Journal: International Journal of Molecular Sciences
Article Title: SAMHD1 Enhances Chikungunya and Zika Virus Replication in Human Skin Fibroblasts
doi: 10.3390/ijms20071695
Figure Lengend Snippet: SAMHD1 over-expression enhances CHIKV and ZIKV replication. ( A ) HFF1 cells were transduced by either control (Ctrl)- or SAMHD1-GFP lentiviral vector. Transduced cells were sorted by a FACSAria III cytometer and obtained cell lines were validated for GFP expression by flow cytometry (10,000 single cell events in the analysis gate) and for SAMHD1 expression using Western blotting analysis. ( B , C ) Untreated (−), control transfected (Ctrl) or SAMHD1-(SAMHD1) transfected HFF1 cells were infected with CHIKV or ZIKV at MOI 1. At 48 hpi, intracellular viral RNA was quantified by RT-PCR (black bars); virus titers were measured using a plaque assay (gray bars). ( D , E ) Wild type U937 cells (WT) or U937 cells expressing SAMHD1, were infected with CHIKV or ZIKV at MOI 1. At 8, 24 and 48 hpi, the intracellular viral RNAs was quantified by RT-PCR (black bars); virus titers were measured by plaque assay (gray bars). ( F , G ) Amounts of CHIKV and ZIKV RNA and infectious virion production were determined at 24 hpi in HFF1 cells that were mock-transfected (Cont) or transfected, either with control siRNA (siCont) or siRNA specific for SAMHD1 (siSAMHD1). Expression of viral RNA and viral titers were quantified using real time RT-PCR (black bars) or plaque assay (gray bars), respectively. Percentage of reduction was calculated for each of the experimental conditions (siCont and siSAMHD1) using the formula [1 − (R/C)] × 100, where C and R designate experimental values (RNA copy numbers or plaque numbers) in untreated cells and siCont- or siSAMHD1-treated cells, respectively. The data represent the mean values ± SD from three independent experiments. ** p -value < 0.01 as compared to control cells.
Article Snippet: The human skin fibroblast cell line HFF1 (ATCC ® SCRC-1041TM) and U937 (ATCC ® CRL-1593.2TM) cells were cultured in DMEM, supplemented with 15% and 10%, respectively.
Techniques: Over Expression, Control, Plasmid Preparation, Cytometry, Expressing, Flow Cytometry, Western Blot, Transfection, Infection, Reverse Transcription Polymerase Chain Reaction, Virus, Plaque Assay, Quantitative RT-PCR
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![IFITs modulate CHIKV and ZIKV replication in HFF1 cells. ( A ) HFF1 cells were infected with CHIKV at a multiplicity of infection (MOI) of 1.0. At 24 and 48 hpi, infected cells were lysed with TETN-150 and analyzed by immunoblotting for the expression of IFIT1, IFIT2, IFIT3, MX1 and β-actin. ( B ) Similar to ( A ) but using ZIKV at a MOI of 1.0. For both ( A , B ), mock-infected cells were used as control. ( C ) CHIKV and ( D ) ZIKV RNA and infectious virus production were determined at 48 hpi in HFF1 cells transfected with an empty plasmid (pCtrl) or the same plasmid encoding IFIT proteins (pIFIT1, pIFIT2 and pIFIT3) by quantitative RT-PCR (black bars) and plaque assay (gray bars), respectively. The percentage of reduction as a function of the presence of each plasmid was calculated using the formula [1 − (R/C)] × 100, where C and R designate experimental values (RNA copy numbers or plaque numbers) in the presence of pCtrl and pIFIT, respectively. ( E ) CHIKV and ( F ) ZIKV RNA and infectious virus production were determined at 48 hpi in cells transfected with control siRNA (siCtrl) or siRNA, specific for IFIT (pIFIT1, pIFIT2 and pIFIT3) by real time RT-PCR (black bars) and plaque assay (gray bars), respectively. The data represent the mean values ± SD from three independent experiments. ** p < 0.01, as compared to siCtrl transfected cells.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0247/pmc06480247/pmc06480247__ijms-20-01695-g002.jpg)
![( A ) Vpx-induced SAMHD1 degradation decreases CHIKV and ZIKV replication. Mock- (−) or VLP/Vpx-treated (+) HFF1 cells were infected with CHIKV for 48 h. Infected cells were lysed with TETN-150 and analyzed by immunoblotting using antibodies against SAMHD1 and β-actin. ( B , C ) Vpx-treated and mock-treated HFF1 cells were infected with CHIKV or ZIKV at MOI 1. After 48 h, intracellular viral RNA levels and infectious virus production were measured by real time RT-PCR (black bars) and plaque assay (gray bars), respectively. The percentage of reduction in the presence of Vpx was calculated using the formula [1 − (R/C)] × 100, where C and R designate experimental values (RNA copy numbers or plaque numbers) as a function of Mock- or VLP/Vpx-treated cells, respectively. ( D ) Stable Huh-7 cells harboring the CHIKV-NCT replicon were untreated or treated with VLP/Vpx. Twenty-four and 48 h post-treatment, cells were lysed and Renilla Luciferase ( Rluc ) activity was measured. Relative Rluc activity expressed by the CHIKV replicon represents the magnitude of CHIKV RNA replication. Values are normalized according to protein content of the cell extract and correspond to the mean of triplicates ± SD. The data represent the mean values ± SD from three independent experiments. ** p -value < 0.01, as compared to untreated cells.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0247/pmc06480247/pmc06480247__ijms-20-01695-g004.jpg)
![SAMHD1 over-expression enhances CHIKV and ZIKV replication. ( A ) HFF1 cells were transduced by either control (Ctrl)- or SAMHD1-GFP lentiviral vector. Transduced cells were sorted by a FACSAria III cytometer and obtained cell lines were validated for GFP expression by flow cytometry (10,000 single cell events in the analysis gate) and for SAMHD1 expression using Western blotting analysis. ( B , C ) Untreated (−), control transfected (Ctrl) or SAMHD1-(SAMHD1) transfected HFF1 cells were infected with CHIKV or ZIKV at MOI 1. At 48 hpi, intracellular viral RNA was quantified by RT-PCR (black bars); virus titers were measured using a plaque assay (gray bars). ( D , E ) Wild type U937 cells (WT) or U937 cells expressing SAMHD1, were infected with CHIKV or ZIKV at MOI 1. At 8, 24 and 48 hpi, the intracellular viral RNAs was quantified by RT-PCR (black bars); virus titers were measured by plaque assay (gray bars). ( F , G ) Amounts of CHIKV and ZIKV RNA and infectious virion production were determined at 24 hpi in HFF1 cells that were mock-transfected (Cont) or transfected, either with control siRNA (siCont) or siRNA specific for SAMHD1 (siSAMHD1). Expression of viral RNA and viral titers were quantified using real time RT-PCR (black bars) or plaque assay (gray bars), respectively. Percentage of reduction was calculated for each of the experimental conditions (siCont and siSAMHD1) using the formula [1 − (R/C)] × 100, where C and R designate experimental values (RNA copy numbers or plaque numbers) in untreated cells and siCont- or siSAMHD1-treated cells, respectively. The data represent the mean values ± SD from three independent experiments. ** p -value < 0.01 as compared to control cells.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0247/pmc06480247/pmc06480247__ijms-20-01695-g005.jpg)