonidet P-40, and protease inhibitors, and cell debris was removed by centrifugation in a microcentrifuge at 14,000 rpm for 5 min. Cell lysates were diluted with PBS so that the final concentration of Nonidet P-40 was 0.2% and incubated with a specific antibody at 4 C overnight. Then 30 l of protein A/G-agarose beads were mixed with the lysate for 1 h at 4 C and washed with PBS, 0.1% Nonidet P-40 three times, 10 min each time. Finally the beads were resuspended in sample buffer and boiled, before subjecting to SDS-PAGE. Co-precipitated proteins were identified by Western blotting using a femto Supersignal detection kit (Thermo Scientific). Immunofluorescence Microscopy Natural 267.4 cells produced on coverslips were transfected with pFMH-Vpx and pGFP-IRF5. THP-1 cells were transduced with the lentiviral vector encoding for 72 h and then treated with 100 nm PMA. After 24 h, cells were treated with 10 m R848 for 6 h and fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and stained with monoclonal anti-FLAG antibody and then with Alexa Fluor 954-conjugated goat anti-mouse IgG (Molecular Probes). Nuclei were visualized using 0.5 g/ml Hoechst 33258 pentahydrate (Molecular Probes). Cells were visualized with a Tis epifluorescence microscope (Nikon) equipped with Metamorph software (objective 40). Luciferase Assays 293T or HT1080 cells in 12-well plates were transiently transfected with an IL12p40 promoter-firefly luciferase reporter plasmid.