Evidence that HIV-1 restriction factor SAMHD1 facilitates differentiation of myeloid THP-1 cells.

BACKGROUND: SAMHD1 counteracts HIV-1 or HIV-2/SIVsmm that lacks Vpx by depleting the intracellular pool of nucleotides in myeloid cells and CD4+ quiescent T cells, thereby inhibiting the synthesis of retroviral DNA by reverse transcriptase. Depletion of nucleotides has been shown to underline the establishment of quiescence in certain cellular systems. These observations led us to investigate whether SAMHD1 could control the transition between proliferation and quiescence using the THP-1 cell model. FINDINGS: The entry of dividing THP-1 myeloid cells into a non-dividing differentiated state was monitored after addition of phorbol-12-myristate-13-acetate (PMA), an inducer of differentiation. Under PMA treatment, cells overexpressing SAMHD1 display stronger and faster adhesion to their support, compared to cells expressing a catalytically inactive form of SAMHD1, or cells depleted of SAMHD1, which appear less differentiated. After PMA removal, cells overexpressing SAMHD1 maintain low levels of cyclin A, in contrast to other cell lines. Interestingly, SAMHD1 overexpression slightly increases cell adhesion even in the absence of the differentiation inducer PMA. Finally, we found that levels of SAMHD1 are reduced in proliferating primary CD4+ T cells after T cell receptor activation, suggesting that SAMHD1 may also be involved in the transition from a quiescent state to a dividing state in primary T cells. CONCLUSIONS: Altogether, we provide evidence that SAMHD1 may facilitate some aspects of THP-1 cell differentiation. Restriction of HIV-1 by SAMHD1 may rely upon its ability to modify cell cycle parameters, in addition to the direct inhibition of reverse transcription.

Interferon block to HIV-1 transduction in macrophages despite SAMHD1 degradation and high deoxynucleoside triphosphates supply.

BACKGROUND: Interferon-α (IFN-α) is an essential mediator of the antiviral response, which potently inhibits both early and late phases of HIV replication. The SAMHD1 deoxynucleoside triphosphate (dNTP) hydrolase represents the prototype of a new antiviral strategy we referred to as "nucleotide depletion". SAMHD1 depletes dNTP levels in myeloid cells below those required for optimal synthesis of HIV viral DNA. HIV-2 and its SIVsm and SIVmac close relatives encode a protein termed Vpx, which counteracts SAMHD1. The potentiality of IFN-α to cooperate with nucleotide depletion has been poorly investigated so far. Here we wondered whether IFN-α affects SAMHD1 expression, Vpx-induced SAMHD1 degradation, Vpx-mediated rescue of HIV-1 transduction and the dNTP supply in monocyte-derived macrophages (MDMs). RESULTS: IFN-α inhibited HIV-1 transduction in monocytes and in MDMs while SAMHD1 expression was not up-regulated. Vpx triggered SAMHD1 degradation in IFN-α treated cells, and weakly restored HIV-1 transduction from the IFN-α block. Vpx helper effect towards HIV-1 transduction was gradually inhibited with increasing doses of IFN-α. dNTP levels were not significantly affected in MDMs and CD4+ primary activated T lymphocytes by IFN-α and, in correlation with SAMHD1 degradation, restoration of dNTP levels by Vpx was efficient in MDMs treated with the cytokine. In contrast, IFN-α inhibited Vpx-mediated SAMHD1 degradation in THP-1 cells, where, accordingly, Vpx could not rescue HIV-1 transduction. CONCLUSION: Our results suggest that the early antiviral effect of IFN-α results from a mechanism independent of nucleotide depletion in MDMs. In addition, they indicate that the macrophage-like THP-1 cell line may provide a system to characterize an IFN-α-induced cell response that inhibits Vpx-mediated SAMHD1 degradation.

HLA-B27 subtype oligomerization and intracellular accumulation patterns correlate with predisposition to spondyloarthritis.

OBJECTIVE: Mechanisms underlying the striking association of spondyloarthritis (SpA) with the class I major histocompatibility complex molecule HLA-B27 remain poorly understood. SpA-like disease develops spontaneously in B*2705-transgenic rats, in conjunction with high HLA-B27 expression levels. This study was undertaken to examine the effects of increased expression of HLA-B27 alleles that are differentially associated with SpA on oligomerization and intracellular redistribution. METHODS: HeLa cells were transfected with complementary DNA encoding for HLA-B proteins fused to yellow fluorescent protein and/or Renilla luciferase and harvested at an early phase and a later phase of expression. We monitored HLA-B intracellular trafficking and localization by means of microscopy and live-cell imaging. Bioluminescence resonance energy transfer (BRET) and Western blotting were used to monitor HLA-B oligomerization. RESULTS: At low expression levels, BRET signals were similarly elevated for all SpA-associated HLA-B27 alleles tested, but were lower for the nonassociated B*2706. Of note, at higher expression levels, HLA-B27 signals remained steady while signal for HLA-B7 decreased sharply, reaching the level observed for B*2706. This was due at least in part to a decreased oligomer proportion without unfolded protein response outbreak. Such differential behavior was not abrogated by proteasome inhibition. With increased expression, all HLA-B proteins accumulated to a high density in cytoplasmic vesicles with labile form and size. The extent of this phenomenon was closely correlated with the level of association with predisposition to SpA. CONCLUSION: To our knowledge, this is the first report of a correlation between the level of predisposition to SpA conferred by HLA-B27 alleles and their biochemical behavior. These findings open new perspectives for understanding the pathogenicity of HLA-B27.

HIV-1 Vpr induces the degradation of ZIP and sZIP, adaptors of the NuRD chromatin remodeling complex, by hijacking DCAF1/VprBP.

The Vpr protein from type 1 and type 2 Human Immunodeficiency Viruses (HIV-1 and HIV-2) is thought to inactivate several host proteins through the hijacking of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Here, we identified two transcriptional regulators, ZIP and sZIP, as Vpr-binding proteins degraded in the presence of Vpr. ZIP and sZIP have been shown to act through the recruitment of the NuRD chromatin remodeling complex. Strikingly, chromatin is the only cellular fraction where Vpr is present together with Cul4A ubiquitin ligase subunits. Components of the NuRD complex and exogenous ZIP and sZIP were also associated with this fraction. Several lines of evidence indicate that Vpr induces ZIP and sZIP degradation by hijacking DCAF1: (i) Vpr induced a drastic decrease of exogenously expressed ZIP and sZIP in a dose-dependent manner, (ii) this decrease relied on the proteasome activity, (iii) ZIP or sZIP degradation was impaired in the presence of a DCAF1-binding deficient Vpr mutant or when DCAF1 expression was silenced. Vpr-mediated ZIP and sZIP degradation did not correlate with the growth-related Vpr activities, namely G2 arrest and G2 arrest-independent cytotoxicity. Nonetheless, infection with HIV-1 viruses expressing Vpr led to the degradation of the two proteins. Altogether our results highlight the existence of two host transcription factors inactivated by Vpr. The role of Vpr-mediated ZIP and sZIP degradation in the HIV-1 replication cycle remains to be deciphered.