SAMHD1-Deficient CD14+ Cells from Individuals with Aicardi-Goutières Syndrome Are Highly Susceptible to HIV-1 Infection

Myeloid blood cells are largely resistant to infection with human immunodeficiency virus type 1 (HIV-1). Recently, it was reported that Vpx from HIV-2/SIVsm facilitates infection of these cells by counteracting the host restriction factor SAMHD1. Here, we independently confirmed that Vpx interacts with SAMHD1 and targets it for ubiquitin-mediated degradation. We found that Vpx-mediated SAMHD1 degradation rendered primary monocytes highly susceptible to HIV-1 infection; Vpx with a T17A mutation, defective for SAMHD1 binding and degradation, did not show this activity. Several single nucleotide polymorphisms in the SAMHD1 gene have been associated with Aicardi-Goutières syndrome (AGS), a very rare and severe autoimmune disease. Primary peripheral blood mononuclear cells (PBMC) from AGS patients homozygous for a nonsense mutation in SAMHD1 (R164X) lacked endogenous SAMHD1 expression and support HIV-1 replication in the absence of exogenous activation. Our results indicate that within PBMC from AGS patients, CD14+ cells were the subpopulation susceptible to HIV-1 infection, whereas cells from healthy donors did not support infection. The monocytic lineage of the infected SAMHD1 -/- cells, in conjunction with mostly undetectable levels of cytokines, chemokines and type I interferon measured prior to infection, indicate that aberrant cellular activation is not the cause for the observed phenotype. Taken together, we propose that SAMHD1 protects primary CD14+ monocytes from HIV-1 infection confirming SAMHD1 as a potent lentiviral restriction factor

How Soluble GARP Enhances TGFβ Activation.

GARP (glycoprotein A repetitions predominant) is a cell surface receptor on regulatory T-lymphocytes, platelets, hepatic stellate cells and certain cancer cells. Its described function is the binding and accommodation of latent TGFβ (transforming growth factor), before the activation and release of the mature cytokine. For regulatory T cells it was shown that a knockdown of GARP or a treatment with blocking antibodies dramatically decreases their immune suppressive capacity. This confirms a fundamental role of GARP in the basic function of regulatory T cells. Prerequisites postulated for physiological GARP function include membrane anchorage of GARP, disulfide bridges between the propeptide of TGFβ and GARP and connection of this propeptide to αvβ6 or αvβ8 integrins of target cells during mechanical TGFβ release. Other studies indicate the existence of soluble GARP complexes and a functionality of soluble GARP alone. In order to clarify the underlying molecular mechanism, we expressed and purified recombinant TGFβ and a soluble variant of GARP. Surprisingly, soluble GARP and TGFβ formed stable non-covalent complexes in addition to disulfide-coupled complexes, depending on the redox conditions of the microenvironment. We also show that soluble GARP alone and the two variants of complexes mediate different levels of TGFβ activity. TGFβ activation is enhanced by the non-covalent GARP-TGFβ complex already at low (nanomolar) concentrations, at which GARP alone does not show any effect. This supports the idea of soluble GARP acting as immune modulator in vivo

How soluble GARP enhances TGFβ activation

GARP (glycoprotein A repetitions predominant) is a cell surface receptor on regulatory T-lymphocytes, platelets, hepatic stellate cells and certain cancer cells. Its described function is the binding and accommodation of latent TGFβ (transforming growth factor), before the activation and release of the mature cytokine. For regulatory T cells it was shown that a knockdown of GARP or a treatment with blocking antibodies dramatically decreases their immune suppressive capacity. This confirms a fundamental role of GARP in the basic function of regulatory T cells. Prerequisites postulated for physiological GARP function include membrane anchorage of GARP, disulfide bridges between the propeptide of TGFβ and GARP and connection of this propeptide to αvβ6 or αvβ8 integrins of target cells during mechanical TGFβ release. Other studies indicate the existence of soluble GARP complexes and a functionality of soluble GARP alone. In order to clarify the underlying molecular mechanism, we expressed and purified recombinant TGFβ and a soluble variant of GARP. Surprisingly, soluble GARP and TGFβ formed stable non-covalent complexes in addition to disulfide-coupled complexes, depending on the redox conditions of the microenvironment. We also show that soluble GARP alone and the two variants of complexes mediate different levels of TGFβ activity. TGFβ activation is enhanced by the non-covalent GARP-TGFβ complex already at low (nanomolar) concentrations, at which GARP alone does not show any effect. This supports the idea of soluble GARP acting as immune modulator in vivo

Transient Expression of the three recombinant GARP variants in HEK 293H cells.

<p>HEK 293H cells were transfected with plasmids containing the cDNA of the constructs GARP_FL, GARP_TS and GARP_ΔTM, respectively. 48h after transfection, the culture medium was exchanged for FCS-free DMEM supplemented with NEAA. Supernatants (S) and cell lysates (L) were obtained after another 48h of incubation. 1 ml of supernatant was precipitated using 2% (w/v) Na-deoxycholate solution (1:100) and 100% TCA (1:10). Cell lysates were prepared using 200 μl RIPA buffer per 1x10⁶ cells. Samples were separated on a 10% PAA SDS-PAGE followed by western blotting on a PVDF membrane. For molecular size determination the magic mark XP marker (Invitrogen; Darmstadt, Germany) was used. For detection the blot was probed with α-Strep-tag and α-His-tag antibodies, respectively (Quiagen; Hilden, Germany). As secondary antibody a peroxidase coupled anti-mouse-IgG antibody (Dianova; Hamburg, Germany) was used.</p

Electrophoretic analysis of GARP_TS fractions from the final Ni-NTA chromatography.

<p>(A) Elution fractions of the Affigel Blue column were pooled and proteins bound on the Ni-NTA matrix overnight. Lane 1–4: Washing fractions, containing 50 mM imidazole; Lane 5: Protein Marker Broad Range (NEB; Ipswich, USA); Lane 6+7: Elution fractions containing 100 mM imidazole. The arrowhead marks the soluble form of GARP_TS at a molecular weight of 75 kDa. Fractions were analyzed by 10% PAA SDS-PAGE und reducing conditions, followed by staining with coomassie brilliant blue. (B) The supernatant of TRP2_TS transfected Expi293F cells (Invitrogen; Darmstadt, Germany) were concentrated and applied to a Strep-Tactin matrix. SM: Starting Material; M: Protein Marker SeeBlue Plus 2 (Invitrogen; Darmstadt, Germany); FT: Flowthrough; Elution: Eluted fractions with 2.5 mM D-Desthiobiotin. The arrowhead marks the soluble TRP2_TS at a molecular weight of 75 kDa. Fractions were analyzed by 10% PAA SDS-PAGE und reducing conditions followed by western blotting on a PVDF membrane. For detection the blot was probed with anti-TRP2 antibody (Abcam; Cambridge, UK). As secondary antibody a peroxidase coupled anti-rabbit-IgG antibody (GE Healthcare; Solingen, Germany) was used.</p

<i>In vivo</i> coupling of GARP_TS to recombinant TGFβ.

<p>HEK 293H cells were transfected with plasmids containing the cDNA of the constructs GARP_FL, TGFβ_Strep or both in combination. 48h after transfection, the culture medium was exchanged for FCS-free DMEM supplemented with NEAA. Cell lysates were prepared using 200 μl RIPA buffer per 1x10⁶ cells. Samples were separated on a 10% PAA SDS-PAGE followed by western blotting on a PVDF membrane. For molecular size determination the magic mark XP marker (Invitrogen; Darmstadt, Germany) was used. For detection the blot was probed with anti-Strep-tag and anti-His-tag antibodies, respectively (Quiagen; Hilden, Germany). As secondary antibody a peroxidase coupled anti-mouse-IgG antibody (Dianova; Hamburg, Germany) was used.</p

<i>In vitro</i> coupling of GARP_TS to recombinant TGFβ.

<p>(A) 300 ng GARP_TS and 600 ng recombinant latent TGFβ per lane were incubated overnight in the presence or absence of a redox-buffer containing 0.05 mM GSSH and 2 mM free cysteine at RT to allow the formation of GARP-LAP interactions. Then the samples were incubated for 4 hours at 4°C together with magnetic Ni-NTA beads to bind GARP_TS and putative GARP-LAP complexes at the beads. Proteins bound to the beads were analyzed by western blot with anti-Strep-tag antibodies, which could be used to detect both GARP_TS and recombinant TGFβ. (B) Equally treated controls were analyzed on a 10% PAA SDS-PAGE under non-reducing conditions but without the pull-down procedure. After gel electrophoresis, proteins were blotted onto a PVDF membrane and probed with anti-Strep-tag (left) or anti-His-tag antibodies (right), respectively.</p