HIV type 1 infection of plasmacytoid and myeloid dendritic cells is restricted by high levels of SAMHD1 and cannot be counteracted by Vpx

Abstract

Dendritic cells are professional antigen-presenting cells of the immune system and are major producers of type-I interferon. Their role in HIV-1 infection is not well understood. They express CD4 and CCR5 yet appear to be resistant to infection. In culture, infection of the cells with HIV-1 is inhibited by the host cell restriction factor SAMHD1. Lentiviruses such as HIV-2/SIVmac counteract the restriction by encoding Vpx, a virion-packaged accessory protein that induces the proteasomal degradation of SAMHD1. In this study we investigated SAMHD1-mediated restriction in the two major dendritic cell subsets: plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC). The cells were highly resistant to HIV-1 and expressed high levels of SAMHD1. SAMHD1 amino acid residue T592, a target of CDK1 phosphorylation, was unphosphorylated, corresponding to the antiviral form of the enzyme. The resistance to infection was not counteracted by Vpx and SAMHD1 was not degraded in these cells. Treatment of pDCs with a cocktail of antibodies that blocked type-I interferon signaling partially restored the ability of Vpx to induce SAMHD1 degradation and caused the cells to become partially permissive to infection. pDCs and mDCs responded to HIV-1 virions by inducing an innate immune response but did not appear to sense newly produced Gag protein. The findings suggest that in vivo, dendritic cells serve as sentinels to alert the immune system to the virus but do not themselves become infected by virtue of high levels of SAMHD1.

FIG. 1.

Myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) express high levels of SAMHD1 and are resistant to HIV-1 infection. (A) SAMHD1 in mDCs, pDCs, and monocyte-derived dendritic cells (MDDCs) from three healthy donors were quantified by immunoblot analysis. The relative abundance of SAMHD1 is expressed as the ratio of SAMHD1 to GAPDH signal intensity under the immunoblot and is represented as a histogram on the right as the average of three donors. (B) mDCs, pDCs, and MDDCs were mock infected or infected with HIV-1 GFP reporter virus lacking or containing Vpx. Three days later, the cells were analyzed by flow cytometry. Shown are the FACS plots from a single representative donor (left) and the data in histogram format for the average of three donors (right). Statistical analysis of two (A) or three (B) independent experiments was performed using two-tailed Student's t tests. *Represents a p-value<0.05 and was considered significant.

FIG. 2.

SAMHD1 in mDCs and pDCs is resistant to Vpx-induced degradation. mDCs, pDCs, and MDDCs were mock infected (m) or infected with HIV-1 GFP reporter virus containing (+) or lacking (–) Vpx at an MOI of 2 or 4. Cell lysates were prepared and SAMHD1 and GAPDH were quantified on an immunoblot. The relative abundance of SAMHD1 is shown below as the ratio of SAMHD1 signal intensity to GAPDH. These data are representative of two independent experiments.

FIG. 3.

SAMHD1 in mDCs and pDCs is neddylated but not phosphorylated on T592. Resting CD4 T cells or CD4 T cells activated with phytohemagglutinin (PHA) and interleukin (IL)-2 or anti-CD3-CD28, mDCs, pDCs, and MDDCs were incubated overnight with or without interferon (IFN)-α_2a and IFN-β_1a Cell lysates were prepared and the proteins were analyzed on an immunoblot probed with antibodies to SAMHD1, T592-phosphorylated SAMHD1, CUL4A, and GAPDH. The relative neddylation of CUL4A is shown as the ratio of neddylated CUL4A signal intensity to nonneddylated CUL4A. These data are representative of two independent experiments.

FIG. 4.

HIV-1 virions trigger IFN release by pDCs, which prevents Vpx-induced SAMHD1 degradation. (A) pDCs from three healthy donors were incubated for 24 h with type-I IFN-R signaling inhibitory antibody cocktail or isotype control IgG. The cells were then mock infected (m) or infected with HIV-1 GFP reporter virus containing or lacking Vpx. IFN-α was quantified in the supernatant 24 h postinfection by ELISA. (B) pDCs were incubated for 24 h with type-I IFN-R signaling inhibitory antibody cocktail or isotype control IgG. Cell lysates were prepared, and SAMHD1 and GAPDH were quantified on an immunoblot. (C, top) pDCs from six donors were incubated with IFN-R signaling inhibitory antibody cocktail and then infected as in (A). After 3 days, the percentage of GFP⁺ cells was determined by flow cytometry. Each symbol represents one donor. (C, bottom) Sixteen hours postinfection, cell lysates were prepared from one donor and SAMHD1 and GAPDH were quantified on an immunoblot. In (B) and (C), the relative abundance of SAMHD1 is shown below the blot defined as the ratio of SAMHD1 signal intensity to GAPDH. Statistical analysis of three (A) or six (C) independent experiments was performed using two-tailed Student's t tests. *Represents a p-value<0.05 and was considered significant.

FIG. 5.

Productive infection of mDCs and pDCs by HIV-1 fails to induce innate immune activation. (A) mDCs from three healthy donors were mock infected, infected with HIV-1 GFP reporter virus containing (HIV X⁺) or lacking (HIV X⁻) Vpx at an MOI=10, or were treated with poly(I:C) TLR agonist. The number of infected cells was quantified by flow cytometry (left). After 24 h and 72 h, supernatant IFN-β was quantified by ELISA (right). (B) pDCs were infected and analyzed as in (A) except that CpGB was used as the TLR agonist and IFN-α was quantified. (C) pDCs were infected with control or Vpx-containing virus or were treated with CpGB. After 3 days the cells were analyzed by flow cytometry for the maturation marker CD86. Histograms are shown for one representative donor (top). Below, the cells were gated for all GFP⁺ cells, high GFP expressors, and low GFP expressors. The average mean fluorescence intensity (MFI) of three donor cell preparations is shown. Statistical analysis was performed using two-tailed Student's t tests. *Represents a p-value<0.05 and was considered significant.