Ector RNAs analyzed.proteins are expressed from this viral vector. To

Ector RNAs analyzed.proteins are expressed from this viral vector. To clone VHenv and VHnef cytoplasmic RNA was extracted from HEK293T cells transfected with VHgenomic and spliced transcripts were amplified by RT-PCR (QuantiTect Kit, Qiagen) as detailed in Materials and Methods S1. The resulting fragments as well as VHgenomic were digested with KasI/NotI 25033180 or KasI/EcoRI and ligated. In the resulting plasmid VHenv and VHnef DNA sequences of SD1 and SA5 or SD1 and SA5 together with SD4 and SA7 are fused, respectively. All generated plasmids and PCR fragments were controlled by sequencing.Cell culture, transfections, Western Blot analyses and determination of infectious titersHEK293 and HEK293T cells were ITI-007 web cultured in Dulbecco’s modified Eagle Medium (DMEM) with 10 fetal calf serum and appropriate antibiotics. Two days after cotransfection of HEK293T cells by the calcium phosphate coprecipitation method [18,39] with the lentiviral vectors and tat, VSV-G and gag/gagpol expression plasmids with or without a rev expression plasmid the precleared (centrifugation for 5 min at 1,000 rpm) and filtered (0.45 mm filter) cell culture supernatant was used to infect HEK293 cells. Two days later green fluorescent cells were counted to obtain the infectious titer as GFP forming units per ml of supernatant (GFU/ml). Western Blot analyses were done essentially as described before [18] with the anti-CA antibody 183H12-5C (NIH AIDS Research and Reference Reagent Program).Encapsidation assay and RT-qPCRDetermination of cytoplasmic and virion-associated RNA copy numbers of transfected HEK293T cells was done as described previously [12,13,18]. In brief, two days after transfection HEK293T cells were MedChemExpress Tunicamycin washed in cold PBS (Invitrogen) and cytoplasmic fractions were obtained by lysing the cells 5 min on ice in cold RLN buffer (50 mM Tris-Cl [pH 8.0], 140 mM NaCl, 1.5 mM MgCl2, 0.5 [vol/vol] Nonidet P-40, 1,000 U/ml RNase inhibitor, 1 mM dithiothreitol). Nuclei were pelleted by centrifugation for 2 min at 300xg and 4uC followed by careful collection of the supernatant as cytoplasmic fraction. Subsequently, the RNeasy Mini kit (Qiagen) was used to extract cytoplasmic RNA. Particle-associated RNA was prepared with the QIAamp Viral RNA Mini kit (Qiagen) after ultracentrifugation of the supernatants of transfected cells through a 30 sucrose cushion. All RNA samples were stored at 275uC until RT-qPCR analyses. Detection of unspliced RNA derived from VHgenomic was facilitated by sense primer p2 binding upstream of SD1 (59gtggaaaatctctagcagtggcgc-39) and antisense primer p4 binding directly downstream of SD1 (59-tctttccccctggccttaaccg-39). Detection of singly-spliced SD1-SA5 RNA of VHgenomic and the unspliced Msd1-sa5 RNA of VHenv was possible by sense primer p3- overlapping SD1 and SA5 (59-ggggcggcgactggaagaa-39) and antisense primer p8+ binding directly downstream of SD4 (59tgattactatggaccacacaactattgc-39). The fully-spliced SD1SA5+SD4-SA7 transcript of VHgenomic and the corresponding transcripts with identical sequences Msd1-sa5+SD4-SA7 and Msd1-sa5+Msd4-sa7 of VHenv and VHnef, respectively, were detected using sense primer p3- together with antisense primer p10 binding downstream of SA7 (59-ccgttcactaatcgaatggatctgtc-39). All primers are identical or very similar to those used by Houzet et al. [9]. RT-PCRs were done with 500 ng of cytoplasmic RNA and 1 ml from a 50 ml particle RNA fraction isolated from 5 ml of cell culture supernatant as described before [12,13,18]. F.Ector RNAs analyzed.proteins are expressed from this viral vector. To clone VHenv and VHnef cytoplasmic RNA was extracted from HEK293T cells transfected with VHgenomic and spliced transcripts were amplified by RT-PCR (QuantiTect Kit, Qiagen) as detailed in Materials and Methods S1. The resulting fragments as well as VHgenomic were digested with KasI/NotI 25033180 or KasI/EcoRI and ligated. In the resulting plasmid VHenv and VHnef DNA sequences of SD1 and SA5 or SD1 and SA5 together with SD4 and SA7 are fused, respectively. All generated plasmids and PCR fragments were controlled by sequencing.Cell culture, transfections, Western Blot analyses and determination of infectious titersHEK293 and HEK293T cells were cultured in Dulbecco’s modified Eagle Medium (DMEM) with 10 fetal calf serum and appropriate antibiotics. Two days after cotransfection of HEK293T cells by the calcium phosphate coprecipitation method [18,39] with the lentiviral vectors and tat, VSV-G and gag/gagpol expression plasmids with or without a rev expression plasmid the precleared (centrifugation for 5 min at 1,000 rpm) and filtered (0.45 mm filter) cell culture supernatant was used to infect HEK293 cells. Two days later green fluorescent cells were counted to obtain the infectious titer as GFP forming units per ml of supernatant (GFU/ml). Western Blot analyses were done essentially as described before [18] with the anti-CA antibody 183H12-5C (NIH AIDS Research and Reference Reagent Program).Encapsidation assay and RT-qPCRDetermination of cytoplasmic and virion-associated RNA copy numbers of transfected HEK293T cells was done as described previously [12,13,18]. In brief, two days after transfection HEK293T cells were washed in cold PBS (Invitrogen) and cytoplasmic fractions were obtained by lysing the cells 5 min on ice in cold RLN buffer (50 mM Tris-Cl [pH 8.0], 140 mM NaCl, 1.5 mM MgCl2, 0.5 [vol/vol] Nonidet P-40, 1,000 U/ml RNase inhibitor, 1 mM dithiothreitol). Nuclei were pelleted by centrifugation for 2 min at 300xg and 4uC followed by careful collection of the supernatant as cytoplasmic fraction. Subsequently, the RNeasy Mini kit (Qiagen) was used to extract cytoplasmic RNA. Particle-associated RNA was prepared with the QIAamp Viral RNA Mini kit (Qiagen) after ultracentrifugation of the supernatants of transfected cells through a 30 sucrose cushion. All RNA samples were stored at 275uC until RT-qPCR analyses. Detection of unspliced RNA derived from VHgenomic was facilitated by sense primer p2 binding upstream of SD1 (59gtggaaaatctctagcagtggcgc-39) and antisense primer p4 binding directly downstream of SD1 (59-tctttccccctggccttaaccg-39). Detection of singly-spliced SD1-SA5 RNA of VHgenomic and the unspliced Msd1-sa5 RNA of VHenv was possible by sense primer p3- overlapping SD1 and SA5 (59-ggggcggcgactggaagaa-39) and antisense primer p8+ binding directly downstream of SD4 (59tgattactatggaccacacaactattgc-39). The fully-spliced SD1SA5+SD4-SA7 transcript of VHgenomic and the corresponding transcripts with identical sequences Msd1-sa5+SD4-SA7 and Msd1-sa5+Msd4-sa7 of VHenv and VHnef, respectively, were detected using sense primer p3- together with antisense primer p10 binding downstream of SA7 (59-ccgttcactaatcgaatggatctgtc-39). All primers are identical or very similar to those used by Houzet et al. [9]. RT-PCRs were done with 500 ng of cytoplasmic RNA and 1 ml from a 50 ml particle RNA fraction isolated from 5 ml of cell culture supernatant as described before [12,13,18]. F.