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 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 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.