Figure 1.
Map of HIV-1 and SIV Vector Constructs
Opposing arrows mark primer binding sites for the quantitative real-time PCR. Predicted secondary structures for wild-type (wt RRE) and mutated RRE are shown, with the grey stem loop representing the MS2-derived RNA motif. (A) HIV-1 vector constructs, (B) SIV vector constructs.
Blas-EGFP, EGFP cDNA fused in frame to the Blasticidin resistance gene; CMV, human cytomegalovirus immediate early promoter; EGFP, gene for the enhanced green fluorescent protein; MLV, murine leukemia virus promoter; R, direct repeat sequence; SA, splice acceptor; SD, splice donor; U3, 3′ unique sequence; U5, 5′ unique sequence.
Figure 2.
Effect of Rev on Encapsidation of HIV-1 and SIV Vector RNA
(A) The HIV-1 vector plasmid VH was cotransfected with expression plasmids for HIV-1 gag-pol, VSV-G, and HIV-1 tat in the presence (+Rev) or absence (−Rev) of an HIV-1 rev expression plasmid. Cytoplasmic RNA levels, packaging efficiency, and vector titers are presented relative to the values obtained in the same transfection experiment with VH in the presence of Rev. The mean value and standard deviation of three to eight independent transfection experiments are shown.
(B) The SIV vector plasmid VS was cotransfected with expression plasmids for SIV gag-pol, VSV-G, and HIV-1 tat in the absence (−Rev) or presence of an SIV (+SIV-Rev) or HIV-1 rev (+Rev) expression plasmid. The mean value and standard deviation of four independent transfection experiments are shown. Numbers above the bars indicate fold induction by Rev.
(C) Using a real-time PCR for preGAPDH RNA, threshold cycle numbers (Ct) were determined for four nuclear (N) and cytoplasmic (C) RNA preparations in three independent experiments. Relative amounts of preGAPDH RNA levels were calculated as arbitrary units (AU) per microgram extracted RNA by including 10-fold serial dilutions of standard nuclear RNA preparations. Ct values of a typical experiment are shown in the left panel, and the table summarizes the results.
(D) Western blot analysis of total cell lysate (T), and cytoplasmic (C) and nuclear (N) fractions for Lamin B.
Figure 3.
Effect of Rev on Gag-Pol Particle Formation
Viral particles were pelleted from the supernatant of cells transfected in the presence (+) or absence (-) of an HIV-1 rev expression plasmid with codon-optimized gag-pol expression plasmids of SIV (Sgpsyn) and HIV-1 (Hgpsyn) (A) or a wild-type HIV-1 gag-pol expression plasmid (UTRgp-RRE) (B). Western blot analyses were performed with pelleted particles and a CA-specific monoclonal antibody (upper panel). To control for transfection efficiency, a GFP expression plasmid had been included during transfection, and lysates of transfected cells were analyzed using an anti-GFP antibody (lower panel).
(C) Cytoplasmic and particle-associated vector RNA copy numbers were determined in cell cultures transfected with different amounts of the VH vector plasmid in the presence or absence of Rev expression plasmid. In addition, pelletable p24CA levels were determined and used to calculate the packaging efficiency as particle-associated vector RNA copies per nanogram p24CA.
Figure 4.
Effect of Rev on RNA Packaging in Cells Infected with HIV-1- or SIV-Based Vectors
(A) HIV-1-based vectors. 293T cells stably infected with the VH vector were transfected with Hgpsyn and expression plasmids for VSV-G and HIV-1 tat in the presence (+Rev) or absence (−Rev) of the HIV-1 rev expression plasmid.
(B) SIV-based vectors. 293T cells stably infected with VS-Blas were cotransfected with Sgpsyn and VSV-G and tat expression plasmids in the presence or absence of the rev expression plasmid. Cytoplasmic RNA levels, packaging efficiencies, and vector titers are presented relative to those obtained in the same transfection experiment in the presence of Rev.
The mean value and standard deviation of three independent transfection experiments are shown. Numbers above the bars indicate fold induction by Rev. 1, result of single transfection experiment.
Figure 5.
Relevance of the Rev–RRE Interaction
(A) An HIV-1 vector plasmid in which stem loop II of the RRE was replaced by the MS2 stem loop was cotransfected with expression plasmids for HIV-1 gag-pol, VSV-G, and tat in the absence (−Rev) or presence of a rev (+Rev) expression plasmid, or in the presence of an expression plasmid encoding a fusion protein of Rev and the MS2 coat protein (+Rev-MSC). (B) An SIV vector with a deletion (VSΔSL in Figure 1B) of stem loop II of the RRE was cotransfected with expression plasmids for SIV gag-pol, VSV-G, and tat in the absence (−Rev) or presence of an HIV-1 (+Rev) or SIV rev expression plasmid. Cytoplasmic RNA levels, packaging efficiency, and vector titers were determined as described, and all values are expressed relative to the values obtained in the same transfection experiment for the parental VH (A) and VS (B) vectors in the presence of Rev, which were set as 100%.
Mean value and standard deviation of at least three independent transfection experiments are shown. Numbers above the short vertical lines indicate fold induction by Rev.