Figure 1.
Characterization of Vpr+ and Vpr− single-cycle, VSV-G-pseudotyped HIV-1 stocks produced from HEK293T cells.
(A) Quantification of luciferase (Luc) expression from the pNL-Luc-E−R+ (HIV-1 Vpr+) and pNL-Luc-E−R− (HIV-1 Vpr−) proviral DNA constructs in HEK293T cells. Luciferase activity was determined 48 h following plasmid transfection and normalized to protein content. Error bars represent the standard deviation of the mean of three independent experiments. (B) The VSV-G pseudotyped, HIV-1 Vpr+ and HIV-1 Vpr− stocks generated from pNL-Luc-E−R+ and pNL-Luc-E−R− were analyzed by immunoblotting for the incorporation of Vpr into virion particles.
Table 1.
Titration of infectivity of single-cycle and replication-competent HIV-1 stocks.
Figure 2.
Vpr enhances single-cycle HIV-1 infection of activated PBMCs, primary CD4+ T cells, and MDDCs.
(A) PHA-activated peripheral blood mononucleocytes (PBMCs), (B) PHA-activated CD4+ T cells, and (C) Monocyte-derived dendritic cells (MDDCs) were infected at an MOI of 1.0 with single-cycle HIV-1 Vpr+/VSV-G and HIV-1 Vpr−/VSV-G to test the role of Vpr in HIV-1 infection. Luciferase expression from the integrated provirus in the infected cells was assessed at the indicated time and normalized to protein content (10 µg/sample). The data shown represents one of three independent experiments carried out for each cell type from three different donors. Error bars represent the standard deviation of the mean of triplicate samples. Statistically significant differences are indicated by the asterisks (P<0.05).
Figure 3.
Vpr-mediated enhancement of single-cycle HIV-1 infection is independent of VSV-G and Ampho envelopes used for virus pseudotyping.
(A) HuT/CCR5 cells were infected at an MOI of 1.0 with single-cycle HIV-1 Vpr+/VSV-G and HIV-1 Vpr−/VSV-G. Luciferase expression from the integrated provirus in the infected cells was assessed at the indicated time and normalized to protein content (10 µg/sample). The data shown represents one of three independent experiments, and error bars represent standard deviation of the mean of triplicate samples. (B) The MLV amphotrophic (Ampho) envelope pseudotyped, HIV-1 Vpr+ and HIV-1 Vpr− stocks generated from pNL-Luc-E−R+ and pNL-Luc-E−R− proviral constructs were analyzed by immunoblotting for the presence of Vpr. (C) HuT/CCR5 cells were infected at an MOI of 1.0 with HIV-1 Vpr+/Ampho and HIV-1 Vpr−/Ampho. Luciferase expression from the integrated provirus in the infected cells was assessed 3 days post infection and normalized to protein content. Error bars represent standard deviation of the mean of triplicate samples. Statistically significant differences are indicated by the asterisks (P<0.05) and the P value.
Figure 4.
Comparison of the viral DNA profiles in single-cycle HIV-1 Vpr+/VSV-G and HIV-1 Vpr−/VSV-G infected cells.
Cellular DNA was isolated from single cycle HIV-1 Vpr+/VSV-G and HIV-1 Vpr−/VSV-G infected HuT/CCR5 cells (A-C) and MDDCs (D-F) at 24, 48 and 72 h post-infection and subjected to real-time quantitative PCR analysis using Taqman-based primer/probe sets specific to quantify the levels of late-reverse transcription (Late-RT) products, 2-LTR circles, and integrated proviral copies. The amounts of genomic DNA used for the PCR are indicated in each panel. Real-time PCR amplification of the glyceraldehyde-3-phosphate dehydrogenase gene was performed for each sample to normalize for the amount of input DNA in each of the amplification reactions. Error bars represent standard error of the mean of duplicate samples. UD; undetectable under current experimental conditions. Statistically significant differences are indicated by P values. The MDDC data shown represents one of three independent experiments using cells from three different donors.
Figure 5.
Vpr significantly enhances HIV-1 gag mRNA levels in HuT/CCR5 cells and MDDCs.
Total cellular RNA was isolated from single cycle HIV-1 Vpr+/VSV-G and HIV-1 Vpr−/VSV-G infected HuT/CCR5 cells (A) and MDDCs (B) at 3 and 4 days post-infection, respectively, and subjected to RT-PCR to quantify the levels of HIV-1 gag mRNA copies in each cell type. The amplification of the glyceraldehyde-3-phosphate dehydrogenase gene was also performed for each sample to normalize for the amount of input cDNA in each of the amplification reactions. The data is represented as the fold change in the number of gag mRNA copies relative to the HIV-1 Vpr−/VSV-G infected sample in each cell type. Statistically significant differences are indicated by the asterisks (P<0.05). The MDDC data shown represents one of two independent experiments using cells from two different donors.
Figure 6.
DCAF1 knockdown in HuT/CCR5 cells does not affect single-cycle HIV-1 infection.
HuT/CCR5 cells were transduced with concentrated lentivirus expressing either shRNA targeting DCAF1 or a scrambled shRNA (control). Three days following transduction a fraction of cells were analyzed by immunoblotting to confirm DCAF1 knockdown (A), and cells each were infected at an MOI of 0.5 with either HIV-1 Vpr+/VSV-G or HIV-1 Vpr−/VSV-G to test whether DCAF1 was involved in the Vpr-mediated enhancement of HIV-1 infection (B). Luciferase expression in the infected cells was assessed at 3 days post infection.
Figure 7.
Vpr complementation does not affect Vpr-defective single-cycle HIV-1 infection of MDDCs.
(A) Vpr incorporation in VSV-G-pseudotyped, single-cycle HIV-Vpr+ and Vpr-complemented HIV-Vpr-. Virion pellets were analyzed by immunoblotting with anti-Vpr and anti-p24, respectively. (B) Vpr complementation does not affect single-cycle HIV-Vpr- infection of MDDCs. Infected cells were lysed at indicated times post infection for the detection of HIV-1 infection by measuring luciferase activity and normalized to protein content (20 µg/sample). cps, counts per second. The data shown represents one of three independent experiments carried out with three individual donors.
Figure 8.
Vpr significantly enhances replication-competent HIV-1NLAD8 infection in MDDCs.
(A) The HIV-1NLAD8 and HIV-1NLAD8ΔVpr virus stocks produced from HEK293T cells were analyzed by immunoblotting for the presence of Vpr. (B) PHA-activated PBMCs and (C) MDDCs were infected with 5 ng and 20 ng of p24, respectively, from HIV-1NLAD8 and HIV-1NLAD8ΔVpr virus and levels of p24 capsid released into the media during virus replication were assayed over a period of 10 days and 7 days post infection for PBMCs and MDDCs, respectively. The data shown represents one of three independent experiments carried out with three individual donors, and error bars represent standard deviation of triplicate infections. Statistically significant differences are indicated by the asterisks (P<0.05).
Figure 9.
Comparison of the viral DNA profiles in HIV-1NLAD8 and HIV-1NLAD8ΔVpr infected cells.
Quantitative PCR analysis was performed to determine levels of late reverse transcription (Late-RT) products, 2-LTR circles, and integrated proviral copies over a period of 7 days following infection of activated PBMCs (A-C) and MDDCs (D-F) following infection with HIV-1NLAD8 and HIV-1NLAD8ΔVpr. Real-time PCR amplification of the glyceraldehyde-3-phosphate dehydrogenase gene was performed for each sample to normalize for the amount of input DNA in each of the amplification reactions. Error bars represent standard error of the mean of duplicate samples. Statistically significant differences are indicated by the asterisks (P<0.05) and P values. The data shown represents one of three independent experiments carried out for each cell type from three different donors.