Down-regulation of cathepsin gene expression in response to HIV-1 occurs in a dose-dependent manner. Day-6 MDDCs were treated with HIV-1_BaL at MOIs ranging from 50 to 0.1, or mock treated. After 48 hours, cathepsins B, C, S, and Z gene expression was determined by QPCR. All data were normalized to GAPDH expression. Representative data from 1 of 2 experiments are shown.

Determination of lysosomal enzyme levels in response to HIV-1 infection by Western blot. Day-6 immature MDDCs were exposed to HIV-1_BaL (MOI 10) or mock treated for 48 hours. The relative quantities of lysosomal enzymes present in HIV-1–treated cells compared with mock were then determined by Western blot. Both GAPDH and beta actin were used as loading controls.

Determination of methods for defining the statistical significance of differentially expressed genes by comparing microarrays and quantitative PCR data. Receiver operating characteristic (ROC) curves associated with using B values (unbroken line) or the absolute fold change (dashed line) to discriminate between genes identified as being differentially expressed or not by QPCR. The area under the curve (AUC) and its standard error (SE) are shown for each variable. The closer the AUC is to 1, the better the variable is at classifying differentially expressed genes.⁴¹

Kinetics of differential DC gene expression induced by HIV-1 uptake and infection. Numbers of differentially expressed genes in response to viable and AT-2–treated HIV-1 are shown. Viable and AT-2–inactivated HIV-1–treated MDDC differentially expressed gene lists were generated after filtering according to B value. (A) Commonly expressed genes in viable and AT-2–inactivated HIV-1–treated MDDCs. The numbers of genes differentially expressed in response to viable and AT-2–inactivated HIV-1 at 6, 24, and 48 hours after treatment are illustrated by the solid and broken lines, respectively. The open histogram shows the numbers of genes differentially expressed in response to viable HIV-1 only, and the shaded area shows the number of genes that were commonly changed in their expression in cells treated with viable virus and those treated with AT-2–inactivated virus. (B) Commonly expressed genes between time points. The numbers of genes differentially expressed in response to viable and AT-2–inactivated HIV-1 at 6, 24, and 48 hours after treatment are illustrated by the solid line. The numbers of specific genes expressed at each time point, or common between 2 time points, are shown by the domes, with the number of genes shown. The dotted line illustrates the numbers of genes differentially expressed at all 3 time points with the number shown in a box with a broken line.

Determination of cathepsin enzyme activity in HIV-1–treated MDDCs. Day-6 immature MDDCs were exposed to HIV-1_BaL (MOI 10) or mock treated for 48 hours and then lysed in a low pH buffer. (A) Affinity labeling of active cysteine proteases. Cell lysates were incubated with the activity-based probe for cysteine cathepsins DCG-0N. Labeled cathepsins then detected by Western blot. Lanes: (1) Mock MDDCs, leupeptin, (2) mock MDDCs, (3) HIV-1_BaL-treated MDDCs, (4) boiled HIV-1_BaL–treated MDDCs, (5) monocyte organelles, (6) macrophage cell lysate. Vertical lines denote a section of the Western blot that was included with a lower exposure time. (B) Fluorogenic substrate measurement of cathepsin activity. The combined catalytic activities of cathepsins X, B, L, and S were determined fluorometrically by cleavage of the common synthetic substrate Z-Phe-Arg-7-amido-4-methylcoumarin. Specific catalytic activities of cathepsins S and C were measured using the substrates Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH₂ and NH₂-Gly-Arg-AMC, respectively. Samples were measured in triplicate.

HIV gp120 binding affects differential expression of T-cell but not DC genes. (A) Binding of HIV-1 envelope proteins to MDDCs. Day-6 immature MDDCs (5 × 10⁶) were incubated with biotinylated monomeric gp120 (BaL strain) and trimeric gp140 (AD8 and UG037 strains) ranging from 0 to 0.15 μg/μL. Biotinylated gp120/140 binding was determined by flow cytometry using PE-conjugated streptavidin. The geometric means of the fluorescent intensity data for the UG037 strain of trimer are displayed in the histogram. (B) Biologic activity of gp120 species. Naive CD4 T cells (5 × 10⁶) were incubated with monomeric gp120 (BaL strain) and trimeric gp140 (AD8 and UG037 strains) at 50 ng per 10⁶ cells for 24 hours. Differential expression of the GUCY2D and CCL3 genes was determined by QPCR. The fold change in gene expression compared with mock-treated cells is displayed in the histogram. (C) Differential MDDC gene expression in response to monomeric and trimeric HIV-1 envelope species. Day-6 immature MDDCs were treated with either recombinant monomeric gp120 (BaL strain) or 1 of 2 trimeric gp140 stocks (AD8 and UG037 strains) for 6, 24, and 48 hours (monomer) or 2, 6, and 24 hours (trimers). Global DC gene expression was determined using Illumina gene expression chips (trimeric gp140s). A representative MA plot is shown with 1.5-fold change lines for trimeric gp140 (UG037 strain) 24 hours after treatment.

Kinetics of differential gene expression corresponds with the 2 phases of HIV-1 processing by dendritic cells (DCs). (A) Routes of entry of HIV-1 into DCs. Following initial binding of HIV-1 to C-type lectin receptors (CLRs) on the cell surface, HIV-1 can enter the DC by 1 of 2 routes. (1) More than 95% of the virus is rapidly endocytosed resulting in processing by the endolysosomal pathway and degradation by acid proteolysis in late endosomes. (2) The remaining less than 5% of attached virus is transferred to cell surface CD4/CCR5 resulting in fusion of the viral envelope with the DC plasma membrane and entry of the capsid into cytoplasm, resulting in reverse transcription, HIV-1 cDNA generation (first detected < 24 hours after infection = second phase of de novo replication), and then the later stages of viral replication and assembly. (B) Corresponding HIV-1 nucleic acid levels in DCs after HIV-1 treatment. The graph shows a representation of the HIV-1 nucleic acid levels after treatment of DCs. As shown by the dotted line, at time 0 the high levels of HIV-1 nucleic acid represents input virions. The nucleic acid levels then rapidly decrease to undetectable levels by 20 hours after infection as the vast majority of HIV-1 enters and is destroyed by late endosomes. As shown by the solid line, 20 hours after infection viral nucleic acid levels begin to slowly rise, representing progeny virions resulting from productive infection by the small amount of virus that was able to fuse with the plasma membrane and initiate a productive infection. (C) Temporal differential gene expression corresponds to the kinetics of the 2 phases of HIV-1 processing by DCs. At 6 hours, a burst of gene expression was seen as a consequence of viral entry into the DC (predominantly via endocytosis). By 24 hours, most of the virus that entered via endocytosis has been degraded and virions that entered by fusion with the plasma membrane have yet to undergo replication. Correspondingly very few genes are differentially expressed at this time point. At 48 hours, a fresh burst of gene expression is detected, representing genes differentially expressed as a result of HIV-1 replication in DCs.