Construction of lentiviral vectors.

<p>A) The HIV-1 provirus NL4.3 and the HIV-1-vector V^Hgenomic are shown. Large parts of the <i>gag, pol</i> and <i>env</i> genes are deleted in V^Hgenomic (see white bars in the deleted regions marked by shaded areas). The remaining <i>gag</i> sequence contains parts of the encapsidation signal (Psi, Ψ) and the <i>env</i> fragments contain splicing regulatory elements as well as the RRE. Due to deletions (shaded squares) and frameshift mutations (black asterisks in <i>gag</i> and <i>rev</i>) no viral genes are expressed from V^Hgenomic. Both vectors are drawn to scale. B) Schematic representation of the lentiviral vectors V^Hgenomic, V^Henv and V^Hnef. The intron between SD1 and SA5 or the introns between SD1 and SA5 and between SD4 and SA7 were deleted from V^Hgenomic in V^Henv or V^Hnef, respectively. Unspliced and spliced transcripts with splice sites (5′ splice sites in green and 3′ splice sites in blue) and <i>cis</i>-acting splicing regulatory elements (in orange) are shown. Please note that the unspliced Msd1-sa5 RNA of V^Henv is identical in sequence to the singly-spliced SD1-SA5 RNA of V^Hgenomic. Furthermore, the unspliced Msd1-sa5+Msd4-sa7 RNA of V^Hnef is identical to the fully-spliced SD1-SA5+SD4-SA7 RNA of V^Hgenomic and the singly-spliced Msd1-sa5+SD4-SA7 RNA of V^Henv. Arrowheads represent RT-PCR primers. C) and D) After cotransfection of lentiviral vectors with <i>tat</i> and <i>rev</i> expression plasmids into HEK293T cells cytoplasmic RNA was isolated and analyzed by RT-PCR with primer pairs depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048688#pone-0048688-g001" target="_blank">figure 1B</a>. Agarose gel electrophoretic analyses of PCR products are shown. The amplification products were sequenced to verify splicing between the indicated splice sites.</p

Rev-dependency of the infectious lentiviral vector titer.

<p>A) Cellular lysates and viral particles were harvested two days after transfection of HEK293T cells and were analyzed by an anti-CA Western Blot. The expression plasmid UTRgpRRE contains wild type <i>gag/gagpol</i> gene sequences combined with a part of the viral 5′UTR and the RRE. The Rev-independent <i>gag/gagpol</i> expression plasmid Hgp^syn encodes proteins with wild type amino acid sequences but the gene sequence is dramatically altered due to codon-optimization. B) HEK293 cells were infected with supernatants containing VSV-G pseudotyped lentiviral vectors produced in the presence or absence of Rev. Constant high Gag/GagPol protein levels were provided during vector production by cotransfection of the Rev-independent codon-optimized expression plasmid Hgp^syn. Two days later green fluorescent cells were counted to obtain the infectious titer as GFP forming units per ml of cell culture supernatant (GFU/ml). Titer of the negative control without VSV-G and Gag/GagPol was below 50 GFU/ml (data not shown). Mean values with SEM (standard error of mean) of log10 transformed results obtained in at least 4 independent experiments are shown. Statistical analysis was performed with an unpaired two-tailed t-test with 95% confidence interval. ***, p≤0.001; **, p≤0.01; *, p≤0.05; n.s., not statistically significant.</p

Encapsidation efficiency in the presence and absence of Rev.

<p>The ratio of virion-associated and cytoplasmic RNA levels defines the encapsidation efficiency for all lentiviral vector transcripts detected. The log10 transformed ratios were calculated for each single data pair obtained in each single experiment for all the different RNA species examined. Mean values with SEM obtained 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.</p

Cytoplasmic and virion-associated lentiviral vector RNA levels in the presence and absence of Rev.

<p>A) Cytoplasmic RNA was extracted two days after transfection and analyzed using quantitative RT-PCR protocols (please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048688#pone.0048688.s001" target="_blank">Materials and Methods S1</a> for experimental details). Transcript copy numbers per µg 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 V^Hgenomic are shown in green. RNA levels of the singly-spliced SD1-SA5 RNA of V^Hgenomic and the unspliced Msd1-sa5 transcript of V^Henv 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 V^Hgenomic, the singly-spliced Msd1-sa5+SD4-SA7 RNA of V^Henv and the unspliced Msd1-sa5+Msd4-sa7 RNA of V^Hnef. 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.</p

Direct infection and exposure of CD8α^neg DCs to LCMV Cl13 differentially contribute to Cl13 persistence.

<p>Naïve DCs, those harvested from uninfected mice, incubated with viral peptide are able to stimulate both LCMV specific CD4 and CD8 T cell proliferation, as well as induce IL-10 expression in naïve polyclonal CD4 T cells. Virally exposed DCs, those isolated from LCMV Cl13 infected mice and negative for surface expression of viral NP, are able to stimulate both LCMV specific CD4 and CD8T cell proliferation and also induce IL-10 expression in naïve polyclonal CD4 T cells. Smarta CD4 T cells cultured with Cl13 exposed DCs produce copious amounts of IL-10. Cl13 infected DC, positive for surface expression of viral NP, express enhanced levels of IL-10 as compared to either naïve or exposed DC, are unable to stimulate LCMV specific CD4 or CD8 T cells. The few CD4 T that are induced to proliferate in the presence of infected DCs express elevated levels of IL-10 as compared to either those cultured alone or in the presence of naïve DCs and LCMV peptide.</p

Direct infection and exposure of CD8α^neg DCs to LCMV Cl13 differentially contribute to Cl13 persistence.

<p>Naïve DCs, those harvested from uninfected mice, incubated with viral peptide are able to stimulate both LCMV specific CD4 and CD8 T cell proliferation, as well as induce IL-10 expression in naïve polyclonal CD4 T cells. Virally exposed DCs, those isolated from LCMV Cl13 infected mice and negative for surface expression of viral NP, are able to stimulate both LCMV specific CD4 and CD8T cell proliferation and also induce IL-10 expression in naïve polyclonal CD4 T cells. Smarta CD4 T cells cultured with Cl13 exposed DCs produce copious amounts of IL-10. Cl13 infected DC, positive for surface expression of viral NP, express enhanced levels of IL-10 as compared to either naïve or exposed DC, are unable to stimulate LCMV specific CD4 or CD8 T cells. The few CD4 T that are induced to proliferate in the presence of infected DCs express elevated levels of IL-10 as compared to either those cultured alone or in the presence of naïve DCs and LCMV peptide.</p

Cl13 exposed DCs induce IL-10 expression in naïve polyclonal CD4T cells.

<p>Adult C57BL/6 females were infected with Cl13 or Arm and naïve spleens harvested 7 days post-infection. Splenic DCs were sorted based on infected state (NP^pos infected, NP^neg exposed) and CD8α expression (CD8α^neg used exclusively). (A) 1×10⁴ sorted DCs were cultured with polyclonal CD4T cells from wildtype or IL-10 knock out C57BL/6 mice. Standard deviation is shown. (D) Polyclonal CD4T cells were harvested from OTII transgenic mice and transferred into either naïve B6.SJL or B6.SJL infected with Cl13 four days prior to T cell transfer. Spleens were harvested 3 days post-transfer (7 days post infection), stimulated in vitro with OVA peptide and analyzed by ICCS for IL-10 expression. Shown is data from 2 experiments with n = 3 Cl13 and n = 4 for naïve. Standard error of the mean is shown.</p

DCs from Cl13 infected mice induce IL-10 expression in naïve LCMV specific CD4 T cells.

<p>Adult C57BL/6 were infected with Cl13 or Arm and naïve spleens harvested 7 days post-infection. Splenic DCs were sorted based on infected state (NP^pos infected, NP^neg exposed) and CD8α expression (CD8α^neg used exclusively). (A) Sorted DCs were cultured for 5 days with LCMV TCR transgenic (Smarta) CD4 T cells and IL-10 in culture supernatants was measured by ELISA. (B) Smarta mice on an IL-10 GFP reporter background were co-cultured with sorted DCs and IL-10 expression measured by flow cytometry in gated CD4 T cells 3.5 days post co-culture. The top panels show Smarta CD4 T cultured with Cl13 exposed (NP^neg) or infected (NP^pos) DC (thick black line), or naïve DC (thin grey line) or Smarta CD4 T cells cultured alone (grey filled histogram). The bottom panels show Smarta CD4 T cells cultured with Cl13 infected (NP^pos) DC. Thick black line is gated on total live Smarta CD4 T cells, thin grey line is gated on non-proliferating Smarta CD4 T cells, grey filled histogram depicts proliferating Smarta CD4 T cells (based on dilution of CellTrace^TMViolet proliferation dye, representative CTV gating shown).</p

Directly infected CD8α^neg DCs are unable to stimulate LCMV-specific CD4 and CD8 T cell proliferation.

<p>DCs isolated from C57BL/6 mice infected <i>in vivo</i> with Cl13 7 days prior were sorted based on CD8α and LCMV NP surface expression. Sorted DCs were placed in culture with TCR transgenic LCMV specific CD4 T (Smarta) or CD8 T (P14) cells labeled with CellTrace^TM Violet proliferation dye (CTV) and cultured for 4.5 days. Control cultures contained LCMV peptide (GP33 and GP61) pulsed CD8α^neg DCs from naïve mice. Shown in the black histogram is the relative proliferation of the co-cultured Teff cells (gated on viability, CD4/8 T cell and congenic marker expression). Dashed line histograms are undivided CTV labeled Teff cells cultured alone, filled grey histograms are unlabeled cells. Each condition was set up in triplicate. Representative data from one of three independent experiments are shown.</p

Clone 13 efficiently infects CD8α^neg CD11c^posDC.

<p>Adult C57BL/6 were infected with LCMV Arm, or Cl13 and spleens harvested 7 days post infection. CD11c^pos cells were positively selected by MACS bead isolation following CD19/90 depletion. (A) Isolated cells were stained for CD11c, CD90, CD19, CD8α and cell surface (top two rows) LCMV NP or intracellular (bottom row) LCMV NP. Gating controls are as follows: Naïve DC (dashed line), DCs from Cl13 infected mice incubated with isotype control antibody for LCMV NP Ab (filled grey histogram). (B & C) CD8α^neg DCs were sorted based on cell surface NP expression. (B) RNA was extracted from sorted DCs; reverse transcription was performed and generated cDNA used in Real Time PCR reaction to determine the copy number of LCMV GP, relative to known concentration of control plasmid. ND indicates below detection limits (C) 4×10⁴ sorted DCs were cultured for 2 days in cRPMI. IL-10 levels were measured in culture supernatants of Cl13 infected (NP^pos), exposed (NP^neg) DCs and Naïve CD8α^neg DCs. n = 4–5 mice per treatment per experiment. RealTime and ELISA data from one representative of three independent experiments is shown. Standard deviation is shown.</p