Characterization of human immunodeficiency virus type 1 replication in immature and mature dendritic cells reveals dissociable cis- and trans-infection

Abstract

Dendritic cells (DCs) transmit human immunodeficiency virus type 1 (HIV-1) to CD4(+) T cells through the trans- and cis-infection pathways; however, little is known about the relative efficiencies of these pathways and whether they are interdependent. Here we compare cis- and trans-infections of HIV-1 mediated by immature DCs (iDCs) and mature DCs (mDCs), using replication-competent and single-cycle HIV-1. Monocyte-derived iDCs were differentiated into various types of mDCs by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), and CD40 ligand (CD40L). iDCs and CD40L-induced mDCs were susceptible to HIV-1 infection and mediated efficient viral transmission to CD4(+) T cells. Although HIV-1 cis-infection was partially restricted in TNF-alpha-induced mDCs and profoundly blocked in LPS-induced mDCs, these cells efficiently promoted HIV-1 trans-infection of CD4(+) T cells. The postentry restriction of HIV-1 infection in LPS-induced mDCs was identified at the levels of reverse transcription and postintegration, using real-time PCR quantification of viral DNA and integration. Furthermore, nucleofection of DCs with HIV-1 proviral DNA confirmed that impaired gene expression of LPS-induced mDCs was responsible for the postentry restriction of HIV-1 infection. Our results suggest that various DC subsets in vivo may differentially contribute to HIV-1 dissemination via dissociable cis- and trans-infections.

FIG. 1.

Cell surface markers of iDCs and mDCs. (A) Phenotypic surface markers of iDCs and mDCs. Monocyte-derived iDCs were separately induced into different types of mDCs by treatment with LPS, TNF-α, or CD40L for 2 days. DC surface CD11c, HLA-DR, and CD86 were stained with specific MAbs or isotype-matched IgGs and analyzed by flow cytometry. (B) Relative expression levels of surface markers on iDCs and mDCs. All data show means ± standard deviations for duplicate samples. Data for one representative experiment out of three are shown. (C) Surface CD4 expression on various types of DCs. Staining of DCs was done with either a murine IgG2a isotypic control antibody (thin lines) or CD4 MAb (thick lines). The mean fluorescence intensity of CD4 staining is shown in the top right corner of each histogram.

FIG. 2.

Comparison of VSV-G-pseudotyped HIV-1 infections among various types of DCs reveals a postentry restriction in LPS-induced mDCs. (A and B) HIV-1 infection in mDC-LPS is blocked. (A) Various types of DCs (2 × 10⁵) were separately infected with increasing MOIs of VSV-G-pseudotyped, single-cycle luciferase reporter HIV-1 (HIV-Luc/VSV-G). DCs were lysed at 4 dpi to determine viral infection by measuring the luciferase activities of cell lysates. (B) DCs were infected with HIV-Luc/VSV-G at an MOI of 0.2, and infection was determined at 3, 5, and 7 dpi. The luciferase activities of the infected mDC-LPS were at the levels (≤100) of the mock infection controls (not shown in panels A and B). cps, counts per second. (C) Enhanced entry of HIV-Luc/VSV-G into mDC-LPS. DCs were pulsed with HIV-Luc/VSV-G at an MOI of 0.5 at 37°C for 2 h, washed, trypsinized, and then lysed for Gag p24 detection. All data show the means ± standard deviations for triplicate samples. Data for one representative experiment out of four are shown.

FIG. 3.

Postintegration restriction of VSV-G-pseudotyped HIV-1 in LPS-induced mDCs. (A) DNase I treatment completely removed plasmid DNA in HIV stocks. HIV-Luc/VSV-G stocks were treated with DNase I (lane 2) or mock treated (lane 1) for 1 h at 37°C, inactivated with 1% Triton X-100, and then subjected to PCR detection using specific primers for the R-gag region of HIV proviral DNA. Lane M, 100-bp ladder; lane 3, a negative control PCR using H₂O as a DNA template. The arrow points to a band of specific PCR products (448 bp). (B to D) HIV-1 viral DNA quantification. DCs (2 × 10⁵) were infected with HIV-Luc/VSV-G at an MOI of 2.5, and cellular DNAs were extracted at 2 dpi and subjected to real-time PCR quantification of HIV-1 late RT products (B), 2-LTR circles (C), and integrated proviral DNAs (integrants) (D). All data show means ± standard deviations for triplicate or duplicate samples. Data for one representative experiment out of three are shown. (E) HIV-1 gag mRNA expression in infected DCs. DCs (2 × 10⁵) were infected with HIV-Luc/VSV-G at an MOI of 2.5, and cellular RNAs were extracted at 2 dpi and subjected to RT-PCR detection of gag mRNA. RT-PCR was performed with (+) and without (−) reverse transcriptase (RT). The amplification of GAPDH was used as an internal control for RT-PCR. PCR products were analyzed by 1% agarose gel electrophoresis with ethidium bromide staining (A and E).

FIG. 4.

RT of replication-competent HIV-1 is restricted in LPS-induced mDCs. (A) HIV-1_NLAD8 entry is enhanced in mDC-LPS. DC-associated Gag p24 was measured after incubation with HIV-1_NLAD8 for 2 h at 37°C in the presence or absence of the HIV-1 fusion inhibitor T-20. After extensive washes, HIV-1-pulsed DCs were trypsinized and lysed for Gag p24 detection. (B) Restricted HIV-1 replication in mDC-LPS. DCs were infected with HIV-1_NLAD8 in the presence or absence of T-20. Supernatants of infected DCs were measured for p24 levels at 3, 5, and 7 dpi. (C) Basal levels of HIV-1 late RT products in mDC-LPS. (D) Undetectable 2-LTR circles in mDC-LPS. Cellular DNAs of HIV-1_NLAD8-infected DCs were extracted at 2 dpi and subjected to real-time PCR quantification. All data show means ± standard deviations for triplicate samples. Data for one representative experiment out of three are shown.

FIG. 5.

Endocytosed replication-competent HIV-1 in DCs does not produce significant amounts of late RT products. (A) HIV-1 fusion is required for generating late RT products in DCs. DCs were infected with HIV-1_NLAD8, washed, trypsinized, and cultured for 12 h before the cells were lysed for real-time PCR detection. (B) Enhanced HIV-1 endocytosis and retention in mDC-LPS. DCs were infected with HIV-1_NLAD8, washed, trypsinized, and cultured for 12 h before the cells were lysed for Gag p24 detection. T-20 was present during the viral incubation and the 12-h culture. All data show means ± standard deviations for duplicate or triplicate samples. Data for one representative experiment out of three are shown.

FIG. 6.

Impaired gene expression in mDC-LPS contributes to the restriction of HIV-1 replication. (A) Gag p24 levels in DCs after nucleofection with HIV-1 proviral DNA. DCs were nucleofected with plasmid pNLAD8. Gag p24 in supernatants and cell pellets was measured at 5 days postnucleofection. (B) Nucleofection of mDC-LPS with HIV-1 proviral DNA failed to produce infectious HIV-1. GHOST/CCR5 cells were infected with the supernatants of DCs that had been nucleofected with pNLAD8 (at day 5 postnucleofection from the experiment described for panel A). HIV-1 replication was quantified by measuring GFP expression at 3 dpi by flow cytometry. Culture medium was used as a mock-infected control. (C and D) Impaired gene expression in mDC-LPS. DCs were nucleofected with plasmid pmaxGFP (expression is controlled by a CMV promoter). GFP expression was measured by flow cytometry at 24 h postnucleofection. (D) Mean fluorescence intensities of GFP from 1 × 10⁴ DCs, measured by flow cytometry at 24 h postnucleofection. All data show means ± standard deviations for duplicate or triplicate samples. Data for one representative experiment out of four are shown.

FIG. 7.

DCs efficiently transmit HIV-1 to CD4⁺ T cells. (A) DC-mediated transmission of single-cycle HIV-1. DCs (1 × 10⁵) were pulsed with HIV-Luc/JRFL (MOI, 0.3) for 2 h, washed, and cocultured with CD4⁺ Hut/CCR5 T cells (1 × 10⁵) for 48 h. Infection was determined by measuring luciferase activities of the cell lysates. cps, counts per second. (B) DC-mediated transmission of replication-competent HIV-1. DCs (8 × 10⁴) were pulsed with HIV-1_NLAD8 (4 ng of p24) for 2 h, washed, and cocultured with Hut/CCR5 cells (8 × 10⁴) for 48 h. Infection was determined by measuring Gag p24 levels in supernatants. Samples with DC alone were used as controls for HIV-1 cis-infection. All data show means ± standard deviations for duplicate or triplicate samples. Data for one representative experiment out of three are shown.