Sidation efficiency of the unspliced RNA of VHgenomic (TBHQ site 53-fold) confirms our previous results (figure 2 and 4 and [13]). Therefore, unspliced RNA of VHgenomic exported to the cytoplasm in the 
absence of Rev cannot be efficiently encapsidated resulting in low viral titers. This could be explained by a direct or an indirect role of Rev in the encapsidation 25033180 process. Binding of Rev to the RRE or to the Rev-binding site in the 59UTR [28] could initiate the encapsidation process. However, such a direct effect is difficult to envision, because Rev was never reported to interact with HIV Gag and Rev is not known to be part of the MedChemExpress Hexokinase II Inhibitor II, 3-BP HIV-virion. Therefore, the most plausible explanation is a more indirect effect such as the generation of an inhibitory ribonucleoprotein complex in the nucleus that prevents further cytoplasmic utilization of the introncontaining lentiviral get Madrasin vector RNA. A Rev-mediated nuclear export of this RNA would prevent/disrupt the association of inhibitory factors with the RNA and would allow cytoplasmic packaging to take place. Other possible explanations for a low efficient encapsidation process without Rev could be trapping 
of the RNA at a sub-cytoplasmic localization unfavorable for encapsidation or an inhibitory RNA structure. Nuclear export mediated by Rev could traffic the RNA to productive sub-cytoplasmic sites or prevent formation of inhibitory RNA structures thereby enabling efficient encapsidation by Gag [14]. Similar to the situation observed for VHgenomic, Rev-dependent encapsidation of RRE-containing RNAs from VHenv correlates with an enhanced infectious vector titer in the presence of Rev. However, the titer was increased by a factor of 6 whereas packaging of RRE-containing RNAs was enhanced by two orders of magnitude. Both the infectious titer and encapsidation of MsdRev-Stimulated Encapsidation of Spliced Vector RNAFigure 3. Cytoplasmic and get K162 virion-associated lentiviral vector RNA levels in the presence and absence of Rev. A) Cytoplasmic RNA was extracted two days after transfection and analyzed using quantitative RT-PCR protocols (please see Materials and Methods S1 for experimental details). Transcript copy numbers per mg of cytoplasmic RNA are shown. B) Virion-associated RNA was isolated from cell culture supernatants of cells analyzed in A. Transcript copy numbers per ml of cellular supernatant were obtained after RT-qPCR analyses. Unspliced RNA levels of VHgenomic are shown in green. RNA levels of the singly-spliced SD1-SA5 RNA of VHgenomic and the unspliced Msd1-sa5 transcript of VHenv are depicted in blue. These RNAs represent the class of singly-spliced transcripts. Shown in red are transcript levels of the multiply-spliced SD1-SA5+SD4-SA7 RNA of VHgenomic, the singly-spliced Msd1-sa5+SD4-SA7 RNA of VHenv and the 
unspliced Msd1-sa5+Msd4-sa7 RNA of VHnef. These RNAs correspond to the class of fully-spliced transcripts. Mean values with SEM of log10 transformed RNA copy numbers obtained in 5 independent experiments are shown. Statistical analysis was performed with a one-way ANOVA combined with the Newman-Keuls post-test. ***, p#0.001; **, p#0.01; *, p#0.05; n.s., not statistically significant. doi:10.1371/journal.pone.0048688.gRev-Stimulated Encapsidation of Spliced Vector RNAFigure 4. Encapsidation efficiency in the presence and absence of Rev. The ratio of virion-associated and cytoplasmic RNA levels defines the encapsidation efficiency for all lentiviral vector transcripts detected. The log10 transformed.Sidation efficiency of the unspliced RNA of VHgenomic (53-fold) confirms our previous 
results (figure 2 and 4 and [13]). Therefore, unspliced RNA of VHgenomic exported to the cytoplasm in the absence of Rev cannot be efficiently encapsidated resulting in low viral titers. This could be explained by a direct or an indirect role of Rev in the encapsidation 25033180 process. Binding of Rev to the RRE or to the Rev-binding site in the 59UTR [28] could initiate the encapsidation process. However, such a direct effect is difficult to envision, because Rev was never reported to interact with HIV Gag and Rev is not known to be part of the HIV-virion. Therefore, the most plausible explanation is a more indirect effect such as the generation of an inhibitory ribonucleoprotein complex in the nucleus that prevents further cytoplasmic utilization of the introncontaining lentiviral vector RNA. A Rev-mediated nuclear export of this RNA would prevent/disrupt the association of inhibitory factors with the RNA and would allow cytoplasmic packaging to take place. Other possible explanations for a low efficient encapsidation process without Rev could be trapping of the RNA at a sub-cytoplasmic localization unfavorable for encapsidation or an inhibitory RNA structure. Nuclear export mediated by Rev could traffic the RNA to productive sub-cytoplasmic sites or prevent formation of inhibitory RNA structures thereby enabling efficient encapsidation by Gag [14]. Similar to the situation observed for VHgenomic, Rev-dependent encapsidation of RRE-containing RNAs from VHenv correlates with an enhanced infectious vector titer in the presence of Rev. However, the titer was increased by a factor of 6 whereas packaging of RRE-containing RNAs was enhanced by two orders of magnitude. Both the infectious titer and encapsidation of MsdRev-Stimulated Encapsidation of Spliced Vector RNAFigure 3. Cytoplasmic and virion-associated lentiviral vector RNA levels in the presence and absence of Rev. A) Cytoplasmic RNA was extracted two days after transfection and analyzed using quantitative RT-PCR protocols (please see Materials and Methods S1 for experimental details). Transcript copy numbers per mg of cytoplasmic RNA are shown. B) Virion-associated RNA was isolated from cell culture supernatants of cells analyzed in A. Transcript copy numbers per ml of cellular supernatant were obtained after RT-qPCR analyses. Unspliced RNA levels of VHgenomic are shown in green. RNA levels of the singly-spliced SD1-SA5 RNA of VHgenomic and the unspliced Msd1-sa5 transcript of VHenv are depicted in blue. These RNAs represent the class of singly-spliced transcripts. Shown in red are transcript levels of the multiply-spliced SD1-SA5+SD4-SA7 RNA of VHgenomic, the singly-spliced Msd1-sa5+SD4-SA7 RNA of VHenv and the unspliced Msd1-sa5+Msd4-sa7 RNA of VHnef. These RNAs correspond to the class of fully-spliced transcripts. Mean values with SEM of log10 transformed RNA copy numbers obtained in 5 independent experiments are shown. Statistical analysis was performed with a one-way ANOVA combined with the Newman-Keuls post-test. ***, p#0.001; **, p#0.01; *, p#0.05; n.s., not statistically significant. doi:10.1371/journal.pone.0048688.gRev-Stimulated Encapsidation of Spliced Vector RNAFigure 4. Encapsidation efficiency in the presence and absence of Rev. The ratio of virion-associated and cytoplasmic RNA levels defines the encapsidation efficiency for all lentiviral vector transcripts detected. The log10 transformed.Sidation efficiency of the unspliced RNA of VHgenomic (53-fold) confirms our previous results (figure 2 and 4 and [13]). Therefore, unspliced RNA of VHgenomic exported to the cytoplasm in the absence of Rev cannot be efficiently encapsidated resulting in low viral titers. This could be explained by a direct or an indirect role of Rev in the encapsidation 25033180 process. Binding of Rev to the RRE or to the Rev-binding site in the 59UTR [28] could initiate the encapsidation process. However, such a direct effect is difficult to envision, because Rev was never reported to interact with HIV Gag and Rev is not known to be part of the HIV-virion. Therefore, the most plausible explanation is a more indirect effect such as the generation of an inhibitory ribonucleoprotein complex in the nucleus that prevents further cytoplasmic utilization of the introncontaining lentiviral vector RNA. A Rev-mediated nuclear export of this RNA would prevent/disrupt the association of inhibitory factors with the RNA and would allow cytoplasmic packaging to take place. Other possible explanations for a low efficient encapsidation process without Rev could be trapping of the RNA at a sub-cytoplasmic localization unfavorable for encapsidation or an inhibitory RNA structure. Nuclear export mediated by Rev could traffic the RNA to productive sub-cytoplasmic sites or prevent formation of inhibitory RNA structures thereby enabling efficient encapsidation by Gag [14]. Similar to the situation observed for VHgenomic, Rev-dependent encapsidation of RRE-containing RNAs from VHenv correlates with an enhanced infectious vector titer in the presence of Rev. However, the titer was increased by a factor of 6 whereas packaging of RRE-containing RNAs was enhanced by two orders of magnitude. Both the infectious titer and encapsidation of MsdRev-Stimulated Encapsidation of Spliced Vector RNAFigure 3. Cytoplasmic and virion-associated lentiviral vector RNA levels in the presence and absence of Rev. A) Cytoplasmic RNA was extracted two days after transfection and analyzed using quantitative RT-PCR protocols (please see Materials and Methods S1 for experimental details). Transcript copy numbers per mg of cytoplasmic RNA are shown. B) Virion-associated RNA was isolated from cell culture supernatants of cells analyzed in A. Transcript copy numbers per ml of cellular supernatant were obtained after RT-qPCR analyses. Unspliced RNA levels of VHgenomic are shown in green. RNA levels of the singly-spliced SD1-SA5 RNA of VHgenomic and the unspliced Msd1-sa5 transcript of VHenv are depicted in blue. These RNAs represent the class of singly-spliced transcripts. Shown in red are transcript levels of the multiply-spliced SD1-SA5+SD4-SA7 RNA of VHgenomic, the singly-spliced Msd1-sa5+SD4-SA7 RNA of VHenv and the unspliced Msd1-sa5+Msd4-sa7 RNA of VHnef. These RNAs correspond to the class of fully-spliced transcripts. Mean values with SEM of log10 transformed RNA copy numbers obtained in 5 independent experiments are shown. Statistical analysis was performed with a one-way ANOVA combined with the Newman-Keuls post-test. ***, p#0.001; **, p#0.01; *, p#0.05; n.s., not statistically significant. doi:10.1371/journal.pone.0048688.gRev-Stimulated Encapsidation of Spliced Vector RNAFigure 4. Encapsidation efficiency in the presence and absence of Rev. The ratio of virion-associated and cytoplasmic RNA levels defines the encapsidation efficiency for all lentiviral vector transcripts detected. The log10 transformed.Sidation efficiency of the unspliced RNA of VHgenomic (53-fold) confirms our previous results (figure 2 and 4 and [13]). Therefore, unspliced RNA of VHgenomic exported to the cytoplasm in the absence of Rev cannot be efficiently encapsidated resulting in low viral titers. This could be explained by a direct or an indirect role of Rev in the encapsidation 25033180 process. Binding of Rev to the RRE or to the Rev-binding site in the 59UTR [28] could initiate the encapsidation process. However, such a direct effect is difficult to envision, because Rev was never reported to interact with HIV Gag and Rev is not known to be part of the HIV-virion. Therefore, the most plausible explanation is a more indirect effect such as the generation of an inhibitory ribonucleoprotein complex in the nucleus that prevents further cytoplasmic utilization of the introncontaining lentiviral vector RNA. A Rev-mediated nuclear export of this RNA would prevent/disrupt the association of inhibitory factors with the RNA and would allow cytoplasmic packaging to take place. Other possible explanations for a low efficient encapsidation process without Rev could be trapping of the RNA at a sub-cytoplasmic localization unfavorable for encapsidation or an inhibitory RNA structure. Nuclear export mediated by Rev could traffic the RNA to productive sub-cytoplasmic sites or prevent formation of inhibitory RNA structures thereby enabling efficient encapsidation by Gag [14]. Similar to the situation observed for VHgenomic, Rev-dependent encapsidation of RRE-containing RNAs from VHenv correlates with an enhanced infectious vector titer in the presence of Rev. However, the titer was increased by a factor of 6 whereas packaging of RRE-containing RNAs was enhanced by two orders of magnitude. Both the infectious titer and encapsidation of MsdRev-Stimulated Encapsidation of Spliced Vector RNAFigure 3. Cytoplasmic and virion-associated lentiviral vector RNA levels in the presence and absence of Rev. A) Cytoplasmic RNA was extracted two days after transfection and analyzed using quantitative RT-PCR protocols (please see Materials and Methods S1 for experimental details). Transcript copy numbers per mg of cytoplasmic RNA are shown. B) Virion-associated RNA was isolated from cell culture supernatants of cells analyzed in A. Transcript copy numbers per ml of cellular supernatant were obtained after RT-qPCR analyses. Unspliced RNA levels of VHgenomic are shown in green. RNA levels of the singly-spliced SD1-SA5 RNA of VHgenomic and the unspliced Msd1-sa5 transcript of VHenv are depicted in blue. These RNAs represent the class of singly-spliced transcripts. Shown in red are transcript levels of the multiply-spliced SD1-SA5+SD4-SA7 RNA of VHgenomic, the singly-spliced Msd1-sa5+SD4-SA7 RNA of VHenv and the unspliced Msd1-sa5+Msd4-sa7 RNA of VHnef. These RNAs correspond to the class of fully-spliced transcripts. Mean values with SEM of log10 transformed RNA copy numbers obtained in 5 independent experiments are shown. Statistical analysis was performed with a one-way ANOVA combined with the Newman-Keuls post-test. ***, p#0.001; **, p#0.01; *, p#0.05; n.s., not statistically significant. doi:10.1371/journal.pone.0048688.gRev-Stimulated Encapsidation of Spliced Vector RNAFigure 4. Encapsidation efficiency in the presence and absence of Rev. The ratio of virion-associated and cytoplasmic RNA levels defines the encapsidation efficiency for all lentiviral vector transcripts detected. The log10 transformed.