The G1/S specific D2 acts as a viral restriction factor in primary macrophages #207 Ester Ballana, Roger Badia, Maria Pujantell, Eva Riveira-Muñoz, Bonaventura Clotet and José A. Esté E-mail: [email protected] AIDS Research Institute-IrsiCaixa and Health Research Institute Germans Trias i Pujol (IGTP), Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain Phone: +34 934656374

ABSTRACT CYCLIN D2 AND KNOCKDOWN DIFFERENTLY IMPACT VIRAL REPLICATION DEPENDING ON MDM TYPE EXPRESSION OF CYCLIN D2 RESTRICTED HIV-1 REPLICATION IN GM-CSF MDM p=0.004 BACKGROUND: Macrophages are a heterogenic cell population with properties strongly influenced by stimuli present during their A. p=0.018 A. B. 5 600 *** *** differentiation from monocytes. Distinct differentiation stimuli lead to differential cell activation and proliferation, differential 30 p=0.008 3 p<0.0001 500 ** * expression of proteins controlling progression and differential capacity to support HIV-1 replication. Cell cycle control * M-CSF 4 plays also a fundamental role in SAMHD1-mediated virus restriction, as it is regulated through phosphorylation by cyclin- GM-CSF 20 400 dependent kinases (CDK). Here, we describe a novel HIV-1 restriction pathway determined by the differential expression of the mRNA 3 mRNA 2 G1/S-specific cyclin D2. *** * 300

% GFP % 2 METHODOLOGY: Primary monocytes were transfected with siRNA and differentiated into monocyte-derived macrophages 10 200 TAULA ec50 CCND3 CCND2

* DNA Proviral (MDM). Monocyte differentiation consisted in 4-day incubation with M-CSF or GM-CSF. Susceptibility to HIV-1 infection was 1 * examined by flow cytometry after infection of a VSV-pseudotyped NL4-3 GFP-expressing virus. Total viral DNA formation was 100 1 3 *** *** quantified by qPCR in MDM infected with full-replicative R5 HIV-1 strain BaL. Co-immunoprecipitation (CoIP) was performed in Relative Relative 0 0 lysates from 293T cells overexpressing cyclin D2-HA or Flag- fusion and were incubated with anti-FLAG or anti-HA 0 0 Mock siNT siCCND2 siCCND3 Mock siNT siCCND2 siCCND3 AZT siNT antibodies covalently attached to agarose. expression and phosphorylation and CoIP were analyzed by immunoblotting. Mock AZT RAL siNT siCDK4 Mock siCCND2siCCND3 siCDK4 B. C. siCDKN1A siCCND2siCCND3 RESULTS: Differential expression profile of cell cycle proteins was observed in GM-CSF vs. M-CSF MDM, being the most M-CSF GM-CSF siCDKN1A 450 * C. relevant the upregulation in GM-CSF MDM of all D-type (D1, 2-fold, p=0.0006; D2, 40-fold, p=0.0045; and D3, 3-fold siRNA: NT D2 D3 NT D2 D3 M-CSF 400 1.25 750 * p=0.0002, respectively) and the CDK inhibitor p21 (4-fold, p=0.038). Efficient and specific siRNA-mediated downregulation of GM-CSF 4 pSAMHD1 350 * cyclin D2 led to a significant increase of HIV-1 replication and total viral DNA formation only in GM-CSF MDM (3-fold increase; 1.00 ** p=0.0037), but not in M-CSF MDM. CyclinD2 knockdown was linked to an increased phosphorylation of SAMHD1, without 300 * 3

mRNA 500 additional differences in the expression profile of cell cycle proteins. Characterization of the restriction mechanism by CoIP and SAMHD1 250 0.75 additional siRNA experiments led to the identification of a protein complex formed by cyclin D2 and p21 as the responsible of a 200 2 % GFP% CCND2 150 0.50 GFP% novel viral restriction mechanism. CyclinD2 250 100 CONCLUSIONS: The G1/S specific cyclin D2 acts as a restriction factor for HIV-1 in primary macrophages. These results further 0.25 *** 1

** *** DNA proviral Relative demonstrate the importance of cell cycle control in viral replication. 50 *** Relative CyclinD3 *** *** *** 0 0.00 0 0

CELL CYCLE PROGRESSION AND SAMHD1 ACTIVATION WAS DEPENDENT ON DIFFERENTIATION STIMULI IN MDM Hsp90 AZT siNT INF Mock Mock siNT INF AZT siNT AZT RAL siNT B. siCCND2 siCCND3 A. D. siCCND2#1siCCND2#2 M-CSF GM-CSF M-CSF GM-CSF Figure 2. siRNA of Cyclin D2 and D3 have differential effects on HIV-1 replication in M-CSF and GM-CSF MDM. (A) Effective knockdown of siCCND2#1siCCND2#2 siCCND2#1siCCND2#2 S/G2M: 11,5% S/G2M: 1,8% D1 D2 CCND2 and CCND3 expression. Relative mRNA expression of CCND2 and CCND3 in M-CSF or GM-CSF MDM. (B) Protein expression and Figure 4. Cyclin D2 downregulation enhances HIV-1 replication in GM-CSF MDM. (A) HIV replication in siRNA-treated GM-CSF M GM M GM SAMHD1 activation in Cyclin D2 and Cyclin D3 siRNA-treated MDM. SAMHD1 inactivation by phosphorylation is reduced in siCCND3 (D3) MDM. Percentage replication in Mock-treated or siRNA-transfected MDM. (B) Proviral DNA formation 16h post-infection in siRNA- M-CSF MDM and increased in siCCND2 (D2) GM-CSF MDM, compared to the corresponding non-targeting siRNA (NT) MDM. (C) HIV treated GM-CSF MDM. Proviral DNA was normalized to mock-treated MDM. Controls include mock-transfected cells treated with 8,3% 0,6% pSAMHD1 AAD) - replication in siRNA-treated M-CSF or GM-CSF MDM. Percentage replication in M-CSF or GM-CSF MDM, mock transfected or transfected 1µg/ml of AZT or Raltegravir (RAL). (C) Cyclin D2 restricts proviral DNA formation and HIV-1 replication. Inhibition of Cyclin D2 SAMHD1 with corresponding siRNA. Mean  SD of 3 different donors performed in triplicate is shown. * p<0.05; ** p<0.005; *** P<0.0005 expression was confirmed with independent siRNA sequences (siCCND2#1 and siCCND2#2, left panel). Effective downregulation of Counts G1: 51,7% G1: 60,8% CCND2 expression with 2 different siRNAs resulted in enhanced viral replication (middle panel) and proviral DNA formation (right DNA (7 G0: 32,1% G0: 34,9% CDK1 CYCLIN D2 EXPRESSION CONTROLED SAMHD1 ACTIVATION IN GM-CSF MDM, SIMILAR TO p21 panel). Mean  SD of at least 3 independent experiments performed in duplicate is shown. * p<0.05; ** p<0.005; *** P<0.0005 Mock siNT Ki67 RNA (Pyronin Y) A. 1.5 2.5 B. siRNA C. CDK2 S/G2M: 1,15% S/G2M: 1,30% IDENTIFICATION OF A PROTEIN COMPLEX WITH CYCLIN D2 AND p21 IN GM-CSF MDM C. 100 2.0 M NT D2 D3 p21 CDK4 *** CDK4 A. B. C. mRNA mRNA 1.0 WCL IP:Flag WCL IP:HA M-CSF 1.5 pSAMHD1 AAD) WCL B IP:D2 IP:p21 GM-CSF CDK6 - M p21 B M p21 M D2 B M D2 CCND2 CCND3 1.0 10 0.5 Cyclin D2 CyclinA2 SAMHD1 Cyclin D2 Cyclin D2 ** *** 0.5

*** DNA (7 *** Relative Relative G1: 62,6% G1: 74,0% *** CyclinD2 relative folds) relative 0.0 0.0 G0: 30,8% G0: 21,9% Cyclin D2 p21 p21 10 1 p21 Mock siNT Mock siNT RNA (Pyronin Y) mRNA expression expression mRNA CyclinD3 siCDK4 siCDK4 (log 2.0 siCCND2siCCND3 2.0 siCCND2siCCND3 siCDKN1A siCDKN1A Cyclin D3 siCCND2 siCDKN1A p21 S/G2M: 1,23% S/G2M: 1,02% Hsp90 Hsp90 Hsp90 1.5 1.5 mRNA 0.1 mRNA p21 Hsp90 Figure 5. Co-immunoprecipitation (IP) studies of Cyclin D2 and p21. (A) and (B) Co-IP assay of 293T cells transfected with plasmids 1.0 1.0 CDK4 AAD)

CDKN1A ** expressing Flag-tagged p21 and HA-tagged CyclinD2, respectively. Cell lysates were immunoprecipitated with anti-Flag (A) or anti- - *** CDK4 0.5 0.5 HA (B). Whole cell lysates (WCL) and immunoprecipitates were analyzed by immunoblotting for the presence of endogenous Figure 1. M-CSF and GM-CSF MDM show different cell proliferation and cell cycle-related protein expression. (A) and (B) Evaluation Relative CyclinD2 (A) or p21 (B). (C) Co-IP assay of endogenous Cyclin D2 and p21 in GM-CSF MDM. Lysates were incubated with anti-

Relative ***

DNA (7 G1: 61,0% G1: 68,9% of cell proliferation and cell cycle analysis in M-CSF and GM-CSF MDM. (A) Histograms showing Ki67 staining in M-CSF and GM-CSF 0.0 0.0 Hsp90 CyclinD2 or anti-p21 antibodies overnight, followed by incubation with Fast flow Sepharose. WCL and immunoprecipitates were MDM. (B) Dot plots representing cell cycle profile showing DNA (7-AAD) and RNA (Pyronin Y) content in M-CSF and GM-CSF MDM. G0: 35,3% G0: 26,1% siNT analyzed by immunoblotting with indicated antibodies. M; mock-transfected cells, B; Beads. Mock siNT Mock siCDK4 siCDK4 Percentage of positive cells in a representative donor is shown in each case. (C) of cell cycle-related and siCCND2siCCND3 siCCND2siCCND3 RNA (Pyronin Y) siCDKN1A siCDKN1A SAMHD1 restriction pathway. mRNA of SAMHD1, CDK1, CDK2, CDK4 and CDK6, the corresponding cyclins (A2, B1, B2, D1, D2, D3, E1 Figure 3. Knockdown of Cyclin D2, Cyclin D3, p21 and CDK4 in GM-CSF MDM. (A) Effective knockdown of CCND2, CCND3, CDKN1A and DISCUSSION and E2) and CDK2 inhibitor p21 (CDKN1A) was quantified in samples of M-CSF and GM-CSF MDM. Data are normalized to M-CSF CDK4 expression. Relative mRNA expression of CCND2, CCND3 (upper panels), CDKN1A and CDK4 (lower panels) in GM-CSF MDM. (B) • Differential expression and function of type D Cyclins determines the ability to support HIV-1 replication of distinct macrophage relative expression. Mean  SD of 3 independent donors is shown. ** p<0.005; *** P<0.0005. (D) SAMHD1 activation and cell cycle- Protein expression and SAMHD1 activation in siRNA-treated GM-CSF MDM. SAMHD1 inactivation by phosphorylation is increased in models, characterized by different cell proliferation and cell cycle progression. related protein expression in M-CSF and GM-CSF MDM. Western blot showing protein expression of different cell cycle proteins, Cyclin D2 and p21 GM-CSF MDM, compared to Mock-transfected (M) or non-targeting (siNT) MDM. (C) Cell cycle profile showing DNA and • Cyclin D2 restricts HIV-1 in non-proliferating MDM. Complex formation between Cyclin D2 and p21 indicates a common cell cycle SAMHD1 expression and activation and Hsp90 as loadingcontrol. Two representative donor are shown. M; M-CSF, GM; GM-CSF. RNA content of mock-treated or siRNA-transfected GM-CSF MDM, quantified by FACS. Data from a representative donor is shown. regulatory pathway ultimately leading to SAMHD1-mediated inhibition of HIV replication.