Supplementary Information. SAMHD1 Restricts HIV-1 Infection in Resting CD4 + T Cells

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Esmond Hensley
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1 Supplementry Informtion SAMHD Restricts HIV- Infection in Resting CD T Cells Hnn-Mri Blduf,2,, Xioyu Pn,, Elin Erikson,2, Srh Schmidt, Wqo Dddch 3, Mnj Burggrf, Kristin Schenkov, In Amiel,2, Guido Wnitz 5, Thoms Grmerg 6, Sylvi Pnitz 7, Egert Flory 7, Nthniel R. Lndu 8, Serkn Sertel 9, Frnk Rutsch, Felix Lsitschk, Bek Kim 3,2, Rente König, 3, Oliver T Fckler & Oliver T Keppler,2 Deprtment of Infectious Diseses, Virology, University of Heidelerg, Heidelerg, Germny. 2 Institute of Medicl Virology, University of Frnkfurt, Frnkfurt, Germny. 3 Deprtment of Microiology nd Immunology, University of Rochester Medicl Center, Rochester, New York, USA. Reserch Group Host-Pthogen Interctions, Pul-Ehrlich-Institute, Lngen, Germny. 5 Institute of Immunology, University of Heidelerg, Heidelerg, Germny. 6 Virologisches Institut, Klinische und Molekulre Virologie, Universität Erlngen-Nürnerg, Erlngen, Germny. 7 Division of Medicl Biotechnology, Pul-Ehrlich-Institute, Lngen, Germny. 8 Deprtment of Microiology, New York University School of Medicine, New York, New York, USA. 9 Deprtment of Otolryngology, Hed nd Neck Surgery, University of Heidelerg, Heidelerg, Germny. Deprtment of Generl Peditrics, Münster University Children's Hospitl, Münster, Germny. Institute of Pthology, University of Heidelerg, Heidelerg, Germny. 2 Deprtment of Phrmcy, College of Phrmcy, Kyung-Hee University, Seoul, South Kore. 3 Infectious & Inflmmtory Disese Center, Snford-Burnhm Medicl Reserch Institute, L Joll, CA, USA. These uthors contriuted eqully to this work. Nture Medicine doi:.38/nm.296

2 Supplementry Fig. Mrkers of T cell ctivtion nd prolifertion in resting nd ctivted CD T cells 3 Resting CD T cells 2 Dy.8% R Dy 3 Side sctter CellTrce (Fr Red) Counts.%.6% Dy 5.6% M 3 Forwrd sctter CD25/CD69-PE BrdU-APC 3 Activted CD T cells 2 Dy R 2.97% Dy 2 Side sctter CellTrce (Fr Red) Counts 8.9% 75.35% Dy 3 Forwrd sctter CD25/CD69-PE BrdU-APC Vile (gte R, left pnels) () resting nd () PHA/IL2-ctivted CD T cells were leled with CellTrce (Fr Red) t dy nd, t the indicted time points, (middle pnels) costined with CD25/CD69-PE. Right pnels: A prllel smple of cells ws leled with 5- romo-2'-deoxyuridine (BrdU) to ssy its incorportion into cellulr DNA during cell prolifertion with n APC-conjugted nti-brdu ma. The percentge of cells tht hve incorported BrdU is indicted. In, note tht the intensity of CellTrce stining (y-xis) is grdully decresing in ctivted cells, while it remins lrgely unltered in resting cells shown in. Nture Medicine doi:.38/nm.296

3 Supplementry Fig. 2 Neither virion-medited Vpx delivery nor dn tretment lter the cell cycle of resting CD T cells SAMHD-66 Resting CD T cells HIV-* GFP HIV- GFP Vpx Solvent dns Reference chnnel 7-AAD (DNA) Pyronin (RNA) Activted CD T cells HIV-* GFP HIV- GFP Vpx Solvent dns SAMHD-66 Reference chnnel 7-AAD (DNA) Pyronin (RNA) () Resting nd () PHA/IL-2-stimulted CD T cells were chllenged with equivlent infectious units of either HIV-* GFP with (Vpx) or without () incorported Vpx from SIV mc239 or HIV- GFP in the presence (dns) or sence (Solvent) of deoxy-nucleosides. One dy post-infection, intrcellulr SAMHD levels were monitored nd, in prllel, cells stined with 7-AAD (for DNA content) nd pyronin Y (for RNA content) nd nlyzed y flow cytometry. Shown re results from representtive donor. Nerly ll resting CD T cells were in G/G, wheres sustntil frction of ctivted CD T cells resided in G (lower right qudrnt) or S/G2/M (upper right qudrnt). (,) Importntly, neither infection with Vpx-crrying HIV-* GFP prticles nor dn tretment significntly ffected the cell cycle profile of primry CD T cells. Nture Medicine doi:.38/nm.296

4 Supplementry Fig. 3 Quntifiction of different HIV- cdna species in resting CD T cells fter infection with HIV-* GFP ± Vpx 2-LTR circles per ng DNA Vpx. RU5/gg per ng DNA Donor 8 Donor 9 Resting CD T cells were chllenged with equivlent infectious units of either 2x DNsetreted HIV-* GFP with (open histogrms) or without (filled histogrms) incorported Vpx from SIV mc239 nd cell liquots were tken on dy nd dy 3 p.i..() reltive 2-LTR circle copy numers in DNA extrcts from dy 3 smples; () reltive RU5/gg copy numers, representing lte cdna product of reverse trnscription, in DNA extrcts from dy (Donor 8) or dy 3 (Donor 9) smples. Levels of RU5/gg in EFV-treted control infections were sutrcted to control for virus input. Shown re mens of triplictes stndrd devition. : elow detection. Nture Medicine doi:.38/nm.296

5 Supplementry Fig. Vpx overcomes the erly restriction to HIV- infection in resting CD T cells, ut does not result in the relese of newly synthesized virus Percentge GFP cells Vpx HIV- p2 (ng ml - ) Vpx Donor Donor Donor Donor Resting Activted (,) Resting nd ctivted CD T cells were chllenged with equivlent infectious units of either HIV-* GFP with (Vpx, open histogrms) or without (, filled histogrms) incorported Vpx from SIV mc239. () Three dys p.i. the percentge of GFP cells ws quntified y flow cytometry. () In prllel, virus production ws ssessed y quntifiction of HIV- p2 in culture superntnts. Note the sence of significnt HIV- relese from efficiently infected cultures. : elow detection. Nture Medicine doi:.38/nm.296

cells, ctivted CD T cells, nd monocyte-derived mcrophges

WCL Cytoplsm Nucleus SAMHD 72 52 52 MW (kd) 3 3 GAPDH B23 3 3 c

μm Mcrophges 5 μm SAMHD immunolot nlysis of nucleo-cytoplsmic

mcrophges (see Fig. 2d for resting CD T cells).

projections) of resting CD T cell (upper pnel), ctivted CD T

Endogenous SAMHD (green) ws co-stined with the nucler envelope

6 Supplementry Fig. 5 SAMHD loclizes to oth cytoplsm nd nucleus in resting CD T cells, ctivted CD T cells, nd monocyte-derived mcrophges Activted CD T cells Mcrophges 72 MW (kd) WCL Cytoplsm Nucleus WCL Cytoplsm Nucleus SAMHD MW (kd) 3 3 GAPDH B c SAMHD Lmin A Merge Resting CD T cells 2 μm Activted CD T cells 2 μm Mcrophges 5 μm SAMHD immunolot nlysis of nucleo-cytoplsmic frctiontion of () ctivted CD T cells nd () monocyte-derived mcrophges (see Fig. 2d for resting CD T cells). (c) Deconvoluted spinning disk confocl microscopy imges (mximum projections) of resting CD T cell (upper pnel), ctivted CD T cell (middle pnel), nd mcrophges (lower pnel). Endogenous SAMHD (green) ws co-stined with the nucler envelope protein Lmin A (red). Note tht in ddition to the nucleus, SAMHD is present to vrile degree diffusely in the cytoplsm of ll primry cell types nlyzed. Nture Medicine doi:.38/nm.296

7 Supplementry Fig. 6 Vpx depletes cytoplsmic s well s nucler SAMHD in resting nd ctivted CD T cells Disply-GFP SAMHD Merge μm Vpx Q76A Vpx WT Activted CD T cells Vpx Q76A Vpx WT Resting CD T cells Spinning disk confocl microscopy imges of CD T cells. Resting (upper pnels) nd ctivted (lower pnels) cells were nucleofected to co-express Disply-GFP nd Vpx WT, the Vpx Q76A mutnt, or n empty vector (). 6 h lter, cells were fixed nd stined for SAMHD expression (red). Note tht expression of Vpx WT, ut not of Vpx Q76A, reduced SAMHD levels elow the detection limit in cytoplsmic nd nucler comprtments Nture Medicine of resting doi:.38/nm.296 nd ctivted CD T cells.

8 Supplementry Fig. 7 Specific detection of intrcellulr SAMHD y flow cytometry Rit control serum Rit nti-samhd SAMHD-66 CD-FITC Resting CD T cells were fixed nd permeilized s descried in Mterils nd Methods nd stined either with rit control serum (left pnel) or rit nti-samhd serum (right pnel). After co-stining with secondry Alex66-conjugted got nti-rit ntiody nd n nti-cd-fitc ma, cells were nlyzed y flow cytometry. Nture Medicine doi:.38/nm.296

9 Supplementry Fig. 8 SAMHD depletion is specific for HIV- virions with incorported Vpx WT or for HIV-2 WT virions Vpx p2 c SAMHD-66 Percentge GFP cells 3 2 WT Q76A Vpx SIVmc239 d SAMHD-66 Vpx HIV-2 GFP WT Vpx WT Donor 6 Donor 7 () Immunolot nlysis of HIV-* GFP virions crrying Vpx WT, Q76A, or no Vpx (). Resting CD T cells were chllenged with identicl infectious units of these HIV-* GFP virions nd nlyzed for () intrcellulr SAMHD levels d p.i. nd (c) for the percentge of GFP cells in CD25 - CD69 - CD T cells 3 d p.i. y flow cytometry. Brs represent mens ± stndrd devition of five donors. (d) In the experiment shown in Fig. i,j, SAMHD levels in resting CD T cells were monitored y flow cytometry 3 dys post-chllenge with either HIV-2 ROD9 GFP (WT) or the Vpx-defective, isogenic counterprt (ΔVpx). Nture Medicine doi:.38/nm.296

Supplementry Fig. 9 Exogenous deoxynucleosides enhnce HIV- infection in resting CD T cells 3 Solvent Deoxynucleosides Side sctter R Forwrd sctter 3 CD25/CD69-APC.5% 2.

10 Supplementry Fig. 9 Exogenous deoxynucleosides enhnce HIV- infection in resting CD T cells 3 Solvent Deoxynucleosides Side sctter R Forwrd sctter 3 CD25/CD69-APC.5% 2.79% GFP Primry FACS dot plots for vile (gte R) resting CD T cells 3 dys fter chllenge with HIV- GFP in the sence (solvent) or presence of deoxynucleosides (.5 mm). CD25 - CD69 - GFP cells define infected resting CD T cells, for which the reltive percentges re shown. Nture Medicine doi:.38/nm.296

11 Supplementry Fig. datp nd dttp levels in primry CD T cells under different experimentl conditions determined y single-nucleotide incorportion Donor 2 Donor 3 datp No dntp dns dns R A R R R R A R R dntp (5 μm) HIV-* GFP HIV-* GFP Vpx R Vpx dttp Primry dt for quntifictions shown in Fig. 3f,g (detils in legend). Methodology s reported 22, 2. R: resting CD T cells; A: ctivted CD T cells. dns: cultivtion in the presence of dns (2mM) for 3h. Chllenge with HIV-* GFP Vpx hd resulted in depletion of SAMHD in 7% (Donor 2) or 3% (Donor 3) of resting CD T cells t 2 h p.i., respectively, t which time cells were hrvested for dntp nlyses. Nture Medicine doi:.38/nm.296

12 Supplementry Fig. sirna-medited silencing of SAMHD in post-ctivtion resting CD T cells CD T cell isoltion stimultion (PHA-L, 2 μg/ml) PHA-L wshed out IL2 (2 IU/ml) st sirna trnsf. IL2 (2 IU/ml) 2nd sirna trnsf. IL2 (7 IU/ml) Medium replcement IL2 (3.5 IU/ml) Cell Trce leling IL2 (.75 IU/ml) HIV- GFP infection knockdown evlution y WB CD25/CD69 stining FACS nlysis d d d5 d8 d d2 d d6 sirna (Con) SAMHD sirna- (si-).5% 5.% CellTrce (Fr Red).5% 5.% c sirna (Con) 7.% GFP SAMHD sirna- (si-) 8.7% CD25/CD69-PE.6% 7.% 8.7% 5.% d Percentge GFP cells GFP 6 8.5x 5 5.x e Con si % Con si-3 SAMHD MAPK Con si- SAMHD 5.% MAPK Con si-3 () Schemtic representtion of experimentl setup. (,c) Flow cytometric nlysis of HIV- GFP infection of post-ctivted resting CD T cells fter sirna-medited silencing of SAMHD. The ctivtion stte of CD T cells ws ssessed y () CellTrce (Fr Red) dilution or (c) y surfce expression of CD25 /CD69. (d) Quntifiction of the percentge of GFP cells nd the reltive increse of HIV- GFP infection in resting CD T cells from two donors fter silencing with SAMHD-specific sirnas (si- or si-3) reltive to non-trgeting control sirna (Con). Mens stndrd devition of infections crried out in triplictes re shown. (e) SAMHD levels in corresponding cell lystes were determined y immunolotting. MAPK: loding control. Nture Medicine doi:.38/nm.296

13 Supplementry Fig. 2 shrna-medited silencing of SAMHD y lentivirl trnsduction of post-ctivtion resting CD T cells relieves lock to HIV- infection CD T cell isoltion stimultion (PHA-P, 5 μg/ml) IL2 ( IU/ml) puromycin selection IL2 (3 IU/ml) Ficoll isoltion puromycin wshed out IL2 (2 IU/ml) shrna vector trnsduction IL2 (5 IU/ml) IL2 (2 IU/ml) IL2 ( IU/ml) IL2 (5 IU/ml) HIV- GFP infection knockdown evlution y FACS Cell Trce leling IL2 (2.5 IU/ml) CD25/CD69 stining FACS nlysis d d3 d5 d7 d8 d d2 d3 d d6 c d CellTrce (Fr Red) Percentge GFP cells Donor.5% 2.2% shrna SAMHD shrna GFP x SAMHD CellTrce (Fr Red) Donor 2 GFP Percentge GFP cells 2 6.9x.2% 2.% SAMHD shrna SAMHD shrna 2 shrna SAMHD shrna SAMHD-66 SAMHD-66 () Schemtic representtion of shrna-medited depletion of SAMHD in post-ctivtion CD T cells. () FACS dot plots for HIV- GFP infection of CD T cells from two donors fter lentivirl trnsduction with (upper pnels) non-trgeting control shrna or (lower pnels) SAMHD-specific shrna or 2-encoding vectors nd puromycin selection. Numers indicte the percentge of vile GFP cells mong the non-proliferting cells. (c) Quntifiction of HIV- GFP infection in SAMHD-silenced nd control shrnatrnsduced cells from the donors shown in. (d) Flow cytometric quntifiction of SAMHD levels in cells nlyzed on dy. Nture Medicine doi:.38/nm.296

TNF-α (pg/ml) IL-6 (ng/ml)

Xio, et l., Supplementry Figure 1 IL-6 (ng/ml) TNF-α (pg/ml) 16 12 8 4 1,4 1,2 1, 8 6 4 2 med Cl / Pm3CSK4 zymosn curdln Poly (I:C) LPS flgelin MALP-2 imiquimod R848 CpG TNF-α (pg/ml) IL-6 (ng/ml) 2 1.6