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HIV-2 infects resting CD4+ T cells but not monocyte-derived dendritic cells.

Chauveau L, Puigdomenech I, Ayinde D, Roesch F, Porrot F, Bruni D, Visseaux B, Descamps D, Schwartz O - Retrovirology (2015)

Bottom Line: HIV-2 particles did not potently fuse with MDDCs and did not lead to efficient viral DNA synthesis, even in the presence of Vpx.In these cells, an entry defect prevents viral fusion and reverse transcription independently of SAMHD1.We propose that HIV-2, like HIV-1, does not productively infect MDDCs, possibly to avoid triggering an immune response mediated by these cells.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Human Immunodeficiency Virus-type 2 (HIV-2) encodes Vpx that degrades SAMHD1, a cellular restriction factor active in non-dividing cells. HIV-2 replicates in lymphocytes but the susceptibility of monocyte-derived dendritic cells (MDDCs) to in vitro infection remains partly characterized.

Results: Here, we investigated HIV-2 replication in primary CD4+ T lymphocytes, both activated and non-activated, as well as in MDDCs. We focused on the requirement of Vpx for productive HIV-2 infection, using the reference HIV-2 ROD strain, the proviral clone GL-AN, as well as two primary HIV-2 isolates. All HIV-2 strains tested replicated in activated CD4+ T cells. Unstimulated CD4+ T cells were not productively infected by HIV-2, but viral replication was triggered upon lymphocyte activation in a Vpx-dependent manner. In contrast, MDDCs were poorly infected when exposed to HIV-2. HIV-2 particles did not potently fuse with MDDCs and did not lead to efficient viral DNA synthesis, even in the presence of Vpx. Moreover, the HIV-2 strains tested were not efficiently sensed by MDDCs, as evidenced by a lack of MxA induction upon viral exposure. Virion pseudotyping with VSV-G rescued fusion, productive infection and HIV-2 sensing by MDDCs.

Conclusion: Vpx allows the non-productive infection of resting CD4+ T cells, but does not confer HIV-2 with the ability to efficiently infect MDDCs. In these cells, an entry defect prevents viral fusion and reverse transcription independently of SAMHD1. We propose that HIV-2, like HIV-1, does not productively infect MDDCs, possibly to avoid triggering an immune response mediated by these cells.

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HIV-2 ROD-GFP and MDDCs. (a) Susceptibility of MDDCs to HIV-2 ROD-GFP infection. MDDCs were exposed to HIV-2 ROD-GFP, pseudotyped or not with VSV-G (50 and 150 ng p27 mL-1, respectively). After 3 days, the levels of GFP were measured by flow cytometry. Results from 6 independent donors are shown (b) HIV-2 ROD-GFP binding. MDDCs were exposed to the indicated doses of HIV-2 ROD-GFP, pseudotyped or not with VSV-G. After 2 h at 4°C, cells were extensively washed and the amount of cell-associated p27 was assessed by ELISA. Data are Mean ± SEM of 4 independent donors. ns: non significant (c) HIV-2 ROD-GFP fusion. MDDCs were exposed to the indicated doses of HIV-2 ROD-GFP, pseudotyped or not with VSV-G, and bearing the chimeric protein β-lactamase-Vpr. After 2 h at 37°C and 2 h at room temperature, viral access to the cytoplasm was assessed by flow cytometry, using the ability of β -lactamase to cleave the cytoplasmic CCF2-AM fluorogenic substrate. A mean ± SEM of 4 independent donors is shown. *: p-value < 0.05. Comparisons were made between the condition indicated and the no VSV condition at the same viral inoculum. (d) HIV-2 ROD-GFP DNA synthesis. MDDCs were exposed to HIV-2 ROD-GFP pseudotyped or not with VSV-G, in the presence or absence of AZT. After 3 days, the cells were harvested for HIV-2 DNA quantification by qPCR. Data are mean ± SEM of 4 independent donors.
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Fig5: HIV-2 ROD-GFP and MDDCs. (a) Susceptibility of MDDCs to HIV-2 ROD-GFP infection. MDDCs were exposed to HIV-2 ROD-GFP, pseudotyped or not with VSV-G (50 and 150 ng p27 mL-1, respectively). After 3 days, the levels of GFP were measured by flow cytometry. Results from 6 independent donors are shown (b) HIV-2 ROD-GFP binding. MDDCs were exposed to the indicated doses of HIV-2 ROD-GFP, pseudotyped or not with VSV-G. After 2 h at 4°C, cells were extensively washed and the amount of cell-associated p27 was assessed by ELISA. Data are Mean ± SEM of 4 independent donors. ns: non significant (c) HIV-2 ROD-GFP fusion. MDDCs were exposed to the indicated doses of HIV-2 ROD-GFP, pseudotyped or not with VSV-G, and bearing the chimeric protein β-lactamase-Vpr. After 2 h at 37°C and 2 h at room temperature, viral access to the cytoplasm was assessed by flow cytometry, using the ability of β -lactamase to cleave the cytoplasmic CCF2-AM fluorogenic substrate. A mean ± SEM of 4 independent donors is shown. *: p-value < 0.05. Comparisons were made between the condition indicated and the no VSV condition at the same viral inoculum. (d) HIV-2 ROD-GFP DNA synthesis. MDDCs were exposed to HIV-2 ROD-GFP pseudotyped or not with VSV-G, in the presence or absence of AZT. After 3 days, the cells were harvested for HIV-2 DNA quantification by qPCR. Data are mean ± SEM of 4 independent donors.

Mentions: We characterized the replicative defect of HIV-2 in MDDCs by comparing the ability of wild-type and VSV-G-pseudotyped virus to bind cells, undergo fusion and perform reverse transcription. To measure viral binding at the cell surface, we incubated MDDCs with increasing doses of GL-AN and GL-AN (VSV) (50 and 150 ng p27 mL−1) for 2 h at 4°C. After extensive washes, p27 Gag levels were measured in cell lysates by ELISA. The viruses bound to the cells in a dose dependent manner (Figure 4a). VSV-G pseudotyping resulted in a slight but non-significant increase in viral binding (Figure 4a). We then performed a viral fusion assay to assess the post-binding step of the viral cycle. HIV-2 Vpr, which is incorporated into viral particles, was fused with ß-lactamase (Blam-Vpr2) [54,55]. The successful cytoplasmic access of Blam-Vpr2, as a result of viral fusion, after 2 h of infection, was monitored by enzymatic cleavage of CCF2-AM, a fluorogenic substrate of ß-lactamase loaded in target cells [54,55]. A representative experiment is shown in Figure 5b. A dose–response analysis of the viral inoculum (8 to 400 ng p27 mL−1) indicated that wild-type HIV-2 fusion in MDDCs was low (Figure 4b). In sharp contrast, a positive fusion signal was detected with GL-AN (VSV), starting at the lowest viral inoculum tested (Figure 4b). A side-by-side comparison indicated that fusion of the VSV-G-pseudotyped HIV-2 with MDDCs was 50 times more efficient than that of the wild-type virus. We then quantified the reverse transcription products at day 3 post-infection. In line with the results obtained in the fusion assay, GL-AN did not permit an efficient synthesis of viral DNA, even at a high viral inoculum. In contrast, infection with VSV-G-pseudotyped HIV-2 was associated with high levels of viral DNA. This signal corresponded to newly synthesized molecules since it was inhibited by AZT (Figure 4c).Figure 4


HIV-2 infects resting CD4+ T cells but not monocyte-derived dendritic cells.

Chauveau L, Puigdomenech I, Ayinde D, Roesch F, Porrot F, Bruni D, Visseaux B, Descamps D, Schwartz O - Retrovirology (2015)

HIV-2 ROD-GFP and MDDCs. (a) Susceptibility of MDDCs to HIV-2 ROD-GFP infection. MDDCs were exposed to HIV-2 ROD-GFP, pseudotyped or not with VSV-G (50 and 150 ng p27 mL-1, respectively). After 3 days, the levels of GFP were measured by flow cytometry. Results from 6 independent donors are shown (b) HIV-2 ROD-GFP binding. MDDCs were exposed to the indicated doses of HIV-2 ROD-GFP, pseudotyped or not with VSV-G. After 2 h at 4°C, cells were extensively washed and the amount of cell-associated p27 was assessed by ELISA. Data are Mean ± SEM of 4 independent donors. ns: non significant (c) HIV-2 ROD-GFP fusion. MDDCs were exposed to the indicated doses of HIV-2 ROD-GFP, pseudotyped or not with VSV-G, and bearing the chimeric protein β-lactamase-Vpr. After 2 h at 37°C and 2 h at room temperature, viral access to the cytoplasm was assessed by flow cytometry, using the ability of β -lactamase to cleave the cytoplasmic CCF2-AM fluorogenic substrate. A mean ± SEM of 4 independent donors is shown. *: p-value < 0.05. Comparisons were made between the condition indicated and the no VSV condition at the same viral inoculum. (d) HIV-2 ROD-GFP DNA synthesis. MDDCs were exposed to HIV-2 ROD-GFP pseudotyped or not with VSV-G, in the presence or absence of AZT. After 3 days, the cells were harvested for HIV-2 DNA quantification by qPCR. Data are mean ± SEM of 4 independent donors.
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Fig5: HIV-2 ROD-GFP and MDDCs. (a) Susceptibility of MDDCs to HIV-2 ROD-GFP infection. MDDCs were exposed to HIV-2 ROD-GFP, pseudotyped or not with VSV-G (50 and 150 ng p27 mL-1, respectively). After 3 days, the levels of GFP were measured by flow cytometry. Results from 6 independent donors are shown (b) HIV-2 ROD-GFP binding. MDDCs were exposed to the indicated doses of HIV-2 ROD-GFP, pseudotyped or not with VSV-G. After 2 h at 4°C, cells were extensively washed and the amount of cell-associated p27 was assessed by ELISA. Data are Mean ± SEM of 4 independent donors. ns: non significant (c) HIV-2 ROD-GFP fusion. MDDCs were exposed to the indicated doses of HIV-2 ROD-GFP, pseudotyped or not with VSV-G, and bearing the chimeric protein β-lactamase-Vpr. After 2 h at 37°C and 2 h at room temperature, viral access to the cytoplasm was assessed by flow cytometry, using the ability of β -lactamase to cleave the cytoplasmic CCF2-AM fluorogenic substrate. A mean ± SEM of 4 independent donors is shown. *: p-value < 0.05. Comparisons were made between the condition indicated and the no VSV condition at the same viral inoculum. (d) HIV-2 ROD-GFP DNA synthesis. MDDCs were exposed to HIV-2 ROD-GFP pseudotyped or not with VSV-G, in the presence or absence of AZT. After 3 days, the cells were harvested for HIV-2 DNA quantification by qPCR. Data are mean ± SEM of 4 independent donors.
Mentions: We characterized the replicative defect of HIV-2 in MDDCs by comparing the ability of wild-type and VSV-G-pseudotyped virus to bind cells, undergo fusion and perform reverse transcription. To measure viral binding at the cell surface, we incubated MDDCs with increasing doses of GL-AN and GL-AN (VSV) (50 and 150 ng p27 mL−1) for 2 h at 4°C. After extensive washes, p27 Gag levels were measured in cell lysates by ELISA. The viruses bound to the cells in a dose dependent manner (Figure 4a). VSV-G pseudotyping resulted in a slight but non-significant increase in viral binding (Figure 4a). We then performed a viral fusion assay to assess the post-binding step of the viral cycle. HIV-2 Vpr, which is incorporated into viral particles, was fused with ß-lactamase (Blam-Vpr2) [54,55]. The successful cytoplasmic access of Blam-Vpr2, as a result of viral fusion, after 2 h of infection, was monitored by enzymatic cleavage of CCF2-AM, a fluorogenic substrate of ß-lactamase loaded in target cells [54,55]. A representative experiment is shown in Figure 5b. A dose–response analysis of the viral inoculum (8 to 400 ng p27 mL−1) indicated that wild-type HIV-2 fusion in MDDCs was low (Figure 4b). In sharp contrast, a positive fusion signal was detected with GL-AN (VSV), starting at the lowest viral inoculum tested (Figure 4b). A side-by-side comparison indicated that fusion of the VSV-G-pseudotyped HIV-2 with MDDCs was 50 times more efficient than that of the wild-type virus. We then quantified the reverse transcription products at day 3 post-infection. In line with the results obtained in the fusion assay, GL-AN did not permit an efficient synthesis of viral DNA, even at a high viral inoculum. In contrast, infection with VSV-G-pseudotyped HIV-2 was associated with high levels of viral DNA. This signal corresponded to newly synthesized molecules since it was inhibited by AZT (Figure 4c).Figure 4

Bottom Line: HIV-2 particles did not potently fuse with MDDCs and did not lead to efficient viral DNA synthesis, even in the presence of Vpx.In these cells, an entry defect prevents viral fusion and reverse transcription independently of SAMHD1.We propose that HIV-2, like HIV-1, does not productively infect MDDCs, possibly to avoid triggering an immune response mediated by these cells.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Human Immunodeficiency Virus-type 2 (HIV-2) encodes Vpx that degrades SAMHD1, a cellular restriction factor active in non-dividing cells. HIV-2 replicates in lymphocytes but the susceptibility of monocyte-derived dendritic cells (MDDCs) to in vitro infection remains partly characterized.

Results: Here, we investigated HIV-2 replication in primary CD4+ T lymphocytes, both activated and non-activated, as well as in MDDCs. We focused on the requirement of Vpx for productive HIV-2 infection, using the reference HIV-2 ROD strain, the proviral clone GL-AN, as well as two primary HIV-2 isolates. All HIV-2 strains tested replicated in activated CD4+ T cells. Unstimulated CD4+ T cells were not productively infected by HIV-2, but viral replication was triggered upon lymphocyte activation in a Vpx-dependent manner. In contrast, MDDCs were poorly infected when exposed to HIV-2. HIV-2 particles did not potently fuse with MDDCs and did not lead to efficient viral DNA synthesis, even in the presence of Vpx. Moreover, the HIV-2 strains tested were not efficiently sensed by MDDCs, as evidenced by a lack of MxA induction upon viral exposure. Virion pseudotyping with VSV-G rescued fusion, productive infection and HIV-2 sensing by MDDCs.

Conclusion: Vpx allows the non-productive infection of resting CD4+ T cells, but does not confer HIV-2 with the ability to efficiently infect MDDCs. In these cells, an entry defect prevents viral fusion and reverse transcription independently of SAMHD1. We propose that HIV-2, like HIV-1, does not productively infect MDDCs, possibly to avoid triggering an immune response mediated by these cells.

Show MeSH
Related in: MedlinePlus