Evidence that HIV-1 restriction factor SAMHD1 facilitates differentiation of myeloid THP-1 cells
© Dragin et al. 2015
Received: 2 July 2015
Accepted: 15 November 2015
Published: 25 November 2015
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.
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.
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.
KeywordsHIV SAMHD1 Restriction factor Differentiation Cell cycle Myeloid cells T cells
Immune quiescent cells display reduced susceptibility to HIV-1 productive infection, due to several quiescence-related phenomena such as cytoskeleton organization or low viral transcription. The activity of several restriction factors also contributes to viral inhibition by blocking specific steps of the viral life cycle. Among them, sterile alpha motif and HD-domain containing protein 1 (SAMHD1) inhibits both HIV-1 and Vpx-deleted HIV-2/SIVsmm viruses at the level of reverse transcription [1, 2]. SAMHD1 reduces the intracellular pool of dNTP through its dNTP hydrolase activity in myeloid and quiescent CD4+ T cells [3–7]. Recent studies report that SAMHD1 also has an RNase activity targeting HIV-1 genomic RNA . The dNTPase activity of the protein is dependent on an intact phosphodiesterase HD domain [1, 2]. Though HIV-1 is sensitive to SAMHD1, it has not developed a viral weapon to counteract this hindrance. In contrast, HIV-2/SIVsmm encodes for the Vpx auxiliary protein, which induces SAMHD1 degradation by hijacking the Cul4A-DDB1 ubiquitin ligase through DCAF1 binding [1, 2].
Several reports point to a role of SAMHD1 in tumorigenesis and in cell cycle progression. In particular, SAMHD1 has been found mutated in chronic lymphocytic leukemia (CLL) . In CLL patient cells, SAMHD1 expression is often reduced as it is the case in several cancer cell lines . Whole sequencing has also suggested that SAMHD1 could be mutated in malignancies . Regarding cell cycle progression, proliferation in HeLa cells is favored by the dNTPase defective SAMHD1 HD/AA mutant . In fibroblasts, SAMHD1 has been shown to be variously expressed along the cell cycle, maximally during quiescence and minimally during S-phase, which correlates with the need for dNTP during DNA replication . Apart from SAMHD1, several reports suggest that suppression of nucleotide metabolism underlines the establishment of a quiescent state and promotes genomic instability [12–14]. Altogether, we brought up the hypothesis that SAMHD1 may facilitate the entry into or the maintenance of quiescence. To test our hypothesis, we used suspension cultures of the monocytic cell line THP-1 that differentiate into a macrophage-like phenotype under phorbol-12-myristate-13-acetate (PMA) treatment and therefore transit from a dividing state to a quiescent state. PMA-induced differentiation of THP-1 cells results, among other things, in cell morphological changes, adhesion to the plastic surface and expression of macrophage surface markers such as CD11a, CD11b and ICAM1 [15, 16]. In addition, THP-1 cells acquire the ability to restrict HIV infection along differentiation, following SAMHD1 dephosphorylation on a specific threonine [17–19].
We established THP-1 myeloid cell lines stably overexpressing HA-tagged wt SAMHD1 or the catalytic mutant form of the protein (HD/AA, referred to SAMHD1μ on the figures) or cells depleted of SAMHD1 (shSAMHD1). As previously reported, the promoter from the pLenti vector used for SAMHD1 expression is sensitive to PMA . In the absence of PMA, exogenous SAMHD1 is poorly expressed, while in PMA-treated cells, levels of SAMHD1 and SAMHD1 HD/AA increased equally (Additional file 1: Figure S1A).
To our surprise, even in the absence of PMA, a small increase in adhesion capability was detected in SAMHD1 overexpressing cells (Fig. 2b and c). This increase was reproducible but in some experiments was transient (Fig. 2b, Exp. 2) and, in any case, less efficient than in PMA-treated cells (Fig. 2c). Nonetheless, overexpression of wt SAMHD1 was not sufficient to induce cell differentiation or to confer a differentiation-associated restriction phenotype against HIV to dividing THP-1 cells (without PMA), while it can increase restriction when the cells were differentiated in the presence of PMA (data not shown and ).
The authors acknowledge the Cytometry and Immunobiology Facility of the Cochin Institute. This work was supported by grants from the “Agence Nationale de la Recherche sur le SIDA et les hépatites virales” (ANRS), SIDACTION, “Fondation de France” and “Fondation pour la Recherche Médicale” (FRM, grant number DEQ20140329528 attributed to FM). LD and JF received a fellowship from the French “Ministère de la Recherche et la Technologie” (MRT) and LD also from SIDACTION, AS from SIDACTION and Fondation de France, SMM from “Fondation pour la Recherche Médicale” (FRM) and HL from ANRS.
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- Hrecka K, Hao C, Gierszewska M, Swanson SK, Kesik-Brodacka M, Srivastava S et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature. 2011;474:658–61.PubMed CentralView ArticlePubMedGoogle Scholar
- Laguette N, Sobhian B, Casartelli N, Ringeard M, Chable-Bessia C, Segeral E et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature. 2011;474:654–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Goldstone DC, Ennis-Adeniran V, Hedden JJ, Groom HC, Rice GI, Christodoulou E et al. HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase. Nature. 2011;480:379–82.View ArticlePubMedGoogle Scholar
- Lahouassa H, Daddacha W, Hofmann H, Ayinde D, Logue EC, Dragin L et al. SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates. Nat Immunol. 2012;13:223–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Powell RD, Holland PJ, Hollis T, Perrino FW. Aicardi-Goutieres syndrome gene and HIV-1 restriction factor SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase. J Biol Chem. 2011;286:43596–600.PubMed CentralView ArticlePubMedGoogle Scholar
- Baldauf HM, Pan X, Erikson E, Schmidt S, Daddacha W, Burggraf M et al. SAMHD1 restricts HIV-1 infection in resting CD4(+) T cells. Nat Med. 2012;18:1682–9.View ArticlePubMedGoogle Scholar
- Descours B, Cribier A, Chable-Bessia C, Ayinde D, Rice G, Crow Y et al. SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4+ T-cells. Retrovirology. 2012;9:87.PubMed CentralView ArticlePubMedGoogle Scholar
- Ryoo J, Choi J, Oh C, Kim S, Seo M, Kim SY et al. The ribonuclease activity of SAMHD1 is required for HIV-1 restriction. Nat Med. 2014;20:936–41.PubMed CentralView ArticlePubMedGoogle Scholar
- Clifford R, Louis T, Robbe P, Ackroyd S, Burns A, Timbs AT et al. SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage. Blood. 2014;123:1021–31.PubMed CentralView ArticlePubMedGoogle Scholar
- Landau DA, Carter SL, Stojanov P, McKenna A, Stevenson K, Lawrence MS et al. Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell. 2013;152:714–26.PubMed CentralView ArticlePubMedGoogle Scholar
- Franzolin E, Pontarin G, Rampazzo C, Miazzi C, Ferraro P, Palumbo E et al. The deoxynucleotide triphosphohydrolase SAMHD1 is a major regulator of DNA precursor pools in mammalian cells. Proc Natl Acad Sci U S A. 2013;110:14272–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Aird KM, Zhang G, Li H, Tu Z, Bitler BG, Garipov A et al. Suppression of nucleotide metabolism underlies the establishment and maintenance of oncogene-induced senescence. Cell Rep. 2013;3:1252–65.View ArticlePubMedGoogle Scholar
- Bester AC, Roniger M, Oren YS, Im MM, Sarni D, Chaoat M et al. Nucleotide deficiency promotes genomic instability in early stages of cancer development. Cell. 2011;145:435–46.PubMed CentralView ArticlePubMedGoogle Scholar
- Kunz BA, Kohalmi SE, Kunkel TA, Mathews CK, McIntosh EM, Reidy JA. International commission for protection against environmental mutagens and carcinogens. Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance of genetic stability. Mutat Res. 1994;318:1–64.View ArticlePubMedGoogle Scholar
- Munoz-Pacheco P, Ortega-Hernandez A, Miana M, Cachofeiro V, Fernandez-Cruz A, Gomez-Garre D. Ezetimibe inhibits PMA-induced monocyte/macrophage differentiation by altering microRNA expression: a novel anti-atherosclerotic mechanism. Pharmacol Res. 2012;66:536–43.View ArticlePubMedGoogle Scholar
- Most J, Schwaeble W, Drach J, Sommerauer A, Dierich MP. Regulation of the expression of ICAM-1 on human monocytes and monocytic tumor cell lines. J Immunol. 1992;148:1635–42.PubMedGoogle Scholar
- Cribier A, Descours B, Valadao AL, Laguette N, Benkirane M. Phosphorylation of SAMHD1 by cyclin A2/CDK1 regulates its restriction activity toward HIV-1. Cell Rep. 2013;3:1036–43.View ArticlePubMedGoogle Scholar
- Welbourn S, Dutta SM, Semmes OJ, Strebel K. Restriction of virus infection but not catalytic dNTPase activity is regulated by phosphorylation of SAMHD1. J Virol. 2013;87:11516–24.PubMed CentralView ArticlePubMedGoogle Scholar
- White TE, Brandariz-Nunez A, Valle-Casuso JC, Amie S, Nguyen LA, Kim B et al. The retroviral restriction ability of SAMHD1, but not its deoxynucleotide triphosphohydrolase activity, is regulated by phosphorylation. Cell Host Microbe. 2013;13:441–51.View ArticlePubMedGoogle Scholar
- Tibaldi L, Leyman S, Nicolas A, Notebaert S, Dewulf M, Ngo TH et al. New blocking antibodies impede adhesion, migration and survival of ovarian cancer cells, highlighting MFGE8 as a potential therapeutic target of human ovarian carcinoma. PLoS One. 2013;8:e72708.PubMed CentralView ArticlePubMedGoogle Scholar
- Dragin L, Nguyen LA, Lahouassa H, Sourisce A, Kim B, Ramirez BC et al. Interferon block to HIV-1 transduction in macrophages despite SAMHD1 degradation and high deoxynucleoside triphosphates supply. Retrovirology. 2013;10:30.PubMed CentralView ArticlePubMedGoogle Scholar
- Di Fiore B, Davey NE, Hagting A, Izawa D, Mansfeld J, Gibson TJ et al. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators. Dev Cell. 2015;32:358–72.View ArticlePubMedGoogle Scholar
- Ruffin N, Brezar V, Ayinde D, Lefebvre C, Schulze Zur Wiesch J, van Lunzen J et al. Low SAMHD1 expression following T-cell activation and proliferation renders CD4+ T cells susceptible to HIV-1. AIDS. 2015;29:519–30.PubMed CentralPubMedGoogle Scholar
- Schmidt S, Schenkova K, Adam T, Erikson E, Lehmann Koch J, Sertel S et al. SAMHD1’s protein expression profile in humans. J Leukoc Biol. 2015. doi:10.1189/jlb.4HI0714-338RR.Google Scholar
- Pauls E, Ruiz A, Badia R, Permanyer M, Gubern A, Riveira-Munoz E et al. Cell cycle control and HIV-1 susceptibility are linked by CDK6-dependent CDK2 phosphorylation of SAMHD1 in myeloid and lymphoid cells. J Immunol. 2014;193:1988–97.View ArticlePubMedGoogle Scholar