Genetic labeling of HIV-1 proteins and its impact on virus infectivity.

<p>(A) Diagram of the HIV-1 <i>gag</i> (red), <i>pol</i> (brown) and <i>env</i> (blue) open reading frames and the positions where the tetracysteine (TC) tag motif was inserted. The majority of the TC motifs were inserted five amino acids downstream (-N tags) or upstream (-C tags) of the protease cleavage site and the first or last five amino acids of the viral protein coding sequence were repeated to prevent alterations in the minimal recognition site for the viral protease. The left insert shows the crystal structure of the RT enzyme and the positions in the “thumb” region where the TC tag was inserted are shown in blue. The right insert shows the crystal structure of the Env gp120 and the positions in the V1/V2 region where the TC tag was inserted are shown in blue. (B) Virion protein processing profiles of 293T cell-derived HIV^MA-C, HIV^CA-N, HIV^CA-C, HIV^NC-N, HIV^NC-C, HIV^PR-C, HIV^RT-N, HIV^RT-C, HIV^RN-N, HIV^RN-C and HIV^IN-N (lanes 1-12, respectively). HIV-1 proteins were detected by Western Blot using pooled HIV-positive patient sera. Data are representative of 2 independent experiments. (C) The infectivity of HIV^MA-C, HIV^CA-N, HIV^CA-C, HIV^NC-N, HIV^NC-C, HIV^PR-C, HIV^RT-N, HIV^RN-N, HIV^RN-C and HIV^IN-N was assessed by comparing their replication kinetics in peripheral blood mononuclear cells with HIV^wt. Samples were collected at 3, 7, 11 and 14 days post infection, and the levels of virus replication were monitored using an <i>in vitro</i> reverse transcriptase assay that is specific for HIV-1 enzymatic activity. Data are representative of 2-3 independent donors. Error bars represent technical replicates. (D) The capacity of several HIV^TC to enter target cells was assessed by measuring β-lactamase activity in MT-2 cells that have been infected with HIV^TC containing a β-lactamase-Vpr fusion protein. Error bars, s.d. are based on the averages of 2-3 independent experiments. (E) The capacity of HIV^TC to infect target cells was assessed by measuring luciferase activity in the indicator TZM-bl cells that have been infected with the indicated viruses. Error bars, s.d. are based on the averages of 2-3 independent experiments.</p

Improve the infectivity of the <i>gag</i>-TC viruses.

<p>(A) Schematic representation of proviral DNA constructs used in the study. The HIV^TC-ΔRev construct differs from HIV^TC by the introduction of an early termination codon and a frameshift into exon 2 of the Rev sequence, which was removed to inactivate Rev function. This mutation also affects Env expression. HIV^NFS is a full-length HIV-1 construct containing codon modifications in the -1 frameshift slippery sequences and the RNA pseudoknot in the <i>gag</i> reading frame to enable the expression of Gag but not Gag-Pol. (B) The capacity of HIV^TC-ΔRev/NFS to infect target cells was assessed by measuring luciferase activity in the indicator TZM-bl cells that have been infected with the indicated viruses. Error bars, s.d. are based on the averages of 3-5 independent experiments.</p

Overview of the modifications introduced in the HIV-1 genome.

<p>Overview of the modifications introduced in the HIV-1 genome.</p

Primers used for site-directed mutagenesis.

<p>Primers used for site-directed mutagenesis.</p

Labeling of tetracysteine-tagged matrix proteins of an infectious HIV-1 does not impact on virus function.

<p>(A) Production of HIV^wt and HIV^MA-C from 293Ts that have been untreated (no FlAsH) or incubated with 1 µM FlAsH-EDT₂ (FlAsH). The level of virus production was measured with an <i>in vitro</i> reverse transcriptase assay that is specific for HIV-1 enzymatic activity. Data are representative of 2 independent experiments. Error bars represent technical replicates. (B) Quantitative PCR analysis of the kinetics of HIV-1 cDNA synthesis during an infection in MT-2 cells. Cells were infected with HIV^wt or with HIV^wt and HIV^MA-C produced in the presence of 1 µM FlAsH-EDT₂ as described above (HIV^wt-F and HIV^MA-C-F). Cells were harvested at the indicated time points post-infection and analyzed by quantitative PCR for both early ([-]ssDNA) and late (post second strand transfer) HIV-1 reverse transcription products. HIV-1 cDNA copies were normalized by the number of copies of the CCR5 gene in the host cells. Data are representative of 2 independent experiments. (C-D) Specificity of the labeling of tetracysteine-tagged virus with biarsenical dyes. (C) HIV^MA-C-F and HIV^wt-F (cell-free virus) were immunostained with HIV-1 p17-MA and p24-CA mAb followed by donkey anti-mouse Alexa Fluor 647. (D) MT-2 cells were fixed after 20 min of synchronized infection with HIV^MA-C-F or HIV^wt-F and immunostained with HIV-1 p17-MA and p24-CA monoclonal antibodies followed by donkey anti-mouse Alexa Fluor 647 secondary antibody. Nuclei were labeled with Hoechst. FlAsH is shown in green, Alexa Fluor 647 in red and nuclei in blue. The provided images were derived from a volume compression of a z stack of 10-38 images taken at a 0.2-µm step size. Scale bar, 5 µm. Images are representative of more than 3 independent experiments. (E) MT-2 cells were infected with HIV^NC-C or HIV^{NC-C-ΔRev/NFS(4∶1)} produced in the presence of 1 µM FlAsH-EDT₂ as described above (HIV^NC-C-F or HIV^{NC-C-ΔRev/NFS(4∶1)-F}, respectively). The infected cells were fixed after 4 h of synchronized infection, and the nuclei were labeled with Hoechst. FlAsH is shown in green and nuclei are shown in blue. The provided images were derived from a volume compression of a z stack of 26 images taken at a 0.5-µm step size. Scale bar, 5 µm. Images are representative of 2 independent experiments. (G) Quantification of the relative intensity of FlAsH staining using softWoRx software (Applied Precision, Issaquah, WA) following infection with HIV^NC-C-F or HIV^{NC-C-ΔRev/NFS(4∶1)-F}. Each symbol represents a separate field from the same experiment. The black lines show the mean. Data are representative of 2 independent experiments.</p

Changes in gene expression in DC-induced latently infected CD4⁺ T cells.

<p>(<b>A</b>) Fold change scatterplot comparing gene expression between HIV T (+DC) (CD4⁺ T cells cultured with DC and HIV), and Mock T (+DC) (CD4⁺ T cells cultured with only DC) relative to their controls, HIV T (CD4⁺ T cells cultured with HIV) and Mock T (CD4⁺ T cells cultured in media alone) respectively. The solid line indicates absolute 2-fold change. (<b>B</b> and <b>C</b>) Top two gene interaction networks as ranked by Ingenuity Pathway Analysis. The networks were built from the list of differentially expressed genes induced by HIV T (+DC), relative to Mock T (+DC) after subtracting HIV T and Mock T from each group respectively. Genes highlighted in red were up-regulated and those in blue were down-regulated. The different node shapes indicate genes in different functional categories according to the legend. The interactions between the different nodes are shown as solid (direct interaction) or dashed (indirect interaction) lines (edges). (<b>D</b>) Fold change in gene expression values for selected genes from the Illumina BeadArrays plotted against Real-Time PCR (qPCR) deltaCt values for each target gene. PCR targets were mapped to BeadArray probes by matching the official gene symbols.</p

Soluble factors in DC-induced HIV latency.

<p>(<b>A</b>) Resting CD4⁺ T cells were cultured alone (light grey) or with sorted pDC (grey) or mDC (dark grey). At day 5 post-infection, cytokines and chemokines were quantified in culture supernatants using cytometric bead arrays, n = 3. *<i>P</i>&lt;0.05; **<i>P</i>&lt;0.01 (paired t-test). (<b>B</b>) Latent infection was quantified in eFluor670^hiEGFP⁻ resting CD4⁺ T cells that were cultured either alone or with sorted pDC in the presence of media alone (light grey), anti-IgG (grey) or anti-IFN-alpha (dark grey) following stimulation with anti-CD3/CD28 in the presence of L8, n = 5. (<b>C</b>) Resting CD4⁺ T cells were co-cultured with mDC with (grey) or without (light grey) the addition of equal numbers of pDC. Productive infection was determined at day 5 post-infection. Latent infection was determined in sorted eFluor670^hiEGFP⁻ CD4⁺ T cells following stimulation with anti-CD3/CD28 in the presence of L8, n = 5. (<b>D</b>) Latent infection was quantified in eFluor670^hiEGFP⁻ resting memory CD4⁺ T cells that were cultured either alone or with sorted mDC in the presence of media alone (light grey), anti-IgG (grey) or neutralising antibodies (dark grey) to IL-6, IL-10-receptor, CXCR3 or CCL19, n = 5. (<b>E</b>) eFluor670-labelled resting CD4⁺ T cells were cultured either alone (light grey) or with blood mDC (dark grey). Virus was added to (<b>i</b>) CD4⁺ T cells cultured alone; (<b>ii</b>) CD4⁺ T cells co-cultured with mDC; (<b>iii</b>) CD4⁺ T cells cultured with mDC in the presence of a 0.4 µm membrane transwell and latency determined at day 5 post-infection, n = 5. (<b>F</b>) eFluor670-labelled resting CD4⁺ T cells were cultured either (<b>i</b>) alone (light grey) or (<b>ii</b>) with blood mDC (dark grey) and infected. (<b>iii</b>) Following 24 hours, supernatant from infected mDC-T cell co-cultures was added to uninfected resting CD4⁺ T cells and these cells were then infected, n = 3. Columns represent the median of 3–5 donors and error bars indicate the interquartile range. *<i>P</i>&lt;0.05; **<i>P</i>&lt;0.01 (Wilcoxon signed-rank test).</p

Myeloid DC induce post-integration latency in non-proliferating memory CD4⁺ T cells.

<p>SNARF-labelled resting CD4⁺ T cells were cultured alone (light grey) or with syngeneic plasmacytoid (pDC; grey) or myeloid DC (mDC; dark grey). (<b>A</b>) Productive infection (EGFP⁺ cells) was determined by flow cytometry on day 5 post-infection. (<b>B</b>) Latent infection was quantified in SNARF^hiEGFP⁻ cells following either addition of PHA-activated PBMC, n = 5; or (<b>C</b>) direct activation with anti-CD3/CD28 in the presence or absence of the integrase inhibitor L8. (<b>D</b>) Integrated HIV DNA was quantified in the sorted SNARF^hiEGFP⁻ T cells by Alu-LTR real-time PCR, n = 3. (<b>E</b>) Productive and latent infection was determined in SNARF^hiEGFP⁻ CD4⁺ T cells from mDC-T cell co-cultures following infection with nef-deficient (-nef) or nef-competent EGFP HIV. (<b>F</b>) Latent infection was determined in sorted SNARF^hiEGFP^- CD4⁺ T cells, cultured alone or with mDC, following activation with PHA-PBMC. (<b>G</b>) Productive and latent infection was determined in SNARF^hiEGFP⁻ CD4⁺ T cells from mDC-T cell co-cultures with and without Staphylococcus Enterotoxin B (SEB), n = 4. (<b>H</b>) SNARF-labelled resting CD4⁺ T cells were cultured alone (light grey) or with syngeneic mDC (grey) at decreasing DC∶T cell ratios and latent infection quantified in sorted SNARF^hiEGFP⁻ T cells following addition of PHA-activated PBMC, n = 5. (<b>I</b>) Resting CD4⁺ T cells were cultured either alone or in the presence of mDC. At day 5 post-infection, SNARF^hiEGFP⁻ cells were sorted into naïve (light grey) or memory (grey) CD4⁺ T cells and latent infection quantified, n = 5. The lower limit of detection is represented by a dotted line. Columns represent the median of 3–7 donors and error bars indicate the interquartile range. *<i>P</i>&lt;0.05; **<i>P</i>&lt;0.01; ns, not significant (Wilcoxon signed-rank test).</p

DC-induced latency in resting CD4⁺ T cells.

<p>(<b>A</b>) Resting CD4⁺ T cells were isolated from the blood of healthy donors and labelled with the proliferation dye SNARF, which decreases in intensity following each round of cell division allowing identification of non-proliferating cells. SNARF-labelled resting CD4⁺ T cells were cultured either alone or with syngeneic blood DC. Following 24 hours of culture, cells were infected with NL(AD8)-nef/EGFP at an MOI of 0.5. All culture media was supplemented with IL-2 (2 U/mL). (<b>B</b>) At day 5 post-infection, the number of productively infected (EGFP⁺) cells was determined and the non-proliferating (SNARF^hi) cells that were not productively infected (EGFP⁻) were sorted. The sorted SNARF^hiEGFP⁻ cells were stimulated with PHA/IL-2 in the presence of PBMC and cultured for 5 days to amplify any replication competent latent infection. (<b>C</b>) Productive infection and (<b>D</b>) latent infection following infection of T cells cultured alone (light grey) or in the presence of DC (grey) is shown. (<b>E</b>) Latent infection in the presence of DC cultured with (grey) or without (light grey) 0.1 µM Indinavir. (<b>F</b>) Expression of the early (CD69; black) and late (HLA-DR; grey) surface activation markers and the intracellular proliferation marker Ki67 (light grey) was quantified by flow cytometry on sorted SNARF^hiEGFP⁺ CD4⁺ T cells following HIV infection of T cells cultured alone or in the presence of DC. The lower limit of detection of each assay is represented by a dotted line. Columns represent the median of 5 independent experiments and error bars indicate the interquartile range. *<i>P</i>&lt;0.05 (Wilcoxon signed-rank test).</p