doi:0.08/nture078 RNse VifHA VifHA βctin 6 Cell lyste IP: ntiha MG VifHA VifHA β ctin 6 7 Cell lyste IP: ntiha Supplementry Figure. Effect of RNse nd MG tretment on the Vif interction., RNse tretment does not ffect the Vif interction. 9T cells were trnsfected with n expression vector for VifHA or control vector (VR0). Cell lystes were untreted or treted with RNse (for 0 min), then immunoprecipitted with ntiha ntiody conjugted to grose eds. Precipitted smples were seprted y SDSPAGE, trnsferred to nitrocellulose memrnes, nd rected with n ntivif ntiody to detect VifHA. Smples were lso nlyzed using nti ntiody., MG tretment does not ffect the Vif interction. 9T cells were trnsfected with n expression vector for VifHA or control vector (VR0). VifHA trnsfected cells were treted with DMSO or MG overnight, WWW.NATURE.COM/NATURE
then immunoprecipitted with the ntiha ntiodygrose ed conjugte. Precipitted smples were seprted y SDSPAGE, trnsferred to nitrocellulose memrnes, nd rected with ntivif ntiody. Smples were lso nlyzed with n nti ntiody. MG tretment incresed VifHA expression in trnsfected 9T cells (lne ) when compred to tht in DMSOtreted cells (lne ). Consequently, more VifHA ws immunoprecipitted from the MGtreted smple (lne 6) thn from the DMSOtreted smple (lne ). A smple treted with twofold dilution of MG ws lso exmined (lne 7). WWW.NATURE.COM/NATURE
YFPVif Acceptor CFP Donor c merge PreBlech Averge F FRET Efficiency % 0 Unleched (n = ) Bleched ((n = 0)) 0 0 0 YFPCFP dimer (positive control) PostBlech YFPVif CFP CFP β YFP CFP (negtive control) d ntigfp GAPDH Donor PreBlech e YFPVif CFP AGHA pcdnahvif YFPVif Donor PostBlech CFP AGHA pcdnahvif GAPDH 6 7 Supplementry Figure. Live cell imging nd Fluorescence Resonnce Energy Trnsfer (FRET) cceptor photoleching indicte colocliztion nd physicl interction etween YFPVif nd CFP. FRET cceptor photoleching exploits the ility for n cceptor fluorophore (e.g. YFP) to quench donor fluorophore (e.g. CFP) signl when in close proximity (~0 nm). When the cceptor fluorophore is leched, the signl from the donor is incresed if the two fluorophores re in close proximity. Thus, this technique cn e used s proxy for whether two proteins which re fused to two FRET pirs (e.g. YFP nd CFP) physiclly interct. 9T cells were cotrnsfected with YFPVif ( µg) nd CFP (0. µg) in 6well glssottom pltes. Cells were imged 6h posttrnsfection with Zeiss LSM0 Met confocl imging system., A representtive imge of YFPVif (yellow) nd W W W. N A T U R E. C O M / N A T U R E CFP (cyn) expressing cells with overly efore nd fter photoleching the
cotrnsfected with YFPVif ( µg) nd CFP (0. µg) in 6well glssottom pltes. Cells were imged 6h posttrnsfection with Zeiss LSM0 Met confocl imging system., A representtive imge of YFPVif (yellow) nd CFP (cyn) expressing cells with overly efore nd fter photoleching the cceptor fluorophore, YFP. The photoleched region is mrked (red open ox)., Zoomed view of pseudocolored intensity imge for the donor signl, CFP efore nd fter photoleching. CFP intensity incresed fter photoleching YFP, indicting dequenching of the donor signl. c, Averged FRET efficiency (%) compring positive control (YFPCFP dimer), experimentl smple (YFPVif CFP), nd negtive controls (YFP CFP nd CFP), leched versus unleched smples. After photoleching, the FRET efficiency for the experimentl smple ws pproximtely 9.% compred to the positive control (8.7%). Both negtive controls hd FRET efficiency of pproximtely %. As expected, the clculted FRET efficiency for unleched smples ws smll due to miniml chnge in donor signl intensity during unleched cquisition of the collected imges. Error rs represent the stndrd devition from the men (n = or 0, s indicted). d, Immunolot determines expression level nd confirms predicted moleculr weight for ech fluorescent protein. 9T cells were seprtely trnsfected with 0. μg of peyfp (~7 kd), pecfp (~7 kd), CFP (~8 kd), nd μg YFPVif (~9 kd). e, pcdnahvif nd YFPVifmedited AG degrdtion is enhnced y CFP. 9T cells were trnsiently trnsfected with pcdnahvif ( μg), YFPVif ( μg), CFP (0. μg), nd/or AGHA (0. μg). Plsmid concentrtion ws equlized in ech well with empty vector (pcdna.). Cells were hrvested 8h posttrnsfection. YFPVif induced the degrdtion of AGHA (lne 6) when compred to empty vector (lne ). However, YFPVif ws not s efficient s pcdnahvif t degrding AGHA (compre lnes nd 6). CFP enhnced oth pcdnahvif nd YFPVifmedited degrdtion of AGHA (lnes nd 7). WWW.NATURE.COM/NATURE
AGHA VifHA 6 Cell lyste IP: ntiha myc VifHA AGHA AGHA 6 7 8 Cell Lyste IP: ntimyc VifHA myc c 6 7 8 Cell lyste IP: ntiha sirna control sirna VifHA AGmyc MG AGmyc VifHA Supplementry Figure. is not required for the interction etween Vif nd AG., Coimmunoprecipittion of with VifHA, ut not AGHA. 9T cells were trnsfected with n expression vector for VifHA, AGHA, or control vector (VR0). Cell lystes were immunoprecipitted with ntiha ntiody conjugted to grose eds. Precipitted smples were seprted y SDSPAGE, trnsferred to nitrocellulose memrnes, nd rected with ntiha ntiody to detect VifHA or AGHAnd with nti ntiody to detect myc., Coimmunoprecipittion of VifHA, ut not AGHA, with myc. 9T cells were trnsfected with n expression vector for myc or control vector WWW.NATURE.COM/NATURE
(pcdna.) plus n expression vector for VifHA or AGHA. Cell lystes were immunoprecipitted with ntimyc ntiody ound to protein A eds. Precipitted smples were seprted y SDSPAGE, trnsferred to nitrocellulose memrnes, nd rected with ntiha ntiody to detect VifHA or AGHA nd ntimyc ntiody to detect myc. c, Silencing of does not ffect the interction etween Vif nd AG. 9T cells were trnsfected with n expression vector for VifHA or control vector (VR0) plus n expression vector for AGmyc in the presence of control sirna (lnes nd 6) or sirna ginst (lnes nd 78). Cell lystes were immunoprecipitted with ntiha ntiody conjugted to grose eds. Precipitted smples were seprted y SDSPAGE, trnsferred to nitrocellulose memrnes, nd rected with ntiha ntiody to detect VifHA or ntimyc ntiody to detect AGmyc. Smples were lso nlyzed using nti ntiody. 6 WWW.NATURE.COM/NATURE
6 Cell lyste 6 Virus sirna control sirna AFV NL Vif NL Pr Gg AFV Vif Riosoml p9 AFV CAp c e infectivity Reltiv reltive infectivity 0% 00% 80% 60% 0% 0% 0% sirna CBF sirna Control SiRNA SiRNA NLAF NL VifAF Supplementry Figure. Suppression of AF y HIV Vif requires., Vifmedited downregultion of AF requires. 9T cells were cotrnsfected with n expression vector for AFV plus NL, NLΔVif, or control vector (pcdna.) in the presence of control sirna (lnes ) or sirna ginst (lnes 6). The expression of AFV, Vif, nd ws ssessed y immunolot nlysis using ntiv, ntivif, nd nti ntiodies. Riosoml p9 ws used s protein loding control., is required for Vifmedited virion exclusion of AF. Superntnts from trnsfected 9T cells s descried in () were collected nd nlyzed for AFV virion pckging () nd virl infectivity (c). Purified viruses were evluted for AFV pckging y immunolot nlysis using ntiv nd ntip ntiodies. c, Virus infectivity ws ssessed y MAGI ssy. Virus infectivity with the WT HIV (Vifpositive) produced from trnsfected 9T cells in the presence of control sirna ws set to 00%. Error rs represent the stndrd devitions from triplicte wells. WWW.NATURE.COM/NATURE 7
Eorf6myc Riosoml p9 Cell lyste p Eorf 6myc HIVVifmyc sirna control SiRNA P Eorf6myc y 6 IP: ntimyc c Cellll llyste C t d As6HA SOCSHA HIVVifHA HIV Vif HA HIVVifHA y Cell lyste 6 IP: ntiha Cell lyste 6 IP: ntiha Supplementry Figure. Selective interction of HIV Vif, ut not other BCoxcontining proteins, with., Coimmunoprecipittion of with Vifmyc, ut not Eorf6myc. 9T cells were trnsfected with n expression vector for Vifmyc, Eorf6myc, or control vector (VR0). Cell lystes were immunoprecipitted with the ntimyc ntiody. Precipitted smples were seprted y SDSPAGE, trnsferred to nitrocellulose memrnes, nd rected with ntimyc ntiody to detect Vifmyc or Eorf6myc. Smples were lso nlyzed using n nti ntiody. Similr experiments were lso conducted using As6HA (c) nd SOCSHA (d)., Silencing does not ffect Eorf6medited degrdtion of p. 9T cells were trnsfected with n expression vector for p nd Eorf6myc or control vector (VR0) in the presence of control sirna (lnes ) or sirna ginst (lnes ). Cell lystes were nlyzed y immunolotting with ntimyc ntiody to detect Eorf6myc. Smples were lso nlyzed using n nti ntiody nd ntip ntiody. Riosoml p9 ws used s protein loding control. c, Coimmunoprecipittion of with VifHA ut not As6HA. d, Coimmunoprecipittion of with VifHA, ut not SOCSHA. 8 W W W. N A T U R E. C O M / N A T U R E
SIVmc Vif SIVgm Vif HA myc 6 6 Cell lyste IP:ntiHA Cell lyste IP:ntimyc c BIV Vif d shrna Control shrna Bovine AF BIV Vif Bovine AF HA BIV Vif 6 Cell lyste IP:ntiHA 6 βctin e BCox HCCH Cullinox inding HIV Vif SIVmc Vif SIVgm Vif BIV Vif Supplementry Figure 6. Selective interction of lentivirl Vif proteins with., Coimmunoprecipittion of with SIVmc VifHA. 9T cells were trnsfected with n expression vector for HIV VifHA, SIVmc VifHA, or control vector (VR0). Cell lystes were immunoprecipitted with the ntiha ntiody. Precipitted smples were seprted y SDSPAGE, trnsferred to nitrocellulose memrnes, nd rected with ntiha ntiody to detect HIV VifHA or SIVmc VifHA. Smples were lso nlyzed using n nti ntiody. Similr experiments were lso performed using Vif from SIVgm () nd BIV (c)., Coimmunoprecipittion of with SIVgm Vifmyc s well s HIV Vifmyc. Antimyc ntiody ws used to detect the presence of HIV Vifmyc nd SIVgm Vifmyc. c, Coimmunoprecipittion of with HIV VifHA ut not BIV VifHA. d, Silencing does not ffect BIV Vif medited ovine APOBECF degrdtion. e, A summry of the existing motifs nd inding of vrious lentivirl Vif proteins. WWW.NATURE.COM/NATURE 9
Vifmyc. c, Coimmunoprecipittion of with HIV VifHA ut not BIV VifHA. d, Silencing does not ffect BIV Vif medited ovine APOBECF degrdtion. e, A summry of the existing motifs nd inding of vrious lentivirl Vif proteins. RUNXmyc VifHA RUNXmyc VifHA RUNXmyc VifHA RUNXmyc VifHA βctin 6 Cell lyste IP: ntimyc Fo old Activtio on 0 0 0 Supplementry Figure 7. Effect of Vifmyc expression on the RUNX interction nd RUNX trnscriptionl ctivity., Coimmunoprecipittion of with RUNX in the presence or sence of Vif. 9T cells were trnsfected with n expression vector for HIV VifHA (lne ), RUNXmyc (lne ), or RUNXmyc plus HIV VifHA (lne ). Cell lystes were immunoprecipitted with n ntimyc 0 WWW.NATURE.COM/NATURE
ntiody. Precipitted smples (lnes 6) were seprted y SDSPAGE, trnsferred to nitrocellulose memrnes, nd rected with ntimyc ntiody to detect RUNXmyc. Smples were lso nlyzed using nti ntiody. To detect coprecipittion of VifHA with RUNXmyc, precipitted smples (lnes 6) were lso nlyzed y immunolotting using rit ntivif ntiody. Coprecipittion of VifHA with RUNXmyc (lne 6) ws detected. VifHA ws not precipitted in the sence of RUNXmyc (lne )., RUNXmycinduced trnscription of p(cbf) TKLuc (TkLuc) is not ffected y HIV Vif. 9T cells were trnsfected with 00ng of TkLuc, 00ng of TkLuc plus 00ng of RUNXmyc, or 00ng of TkLuc, plus 00ng of RUNXmyc nd 600ng of Vifmyc. Luciferse ctivities were determined fter dys. Luciferse ctivity ws determined using the Dul Luciferse Reporter Assy System (Promeg) ccording to the mnufcturer s protocol, nd luminescence signls were recorded using luminometer. Error rs represent the stndrd devition from the men (n = ). WWW.NATURE.COM/NATURE
HIV Vif WxDRMR7 Kx7YRHHY T7GERxW79 HIV Vif N SOCSBox HCCH zinc inding Motif AF Binding AG Binding AF Binding Virl BCBox CullinBox Cullin Box 9 99 9 c Vifmyc Cul Cul VifHA Vifmyc ElonginB ElonginB ElonginC ElonginC Cell lyste 6 6 7 8 IP:ntimyc Cell lyste IP:ntiHA d Vifmyc 6 7 8 9 0 IP ti IP:ntimyc Supplementry Figure 8. The minoterminl region of HIV Vif is required for the interction of Vif with., Digrm of the functionl domins of HIV Vif nd the Vif trunction mutnt construct., Fulllength HIV Vif, ut not the trunction mutnt HIV VifΔN, cn interct with. 9T cells were trnsfected with n expression vector for VifHA, VifΔNHA, or control vector (VR0). Cell lystes were immunoprecipitted with the ntiha ntiody, nd the precipitted smples were nlyzed y immunolotting with ntiha ntiody to detect VifHA or VifΔNHA. Smples were lso nlyzed using nti, nticul, ntielonginb, or ntielonginc ntiody. c, VifWA nd VifW8A mutnts hve reduced ility to interct with when compred to fulllength Vif. d, Vif mutnts tht re known to e defective for AG or AF interction mintin the ility to interct with. W W W. N A T U R E. C O M / N A T U R E
H9 βctin c NL EEGFP Vif NL EEGFP shrna Control shrna Pr Gg Dy fter infection 0 7 9 AG H9 Control shrna CAp Vif H9 shrna CAp βctin Supplementry Figure 9. Endogenous is criticl for HIV repliction in CD T cells (H9)., Silencing in H9 cells. plko. or plko. (Open Biosystems), together with prsvrev, pmdlg/prre, nd pcmv.vsvg, were cotrnsfected into 9T cells. The ssemled VLPs in the culture superntnts were used to infect H9 cells. Three dys lter, H9 cells were selected with µg/ml puromycin for dys. Cell lystes were nlyzed y immunolotting using n nti ntiody or ntiβctin ntiody., Silencing in H9 cells inhiits HIV repliction. H9 shrna control (plko.) or H9 shrna (plko.) cells were infected with n equl mount of Vif positive HIV (NL) virus. Virl repliction ws monitored y mesuring CAp in the superntnts of infected cells WWW.NATURE.COM/NATURE
y immunolotting with n ntip ntiody. c, Vifmedited downregultion of endogenous AG in H9 cells requires. 9T cells were cotrnsfected with pvsvg plus NLΔEEGFP or NLΔEEGFPΔVif. Two dys fter trnsfection, viruses from the superntnts of trnsfected 9T cells were used to infect H9 shrna control (plko.) or H9 shrna (plko.) cells. Three dys fter infection, H9 cells were ssessed y immunolot nlysis using ntiag, ntip, ntivif, ntiβctin, nd nti ntiodies. HIV infection ws monitored y the detection of intrcellulr PrGg using the ntip ntiody. Endogenous AG in H9shRNA control (plko.) cells ws reduced y the Vif positive NLΔEEGFP virus infection (lne ) compred to tht infected with NLΔEEGFPΔVif (lne ). Silencing endogenous in H9shRNA (plko.) cells resulted in the inhiition of Vif induced AG degrdtion (lne ). WWW.NATURE.COM/NATURE