Corine St. Gelais

SAMHD1 restricts HIV-1 infection in dendritic cells (DCs) by dNTP depletion, but its expression in DCs and primary CD4+T-lymphocytes cannot be upregulated by interferons

BackgroundSAMHD1 is an HIV-1 restriction factor in non-dividing monocytes, dendritic cells (DCs), macrophages, and resting CD4+ T-cells. Acting as a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, SAMHD1 hydrolyzes dNTPs and restricts HIV-1 infection in macrophages and resting CD4+ T-cells by decreasing the intracellular dNTP pool. However, the intracellular dNTP pool in DCs and its regulation by SAMHD1 remain unclear. SAMHD1 has been reported as a type I interferon (IFN)-inducible protein, but whether type I IFNs upregulate SAMHD1 expression in primary DCs and CD4+ T-lymphocytes is unknown.ResultsHere, we report that SAMHD1 significantly blocked single-cycle and replication-competent HIV-1 infection of DCs by decreasing the intracellular dNTP pool and thereby limiting the accumulation of HIV-1 late reverse transcription products. Type I IFN treatment did not upregulate endogenous SAMHD1 expression in primary DCs or CD4+ T-lymphocytes, but did in HEK 293T and HeLa cell lines. When SAMHD1 was over-expressed in these two cell lines to achieve higher levels than that in DCs, no HIV-1 restriction was observed despite partially reducing the intracellular dNTP pool.ConclusionsOur results suggest that SAMHD1-mediated reduction of the intracellular dNTP pool in DCs is a common mechanism of HIV-1 restriction in myeloid cells. Endogenous expression of SAMHD1 in primary DCs or CD4+ T-lymphocytes is not upregulated by type I IFNs.

Evidence for the formation of a heptameric ion channel complex by the hepatitis C virus P7 protein in vitro

The p7 protein of hepatitis C virus functions as an ion channel both in vitro and in cell-based assays and is inhibited by amantadine, long alkyl chain imino-sugar derivatives, and amiloride compounds. Future drug design will be greatly aided by information on the stoichiometry and high resolution structure of p7 ion channel complexes. Here, we have refined a bacterial expression system for p7 based on a glutathione S-transferase fusion methodology that circumvents the inherent problems of hydrophobic protein purification and the limitations of chemical synthesis. Rotational averaging and harmonic analysis of transmission electron micrographs of glutathione S-transferase-FLAG-p7 fusion proteins in liposomes revealed a heptameric stoichiometry. The oligomerization of p7 protein was then confirmed by SDS-PAGE and mass spectrometry analysis of pure, concentrated FLAG-p7. The same protein was also confirmed to function as an ion channel in suspended lipid bilayers and was inhibited by amantadine. These data validate this system as a means of generating high resolution structural information on the p7 ion channel complex.

Identification of Cellular Proteins Interacting with the Retroviral Restriction Factor SAMHD1

ABSTRACT Human and mouse SAMHD1 proteins block human immunodeficiency virus type 1 (HIV-1) infection in noncycling human monocytic cells by reducing the intracellular deoxynucleoside triphosphate (dNTP) concentrations. Phosphorylation of human SAMHD1 at threonine 592 (T592) by cyclin-dependent kinase 1 (CDK1) and cyclin A2 impairs its HIV-1 restriction activity, but not the dNTP hydrolase activity, suggesting that dNTP depletion is not the sole mechanism of SAMHD1-mediated HIV-1 restriction. Using coimmunoprecipitation and mass spectrometry, we identified and validated two additional host proteins interacting with human SAMHD1, namely, cyclin-dependent kinase 2 (CDK2) and S-phase kinase-associated protein 2 (SKP2). We observed that mouse SAMHD1 specifically interacted with cyclin A2, cyclin B1, CDK1, and CDK2. Given the role of these SAMHD1-interacting proteins in cell cycle progression, we investigated the regulation of these host proteins by monocyte differentiation and activation of CD4+ T cells and examined their effect on the phosphorylation of human SAMHD1 at T592. Our results indicate that primary monocyte differentiation and CD4+ T-cell activation regulate the expression of these SAMHD1-interacting proteins. Furthermore, our results suggest that, in addition to CDK1 and cyclin A2, CDK2 phosphorylates T592 of human SAMHD1 and thereby regulates its HIV-1 restriction function. IMPORTANCE SAMHD1 is the first dNTP triphosphohydrolase found in mammalian cells. Human and mouse SAMHD1 proteins block HIV-1 infection in noncycling cells. Previous studies suggested that phosphorylation of human SAMHD1 at threonine 592 by CDK1 and cyclin A2 negatively regulates its HIV-1 restriction activity. However, it is unclear whether human SAMHD1 interacts with other host proteins in the cyclin A2 and CDK1 complex and whether mouse SAMHD1 shares similar cellular interacting partners. Here, we identify five cell cycle-related host proteins that interact with human and mouse SAMHD1, including three previously unknown cellular proteins (CDK2, cyclin B1, and SKP2). Our results demonstrate that several SAMHD1-interacting cellular proteins regulate phosphorylation of SAMHD1 and play an important role in HIV-1 restriction function. Our findings help define the role of these cellular interacting partners of SAMHD1 that regulate its HIV-1 restriction function.

SAMHD1: a new insight into HIV-1 restriction in myeloid cells

Human myeloid-lineage cells are refractory to HIV-1 infection. The Vpx proteins from HIV-2 and sooty mangabey SIV render these cells permissive to HIV-1 infection through proteasomal degradation of a putative restriction factor. Two recent studies discovered the cellular protein SAMHD1 to be this restriction factor, demonstrating that Vpx induces proteasomal degradation of SAMHD1 and enhances HIV-1 infection in myeloid-lineage cells. SAMHD1 functions as a myeloid-cell-specific HIV-1 restriction factor by inhibiting viral DNA synthesis. Here we discuss the implications of these findings in delineating the mechanisms of HIV-1 restriction in myeloid-lineage cells and the potential role of Vpx in lentiviral pathogenesis.

SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells

SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G1/G0 phase and reduces apoptosis. These alterations correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection.

SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

To the Editor: Sterile alpha motif domain– and HD domain–containing protein 1 (SAMHD1) is a cellular dNTP triphosphohydrolase (dNTPase) that restricts HIV-1 replication in myeloid cells and resting CD4+ T cells by degrading dNTPs and limiting viral reverse transcription1–5. Purified recombinant SAMHD1 also has exonuclease activity when synthetic nucleic acids or HIV-1 gag and tat RNAs transcribed in vitro are used as substrates6. Ryoo et al.7 recently suggested that SAMHD1 restricts HIV-1 infection through its ribonuclease (RNase) activity by cleaving the viral RNA genome. By using SAMHD1 mutants purported to specifically retain dNTPase (SAMHD1Q548A) or RNase (SAMHD1D137N) activities, Ryoo et al.7,8 proposed that the RNase activity of SAMHD1, but not its dNTPase activity, is essential for HIV-1 restriction in nondividing cells. They also suggested that SAMHD1 phosphorylation at T592 negatively regulated its RNase activity7. To extend these findings7, we measured HIV-1 protein synthesis and virion production in the presence of SAMHD1 when the requirement for intracellular dNTP-dependent HIV-1 reverse transcription was bypassed. We co-transfected an HIV-1 proviral DNA plasmid (pNL4-3) with a plasmid expressing wild-type (WT) SAMHD1 or a phosphoablative, but dNTPase-active, mutant (SAMHD1T592A; refs. 9–11) into human embryonic kidney (HEK) 293T cells and assessed intracellular HIV-1 Gag protein synthesis and viral particle release in the supernatants. This transfection-based HIV-1 production is independent of reverse transcription requiring intracellular dNTPs as precursors of viral DNA synthesis, but is dependent on HIV-1 mRNA–mediated gene expression. Intracellular HIV-1 Gag protein levels, p24 capsid levels in released HIV-1 virions and infectivity were not reduced by the ectopic expression of WT SAMHD1 or SAMHD1T592A mutant (Supplementary Fig. 1), which suggests that SAMHD1 cannot inhibit HIV-1 production after the reverse transcription step, regardless of its phosphorylation at T592. These findings are consistent with our previous results showing that the expression of WT SAMHD1 or of the SAMHD1T592A mutant in dividing cells does not restrict HIV-1 infection11,12. Our results suggest that SAMHD1 does not have broad nuclease activity, but do not rule out a specific nucleolytic interaction between SAMHD1 and incoming HIV-1 genomic RNA (gRNA). Given the preponderance of previous data implicating the dNTPase activity of SAMHD1 as its primary antiviral mechanism, we reproduced the key experiments of Ryoo et al.7. Their conclusion that SAMHD1 restricts HIV-1 through RNase activity was based on the differential activity of the SAMHD1 mutants, SAMHD1D137N and SAMHD1Q548A (ref. 7). We independently generated these two mutant constructs and confirmed the expected mutations by DNA sequencing to ensure that there were no other disabling mutations in the constructs. We then examined the HIV-1 restriction and intracellular dNTP regulation by these mutants and WT SAMHD1 by following the protocol of Ryoo et al.7. Our results show that both the SAMHD1D137N and SAMHD1Q548A mutants were expressed at similar levels to that of WT SAMHD1, and each efficiently restricted HIV-1 infection and decreased dATP, dGTP and dTTP levels in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells (Fig. 1a–c). SAMHD1 expression did not significantly decrease dCTP levels, as compared to vector control cells (Fig. 1c, right), probably owing to the different biosynthesis pathway of dCTP. Notably, Ryoo et al.7 showed only dCTP levels, but not dATP, dGTP or dTTP levels, in SAMHD1expressing or control cells. Previous studies have used a SAMHD1D137A mutant to explore the effects of the dGTP binding site (D137) on the dNTPase activity, tetramer formation and HIV-1 restriction of SAMHD1 (refs. 13–15). The SAMHD1D137A mutant has no detectable dNTPase activity or HIV-1 restriction in vitro, owing to its inability to form a stable tetramer14,15. It is possible that the SAMHD1D137N mutant might be stabilized in cells to remain in a tetrameric form, thus maintaining its ability to reduce intracellular dNTP levels and restrict HIV-1 infection. It is also possible that an in vitro dNTPase assay using purified recombinant SAMHD1 proteins might not fully reflect the dNTPase activity of SAMHD1 in PMA-differentiated U937 cells. However, these possibilities remain to be examined to explain how the SAMHD1D137N mutant restricts HIV-1 infection if its dNTPase activity is impaired. In contrast to the results of Ryoo et al.7, the SAMHD1D137N and SAMHD1Q548A mutants in our experiments do not have differing anti-HIV-1 activities, and neither lacks the ability to lower cellular dNTP levels. Ryoo et al.7 reported an approximately twofold decrease in HIV-1 gRNA levels in PMA-differentiated U937 cells expressing WT SAMHD1 or SAMHD1D137N, but not the SAMHD1Q548A mutant, as compared to the control cells at 3 h and 6 h postinfection (h.p.i.), which suggests SAMHD1-mediated HIV-1 gRNA degradation7. By contrast, we detected comparable levels of HIV-1 gRNA in PMAdifferentiated U937 cells expressing SAMHD1 (WT, SAMHD1D137N and SAMHD1Q548A mutants) and the vector control cells at 1, 3 and 6 h.p.i., respectively (Fig. 1d), showing that, in our study, SAMHD1 cannot degrade HIV-1 gRNA during early infection. To further support our findings, we measured the levels of HIV-1 late reverse transcription products in infected cells at 12 and 24 h.p.i, which represent viral cDNA synthesis dependent on the intracellular dNTP pool. We found that the expression of WT SAMHD1, SAMHD1D137N and SAMHD1Q548A mutants significantly reduced HIV-1 late reverse transcription products as compared to the vector control cells (Fig. 1e), correlating well with a reduced intracellular dNTP pool (Fig. 1c). Thus, in our study, these two mutants of SAMHD1 cannot distinguish its dNTPase and RNase functions, and dNTP depletion accounts for SAMHD1-mediated HIV-1 restriction. Seamon et al.16 reported that trace exonuclease activities of recombiSAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

Cellular and viral mechanisms of HIV-1 transmission mediated by dendritic cells.

Dendritic cells (DCs) play a key role in the initial infection and cell-to-cell transmission events that occur upon HIV-1 infection. DCs interact closely with CD4(+) T cells, the main target of HIV-1 replication. HIV-1 challenged DCs and target CD4(+) T cells form a virological synapse that allows highly efficient transmission of HIV-1 to the target CD4(+) T cells, in the absence of productive HIV-1 replication in the DCs. Immature and subsets of mature DCs show distinct patterns of HIV-1 replication and cell-to-cell transmission, depending upon the maturation stimulus that is used. The cellular and viral mechanisms that promote formation of the virological synapse have been the subject of intense study and the most recent progress is discussed here. Characterizing the cellular and viral factors that affect DC-mediated cell-to-cell transmission of HIV-1 to CD4(+) T cells is vitally important to understanding, and potentially blocking, the initial dissemination of HIV-1 in vivo.

HIV-1 Nef Enhances Dendritic Cell-Mediated Viral Transmission to CD4+ T Cells and Promotes T-Cell Activation

HIV-1 Nef enhances dendritic cell (DC)-mediated viral transmission to CD4+ T cells, but the underlying mechanism is not fully understood. It is also unknown whether HIV-1 infected DCs play a role in activating CD4+ T cells and enhancing DC-mediated viral transmission. Here we investigated the role of HIV-1 Nef in DC-mediated viral transmission and HIV-1 infection of primary CD4+ T cells using wild-type HIV-1 and Nef-mutated viruses. We show that HIV-1 Nef facilitated DC-mediated viral transmission to activated CD4+ T cells. HIV-1 expressing wild-type Nef enhanced the activation and proliferation of primary resting CD4+ T cells. However, when co-cultured with HIV-1-infected autologous DCs, there was no significant trend for infection- or Nef-dependent proliferation of resting CD4+ T cells. Our results suggest an important role of Nef in DC-mediated transmission of HIV-1 to activated CD4+ T cells and in the activation and proliferation of resting CD4+ T cells, which likely contribute to viral pathogenesis.

Phosphorylation of mouse SAMHD1 regulates its restriction of human immunodeficiency virus type 1 infection, but not murine leukemia virus infection

Human SAMHD1 (hSAMHD1) restricts HIV-1 infection in non-dividing cells by depleting intracellular dNTPs to limit viral reverse transcription. Phosphorylation of hSAMHD1 at threonine (T) 592 by cyclin-dependent kinase (CDK) 1 and CDK2 negatively regulates HIV-1 restriction. Mouse SAMHD1 (mSAMHD1) restricts HIV-1 infection in non-dividing cells, but whether its phosphorylation regulates retroviral restriction is unknown. Here we identified six phospho-sites of mSAMHD1, including T634 that is homologous to T592 of hSAMHD1 and phosphorylated by CDK1 and CDK2. We found that wild-type (WT) mSAMHD1 and a phospho-ablative mutant, but not a phospho-mimetic mutant, restricted HIV-1 infection in differentiated U937 cells. Murine leukemia virus (MLV) infection of dividing NIH3T3 cells was modestly restricted by mSAMHD1 WT and phospho-mutants, but not by a dNTPase-defective mutant. Our results suggest that phosphorylation of mSAMHD1 at T634 by CDK1/2 negatively regulates its HIV-1 restriction in differentiated cells, but does not affect its MLV restriction in dividing cells.

A Putative Cyclin-binding Motif in Human SAMHD1 Contributes to Protein Phosphorylation, Localization and Stability

SAMHD1 (sterile α motif and HD domain-containing protein 1) is a mammalian protein that regulates intracellular dNTP levels through its hydrolysis of dNTPs. SAMHD1 functions as an important retroviral restriction factor through a mechanism relying on its dNTPase activity. We and others have reported that human SAMHD1 interacts with the cell cycle regulatory proteins cyclin A, CDK1, and CDK2, which mediates phosphorylation of SAMHD1 at threonine 592, a post-translational modification that has been implicated in abrogating SAMHD1 restriction function and ability to form stable tetramers. Utilizing co-immunoprecipitation and co-localization approaches, we show that endogenous SAMHD1 is able to interact with the cyclin A-CDK1-CDK2 complexin monocytic THP-1 cells and primary monocyte-derived macrophages. Sequence analysis of SAMHD1 identifies a putative cyclin-binding motif found in many cyclin-CDK complex substrates. Using a mutagenesis-based approach, we demonstrate that the conserved residues in the putative cyclin-binding motif are important for protein expression, protein half-life, and optimal phosphorylation of SAMHD1 at Thr592. Furthermore, we observed that SAMHD1 mutants of the cyclin-binding motif mislocalized to a nuclear compartment and had reduced ability to interact with cyclin A-CDK complexes and to form the tetramer. These findings help define the mechanisms by which SAMHD1 is phosphorylated and suggest the contribution of cyclin binding to SAMHD1 expression and stability in dividing cells.