Identification of a tripartite interaction between the N terminus of HIV 1 Vif and CBFβ that is critical for Vif function

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1 DOI /s Retrovirology RESEARCH Open Access Identifiction of triprtite interction etween the N terminus of HIV 1 nd CBFβ tht is criticl for function Belete A. Desimmie 1, Jessic L. Smith 1, Hiroshi Mtsuo 2, Wei Shu Hu 3 nd Viny K. Pthk 1 Astrct Bckground: HIV-1 intercts with the cellulr core-inding fctor β (CBFβ) nd countercts the protective roles of certin humn APOBEC3 (A3) proteins y trgeting them for protesoml degrdtion. Previous studies hve identified some mino cids importnt for CBFβ interctions, nd recently co-crystl structure of pentmeric complex of HIV-1, CBFβ, Cul5, EloB, nd EloC ws resolved. However, comprehensive nlysis of CBFβ interctions tht re importnt for function hs not een performed. Results: Here, we crried out doule-lnine scnning mutgenesis of the first 6 mino cids of nd determined their effects on interction with CBFβ nd their ility to induce A3G degrdtion s well s rescue HIV-1 repliction in the presence of A3G. We found tht multiple residues re involved in the extensive N-terminl CBFβ interction nd tht the 5 WQVMIVW 11 region of is the mjor determinnt. A minimum of three lnine sustitutions re required to completely rogte the CBFβ interction nd s ility to rescue HIV-1 infectivity in the presence of A3G. Muttionl nlysis of CBFβ reveled tht F68 nd I55 residues re importnt nd prticipte in triprtite hydrophoic interction with W5 of to mintin stle nd functionl CBFβ complex. We lso determined tht CBFβ mino cids 73 WQGEQR 78, which re not resolved in the structure of the pentmeric complex, re not involved in interction with HIV-1. Conclusions: Our results provide detiled insight into the CBFβ interctions tht re criticl for function nd my contriute to the rtionl design of HIV-1 inhiitors tht lock -medited degrdtion of A3 proteins. Keywords: APOBEC3G, CBFβ, HIV-1, Protesoml degrdtion,, Restriction fctor Bckground HIV-1 nd other lentiviruses encode the ccessory protein tht countercts the ntivirl ctivities of APOBEC3 (A3) proteins nd is required for infection nd propgtion in primry CD4 + T cells nd in non-permissive T cell lines (for recent reviews see Refs. [1, 2]). neutrlizes the inhiitory ctivities of A3 proteins y trgeting them for polyuiquitintion nd protesoml degrdtion y hijcking n E3 uiquitin ligse complex [3]. Correspondence: viny.pthk@nih.gov 1 Virl Muttion Section, HIV Dynmics nd Repliction Progrm, Center for Cncer Reserch, Ntionl Cncer Institute, Frederick, MD 2172, USA Full list of uthor informtion is ville t the end of the rticle Jger et l. [4] nd Zhng et l. [5] recently reported tht in ddition to inding to cullin 5 (Cul5) nd elongin B/C (EloB/C), inds to the cellulr core-inding fctor β (CBFβ), nd the CBFβ interction is essentil for inducing efficient degrdtion of A3 proteins, which inhiit HIV-1 repliction in non-permissive cell types. CBFβ is n evolutionrily conserved non-dna inding component of the mmmlin runt-relted trnscription fctors (RUNX 1-3), which re criticl in hemtopoiesis, T cell differentition, nd skeletl development [6, 7]. CBFβ is n llosteric regultor of RUNX proteins nd hs previously een reported to stilize RUNX1 y preventing its uiquitin-medited degrdtion [8]. Recently, CBFβ ws proposed to ply criticl role in stilizing the intrinsiclly unstructured HIV-1 nd promoting formtion of The Author(s) 217. This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pulicdomin/zero/1./) pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Pge 2 of 13 well-ordered sustrte receptor y fcilitting locl folding of the N-terminl region [4, 5, 9 11]. Severl reports hve estlished tht HIV-1 intercts with CBFβ nd hve shown tht CBFβ depletion hmpers virus repliction in cells expressing A3 proteins primrily y interfering with s ility to induce degrdtion of the A3 proteins [4, 5, 9]. HIV-1 ssocites with CBFβ in humn cells nd recominnt CBFβ enhnces s soluility, stility, nd ssocition with n E3 uiquitin ligse complex [9, 1, 12 14]. Initilly, the interction interfce ws mpped to residues of CBFβ [9, 15]; CBFβ mino cid F68 ws then reported to e criticl for stle HIV-1 CBFβ interction [14]. Susequently, n X-ry crystl structure of five pentmeric complexes, ech contining CBFβ EloB/C- Cul5, ws determined [1]. In the pentmeric complex, HIV-1 is tightly ssocited with CBFβ nd intercts with oth EloC nd Cul5. Determining the iologicl relevnce of the criticl residues of tht govern the specificity of the CBFβ interction cn fcilitte our understnding of this importnt host virus interction. Additionlly, elucidting this interction t the moleculr level cn potentilly ssist in the design of inhiitors tht lock CBFβ inding nd therey interfere with function. Previous muttionl nlyses identified severl residues of tht were importnt for its interction with CBFβ [1, 16 18]. Of prticulr relevnce to this work, Friourgh et l. [16] reported tht deletion of the first 13 mino cids of olished the interction of recominnt with CBFβ, consistent with the crystl structure dt, which showed tht there re extensive interctions etween the N-terminl hydrophoic nti-prllel β-strnds of HIV-1 nd CBFβ [1]. To determine the functionl importnce of these extensive N-terminl HIV-1 CBFβ interctions, we nlyzed doule-lnine mutnts of the first 6 mino cids of HIV-1 nd determined the effects of the muttions on CBFβ inding y co-immunoprecipittion. We found tht 5 WQVMIVW 11 region of is importnt for the CBFβ interction, of which W5 is the most criticl determinnt. We lso found tht H27 H28 nd D37 W38 influence CBFβ inding eventhough they re not t the CBFβ interfce in the pentmeric complex, suggesting tht they indirectly ffect the interction with CBFβ. We lso oserved tht CBFβ mino cids 73 WQGEQR 78, which re in close proximity to residues E45 S52 of nd re not resolved in the pentmeric complex crystl structure, do not contriute to the CBFβ interction. Finlly, consistent with the crystl structure, our muttionl nlysis indicted tht CBFβ mino cids I55 nd F68 form triprtite interction with W5 of tht is criticl for function. Results Identifiction of HIV 1 determinnts tht re importnt for interction with CBFβ To identify HIV-1 determinnts tht re essentil for interction with CBFβ, we used previously descried pnel of single or doule-lnine sustitution mutnts of the first 6 mino cids of [19]. Our lnine-scnning mutgenesis screen focused primrily on identifying residues tht rogted the interction etween nd CBFβ s determined y co-immunoprecipittion (co-ip) ssys (Fig. 1). We co-trnsfected 293T cells with plsmids encoding nd untgged mutnts, performed co-ip ssys using nti-flg ntiody nd lystes of the co-trnsfected cells nd estimted the mounts of proteins tht co-immunoprecipitted with using quntittive western lotting nlysis (Fig. 1). The levels of the different mutnt proteins were comprle when they were expressed in the presence of (Fig. 1, ). The efficiency of CBFβ inding to wild-type () ws set to %, nd used to compre the inding efficiencies of the mutnts (Fig. 1, c). The verge inding efficiencies from five independent experiments re shown elow for ech mutnt. Notly, of the mutnts exmined, W5A Q6A, I9A V1A, H27A H28A, nd D37A W38A significntly reduced s ility to ind to CBFβ compred to (Fig. 1; vlues indicted in green). The H27A H28A nd D37A W38A mino cids re not t the CBFβ interfce. The solvent ccessile surfce re (SASA; determined y using getre progrm ville t curie.utm.edu/getre.html) for H28 is only 4.86 Å 2, nd W38 is not surfce exposed (SASA is. Å 2 ), suggesting tht these mino cids re uried in the protein, nd sustitution of these mino cids with lnines my hve disrupted the overll structure of the α-domin of. The loctions of these mino cids re shown in Fig. 1d. In ddition, muttions in the E45 P58 strnd helix loop structure of lso exhiited prtil defects in inding to CBFβ; mutnt E45A S46A, T47 N48A, S53A E54A, V55A H56A, nd I57A P58A showed significntly lower inding thn (Fig. 1c, d; vlues indicted in green). These results led to the identifiction of severl mino cids in the N-terminl region tht re criticlly importnt for inding to CBFβ. The pentmeric structure [1] indicted tht the mino cids from W5 W11 re in close proximity to CBFβ mino cids N63 F68 nd I55; ech of the mino cids is within ngstroms (Å) of the CBFβ mino cids, suggesting tht ny or ll of these CBFβ interctions could e criticl for function. The importnce of the W5 W11 mino cids of is supported y previous study [16] indicting tht deleting the first 13 mino cids of olishes the interction

3 Pge 3 of 13 Cell lyste 1 Region nlyzed 6 Zn domin BC ox A3F A3G Cul5 EloC E2A N3A-R4A Q12A-V13A D14A-R15A M16A-R17A I18A N19A-T2A W21A-K22A R23A-L24A V25A-K26A H27A-H28A M29A-Y3A I31A-S32A R33A-K34A A35A-K36A D37A-W38A F39A-Y4A R41A-H42A H43A-Y44A 192 E45A-S46A T47A-N48A P49A-K5A I51A-S52A S53A-E54A V55A-H56A I57A-P58A L59A-G6A c Flg IP Binding (%) d E2A N3A-R4A Q12A-V13A D14A-R15A M16A-R17A I18A N19A-T2A W21A-K22A R23A-L24A V25A-K26A H27A-H28A M29A-Y3A I31A-S32A R33A-K34A A35D-K36A D37A-W38A F39A-Y4A R41A-H42A H43A-Y44A E45A-S46A T47A-N48A P49A-K5A I51A-S52A S53A-E54A V55A-H56A I57A-P58A L59A-G6A 73 CBFβ 75GEQRQPT81 5WQVMIVW11 45ESTN48 53SEVHIP58 D37-W38 H27-H28 Fig. 1 Identifiction of determinnts tht re criticl for inding to CBFβ. A schemtic of structure nd its interction domins with cellulr proteins APOBEC3F (A3F), APOBEC3G (A3G), cullin 5 (Cul5) nd elongin C (EloC). Doule lnine mutnts of the first 6 mino cids of were nlyzed. Immunolots of cell lystes to detect,, nd tuulin (used s loding control). c Immunolots of nd mutnt s tht co-immunoprecipitted with. The verge inding efficiency of mutnts to CBFβ from five independent trnsfection experiments is shown elow ech lne reltive to (set to %). Sttisticl significnce ws determined using t test; P <.5. The mutnts with significntly reduced CBFβ inding re shown in green; mutnts with incresed CBFβ inding re shown in red. The upper nds of the IP lots of Q12A-V13A nd (the lst pnel) re most likely the light chins of the nti-flg ntiody used for immunoprecipittion. d A rion digrm of HIV-1 ; the highlighted residues 5 WQVMIVW 11, H27 H28, D37 W38, 45 ESTN 48, nd 53 SEVHIP 58 decrese CBFβ inding efficiency s shown in (c). 45 ESTNPKIS 52 residues re in loop nd eginning of β-strnd; these mino cids re ner loop of CBFβ tht ws not resolved in the structure ( 75 GEQRQPT 81, purple dshed line). is shown in red nd CBFβ is shown in cyn (PDB:4N9F)

4 Pge 4 of 13 etween nd CBFβ. Our results indicting tht the doule mutnts W5A Q6A nd I9A V1A disrupt the interction with CBFβ, ut the V7A M8A nd mutnts did not, indicted tht some of these mino cids re criticl for the interction while others re less importnt. Surprisingly, our results showed tht the H27A H28A nd D37A W38A mutnts severely hmpered the interction etween nd CBFβ in our inding ssy; these mino cids re Å wy from the nerest CBFβ mino cid (R151) in the pentmeric structure nd re not t the interction interfce (PDB:4N9F) [1]. Anlysis of the structure of [1] suggested tht W38, I57, I17, Y111, nd F112 re involved in n extensive hydrophoic interction tht mintins the structurl orgniztion of the α-domin of HIV-1 (Additionl file 1: Supplementry Fig. S1), implying tht they indirectly ffect the interction with CBFβ. Our oservtions tht the doule mutnts W5A Q6A nd I9A V1A disrupt the interction with CBFβ in cells provide functionl insights into the recently reported structurl informtion of the pentmeric complex [1], which showed tht mino cids 5 WQVMIVW 11 interct with CBFβ. We lso found tht mino cids E45 P58 of, which forms flexile loop [1], influenced the CBFβ interction. Amino cids E45 S52 re in position to potentilly form direct interctions with mino cids W73 R78 of CBFβ. However, this region of CBFβ ws not resolved in the pentmeric complex [1]; thus, potentil direct interction etween these mino cids of nd CBFβ could not e verified or excluded sed on the crystl structure. Finlly, we lso oserved tht N3A R4A, Q12A V13A, M16A R17A, N19A T2A, nd R23A L24A mutnts exhiited significntly higher efficiency of inding to CBFβ compred to (Fig. 1; vlues indicted in red); the potentil iologicl significnce of incresed inding to CBFβ y these mutnts ws not further exmined in these studies. Effects of CBFβ inding muttions on s ility to induce A3G degrdtion nd rescue HIV 1 infectivity Next, we exmined whether the mutnts tht exhiited reduced inding to CBFβ showed defect in their ility to induce A3G degrdtion nd inhiit HIV-1 repliction. We co-trnsfected 293T cells with HDV-GFP, VSV-G, nd (or mutnts) in comintion with different mounts of A3G expression plsmids nd exmined the ility of the mutnts to medite A3G degrdtion nd rescue infectivity of HIV-1 (Fig. 2). In ddition to the mutnts tht were defective in CBFβ inding, we included V7A M8A nd ecuse they cn potentilly contriute to the extensive hydrophoic interction etween nd CBFβ nti-prllel β-strnds. In ddition, we included doule mutnts P49A K5A c d Flg-A3G Flg-A3G Reltive A3G levels (% no ) Reltive infectivity (% ) W No Q6 No F68 I55 V7 Q67 No M8 L66 A56 H27A-H28A D37A-W38A H27A-H28A D37A-W38A V9 S65 V58 E45A-S46A T47A-N48A E45A-S46A T47A-N48A A3G,.34 µg A3G,.67 µg H27A-H28A D37A-W38A I1 L64 W11 E45A-S46A T47A-N48A N63 P49A-K5A G61 CBFβ I51A-S52A A3G,.34 µg A3G,.67 µg P49A-K5A I51A-S52A P49A-K5A I51A-S52A Fig. 2 Effect of selected doule-lnine muttions in on A3G degrdtion nd HIV-1 infectivity. Stick digrm of mino cids t the CBFβ interfce. 5 WQVMVIW 11 mino cids of nd mino cids of CBFβ, nd the interction etween W5 of with I55 nd F68 of CBFβ re shown. Representtive immunolots of Flg- A3G,, nd tuulin (loding control). Degrdtion of Flg-A3G ws determined y co-trnsfection of either.34 or.67 μg of Flg-A3G expression plsmid. c Quntittion of -induced A3G degrdtion reltive to the no control (which ws set to %) is shown. Br grphs represent men nd stndrd devitions of two or more independent trnsfection experiments nd sttisticl significnce ws determined using t test; P <.5. d Infectivity of viruses in the presence of different mutnts. Dt (men ± SDs from two or more independent experiments done in triplictes) re plotted reltive to viruses produced in the presence of (set to %). Sttisticl significnce ws determined using t test; P <.5

5 Pge 5 of 13 nd I51 S52A, ecuse of their proximity to other doule mutnts tht showed significnt defects in CBFβ inding. mutnts W5A Q6A, H27A H28A, D37A W38A, T47A N48A, P49A K5A, nd I51A S52A exhiited significnt defects in their ility to medite A3G degrdtion compred to (Fig. 2, c). Mutnts V7A M8A, I9A V1A,, nd E45A S46A induced A3G degrdtion s well s. We next compred the infectivity of viruses produced in the presence of these mutnts to (set to %); infectivity ws significntly reduced for ll of the mutnts except V7A M8A (Fig. 2d). Although the stedy-stte levels of A3G in the presence of nd E45A S46A mutnts were similr to the level of A3G in the presence of (Fig. 2, c), there ws two-fold reduction in the infectivity of viruses produced in the presence of these mutnts (Fig. 2d). These results indicte tht when A3G levels re low nd rely detectle on western lots, virus infectivity is more sensitive mesure of the levels of A3G remining thn western lotting. Tken together, these results strongly indicte tht residues W5 Q6, I9 V1, H27 H28, D37 W38, nd the loop region E45 S52 re criticl for s ility to ind to CBFβ, induce A3G degrdtion, nd rescue infectivity. Effect of reducing the level of CBFβ on the ility of mutnts to degrde A3G nd rescue HIV 1 infectivity mino cids 5 WQVMIVW 11 form β-strnd tht is t the CBFβ interfce [1]; however, our lnine-scnning mutgenesis suggested tht only W5A Q6A nd I9A V1A mutnts significntly reduced the interction with CBFβ. We hypothesized tht the other mino cids in the 5 WQVMIVW 11 motif contriute to CBFβ inding, ut ecuse there re high levels of CBFβ in 293T cells, modest reductions in CBFβ inding would not e oserved in our co-ip ssys. We therefore sought to ssess whether reducing CBFβ levels in cells y sirna knockdown would more redily revel minor interctions tht contriute to CBFβ inding. We first determined the effect of CBFβ depletion on A3G degrdtion y trnsfecting either CBFβ-specific sirna or control sirna (Fig. 3, ). The results showed tht the CBFβ-specific sirna-medited knockdown reduced CBFβ protein to % of the levels in the presence of control sirna. We lso determined the comined effect of CBFβ knockdown nd mutnts on infectivity of viruses produced in the presence of.34 μg or.67 μg of A3G plsmid (Fig. 3c). The results showed tht virus produced in cells with reduced levels of CBFβ nd resulted in further reduced infectivity compred to the infectivity in the presence of control sirna (18 nd 6% in the presence of.34 nd.67 μg of A3G, respectively). When CBFβ ws depleted nd the V7A M8A nd mutnts were expressed, we oserved more significnt defect in restoring virus infectivity (8 nd 4% of the in the presence of control sirna, respectively). Thus, in the presence of norml levels of CBFβ, the V7A M8A nd mutnts either showed no defect or modest twofold defect in rescuing virus infectivity compred to (Fig. 3c), ut in CBFβ-depleted cells, the sme mutnts showed significnt defects in rescuing virus infectivity (pproximtely threefold nd 4.5-fold, respectively). Tken together, these dt indicte tht the V7A M8A nd W11 residues do mke minor contriution to CBFβ inding, ut their contriution to CBFβ inding is dependent on the CBFβ protein expression level. Effect of qudruple nd triple lnine sustitution muttions 5 WQVM 8 > 4A nd 9 IVW 11 > 3A on interction with CBFβ nd A3G degrdtion The doule-lnine sustitution muttions in 5 WQVMIVW 11 inding motif of HIV-1 did not completely olish CBFβ inding, suggesting tht multiple residues re involved in the interction etween nd CBFβ [1]. Moreover, single-lnine sustitution mutgenesis in the 5 WQVMIVW 11 motif of reveled tht only the W5A mutnt significntly ffected s ctivity ginst A3G nd filed to rescue infectivity (Additionl file 2: Supplementry Fig. S2). Becuse single mino cid sustitutions 6 QVMIVW 11 did not hve significnt effect on CBFβ inding, we sought to determine whether comining multiple sustitutions in this region would influence interction with CBFβ. We constructed two dditionl mutnts in which four or three mino cids were sustituted with lnines to generte 5 WQVM 8 > A4 nd 9 IVW 11 > A3 mutnts. Consistent with our hypothesis, oth 5 WQVM 8 > A4 nd 9 IVW 11 > A3 mutnts lmost completely eliminted inding to CBFβ with inding efficiencies of 2 nd.3%, respectively (Fig. 4, ). Next, we determined the effects of the qudruple nd triple lnine sustitution muttions in on A3G degrdtion nd infectivity (Fig. 4c e). We found tht oth 5 WQVM 8 > A4 nd 9 IVW 11 > A3 mutnts filed to medite A3G degrdtion (Fig. 4c, d) nd rescue infectivity when viruses were produced in cells tht co-expressed A3G (Fig. 4e). These dt suggest tht the N-terminl interction etween HIV-1 nd CBFβ is stilized y hydrophoic interctions nd t lest three lnine sustitutions re required to olish the interction etween the nti-prllel β-strnds of nd CBFβ [1]. 73 WQGEQR 78 residues of CBFβ re not importnt for stle CBFβ interction After identifying the determinnts tht interct with CBFβ, we sought to determine the role of

6 Pge 6 of 13 Flg-A3G (µg) Flg-A3G CBFβ No CBFβ level (% ve. Ctrl sirna) A3G levels (%no + Ctrl sirna) A3G,.34 µg A3G,.67 µg No 5.4 c Ctrl sirna Ctrl sirna Fig. 3 Effect of comintion of doule-lnine mutnts in CBFβ-depleted cells on ctivity. Representtive western lots showing levels of A3G,, CBFβ, nd tuulin in cells co-trnsfected with Flg-A3G, vrints, nd CBFβ-specific sirna or control sirna. Lystes of virus producer cells were collected 48 h post-trnsfection nd nlyzed y western lotting. ws used s loding control for quntittion A3G levels. Quntittion of CBFβ knockdown efficiency from two independent experiments is depicted elow ech lne. Quntittion of -induced A3G degrdtion reltive to the no control (control sirna; set to %) from two independent experiments. c Infectivity of viruses produced in experiments shown in () is plotted reltive to virus produced in the presence of nd control sirna (set to %; not shown). Infectivity ws determined y mesuring luciferse ctivity in TZM-l cells. Sttisticl significnce ws determined using t test; P < Reltive infectivity (% + Ctrl sirna) A3G,.34 µg 18. A3G,.67 µg CBFβ determinnts tht re in close proximity to in the co-crystl structure nd my contriute to the CBFβ inding [1]. We first investigted whether 73 WQGEQRQTPS 82 residues in flexile strnd-loop region of CBFβ re importnt for interction with residues E45 S52 (Fig. 5). Unfortuntely, the CBFβ residues 75 GEQRQTP 81 re not resolved in the pentmeric crystl structure [1] (Fig. 5; dshed line). After nlysis of the pentmeric structure, we decided to investigte 73 WQGEQR 78 residues, which re potentilly in close proximity to the residues E45 S52. We generted CBFβ mutnts W73A Q74A, G75A E76A, nd Q77A R78A nd determined their ility to interct with (Fig. 5, c). To exmine the effects of these CBFβ doule muttions on inding, we first knocked down the endogenous CBFβ in 293T cells using n sirna tht inds to the 3 untrnslted region of the CBFβ mrna; the efficiency of knockdown ws >9% (Fig. 5). We then expressed wild-type or its mutnts nd determined the effects of the muttions on stedy-stte levels of Flg-A3G. We found tht none of these CBFβ doule mutnts hd mesurle effect on the interction etween CBFβ nd compred to wildtype CBFβ (Fig. 5, c), suggesting tht the 73 WQGEQR 78 residues re not required for the CBFβ interction. A triprtite interction etween I55 nd F68 of CBFβ nd W5 of is criticl for stle CBFβ inding The mjor interction etween CBFβ nd N-terminl residues involves two nti-prllel β-strnds. Bsed on the pentmeric crystl structure, the distnce etween W5 nd I55 is 3.8 Å, W5 nd F68 is 4.4 Å, nd I55 nd

7 Fig. 4 Effect of qudruple nd triple lnine sustitution mutnts of on CBFβ interction nd A3G degrdtion. Comprison of selected doule, qudruple ( 5 WQVM 8 > 4A), nd triple ( 9 IVW 11 > 3A) lnine sustitution mutnts of with respect to CBFβ inding, A3G degrdtion, nd ility to rescue virus infectivity. Representtive immunolots of cell lystes (upper three pnels) nd co-ip (lower pnel) proed for,, nd tuulin (loding control) re shown. Averge efficiency with which mutnts ind to CBFβ from two independent trnsfection experiments reltive to (set to %). Sttisticl significnce ws determined using t test; P <.5. c A representtive immunolot showing the effect of mutnts on A3G degrdtion. d Quntittion of reltive A3G levels compred to the no control (set to %). Error rs indicte stndrd devitions from four independent experiments. Sttisticl significnce ws determined using t test; P <.5. e Infectivity of viruses produced in 293T cells in the presence of Flg-A3G nd the indicted mutnts; men ± SDs re for virus stocks otined from two independent trnsfection experiments; virus infectivity of ech virus stock ws n verge of three infections. All comprisons were sed on viruses produced in the presence of (set to %). Sttisticl significnce ws determined using t test; P <.5 F68 is 4.4 Å. We hypothesized tht W5 of nd I55 nd F68 residues of CBFβ form triprtite interction tht is criticl for CBFβ inding. To confirm tht this interction is importnt in cells, we mutted the CBFβ residues I55 nd F68 nd mesured the impct tht these muttions hd in co-ip ssys in which the endogenous CBFβ ws depleted y sirna. Hultquist et l. previously showed tht the F68D mutnt of CBFβ differentilly rogted the interction etween CBFβ nd while preserving CBFβ s ility to ind to its cellulr interction prtner RUNX1 [14]. In ddition to the F68D mutnt, we generted CBFβ mutnts I55A, I55D, F68A, I55A F68D, nd I55D F68D nd exmined their ility to interct with (Fig. 6). To exmine the effects of the muttions in CBFβ on inding, we first knocked down the endogenous CBFβ in 293T cells using sirnas tht ind to the 3 untrnslted region of the CBFβ; the efficiency of knockdown ws >9%. We then expressed Flgtgged wild-type CBFβ or its mutnts nd determined the effect of the muttions on stedy-stte levels of Flg- A3G (Fig. 6, ). The results showed tht, compred to CBFβ-, the I55A nd I55D mutnts were less efficient t restoring virus infectivity in the presence of.34 nd.67 μg of A3G. The F68A sustitution did not influence CBFβ interction in this ssy nd could restore virus infectivity to the sme extent s CBFβ. In greement with previously reported results [14], the F68D mutnt ws unle to rescue virus infectivity, s were oth the I55A F68D nd I55D F68D doule mutnts (Fig. 6, ). These results indicted tht in ddition to F68, I55 ffects CBFβ interction. Finlly, we exmined the ility of the CBFβ mutnts to ind to HIV-1 (Fig. 7, ). Both I55A F68D nd Cell lyste Flg IP c d e Reltive A3G levels (%no ) mutnts IP (%) Flg-A3G Flg-A3G Reltive infectivity (% ) A3G,.34 µg A3G,.67 µg 5WQVM8>4A 9IVW11>3A No Pge 7 of 13 only 5WQVM8>4A 9IVW11>3A 5WQVM8>4A 9IVW11>3A 5WQVM8>4A 9IVW11>3A No 5WQVM8>4A 9IVW11>3A No

8 Pge 8 of 13 Fig. 5 Effect of muttions in the 73 WQGEQR 78 region of CBFβ on CBFβ interction. A rion digrm of HIV-1 nd CBFβ; residues 45 ESTNPKIS 52 re shown in ornge nd CBFβ residues 73 WQGEQR 78 re shown in purple. The dshed line indictes loop region of CBFβ tht ws not resolved in the structure. is shown in red nd CBFβ is shown in cyn (PDB: 4N9F). After trnsfection of 293T cells with CBFβ-specific or control sirna, the cells were cotrnsfected with CBFβ or doule-lnine sustitution mutnts of CBFβ nd. Immunolots of cell lystes (upper pnel) nd co-ip smples (lower pnel) proed for, endogenous CBFβ,, nd tuulin (loding control) re shown. c The r grphs represent the verge immunoprecipittion efficiency of the different CBFβ mutnts to reltive to CBFβ (set to %). None of the muttions hd mesurle effect on CBFβ levels or on CBFβ inding CBFβ 73WQGEQR78 I55D F68D CBFβ doule mutnts lmost completely filed to ind to, reducing inding efficiency to ~8% of tht oserved for CBFβ- in the co-ip ssy (Fig. 7). Individully, the I55D nd F68D mutnts significntly reduced inding to ~15 nd ~28% compred to tht of CBFβ-, respectively (Fig. 7), suggesting tht disruption of the triprtite interction lmost completely inhiits the CBFβ interction. As expected, sustituting I55 with the less ulky hydrophoic residue (A55) hd only modest effect on inding (Fig. 7, ). Tken together, these results show tht the triprtite interction etween I55 nd F68 of CBFβ nd W5 of is criticl for s ility to induce A3G degrdtion nd restore virus infectivity (Fig. 7c). Discussion The list of host proteins in the interctome is expnding nd mong these re EloB/C, CUL5, nd CBFβ, which re essentil for countercting the ntivirl ctivities of A3 proteins y trgeting them for protesoml degrdtion [4, 5, 1, 2 22]. Thus, the CBFβ interction is potentil trget for development of ntivirl gents tht interfere with s ility to induce degrdtion of A3 proteins. The recently reported pentmeric crystl structure of CBFβ CUL5 EloB/C hs provided vlule structurl informtion out this interction [1], nd hs indicted tht the interfce is stle primrily due to extensive hydrophoic interctions involving lrge surfce re (4797 Å 2 ). Although the extensive nture of this protein protein interction suggests tht smll molecules re unlikely to disrupt the CBFβ interction, it is possile tht specific determinnts of this interction re essentil for function, nd such functionlly relevnt determinnts could provide trgets for ntivirl drug development. In these studies, we sought to c Flg IP Cell lyste 45ESTNPKIS52 IP efficiency (% CBF-β) Endo. CBFβ W73A -Q74A G75A -E76A Q77A -R78A W73A -Q74A G75A -E76A Q77A -R78A Ctrl sirna Ctrl sirna

9 Pge 9 of 13 A3G A3G Endo. CBFβ I55A I55D - I55A I55D F68D F68D F68D F68A - - Ctrl sirna + + Reltive infectivity (% CBFβ + no A3G) A3G - I55A I55D F68A F68D A3G,.34 µg A3G,.67 µg Fig. 6 Identifiction of CBFβ mutnts tht re defective in inding nd restoring infectivity in the presence of A3G. Representtive western lots of 293T cells co-trnsfected with or control sirna, long with,, nd.34 or.67 μg of Flg-A3G. The lots show Flg-A3G,, endogenous CBFβ,, nd tuulin (loding control). Infectivity of viruses produced in experiments shown in () s determined y mesuring luciferse ctivity in TZM-l cells. The r grphs represent reltive infectivity compred to no A3G smples (set to %). All dt represent men ± SDs from two independent trnsfection experiments; virus infectivity for ech virus stock ws n verge of three infections. Sttisticl significnce ws determined using t test; P <.5 I55A F68D I55D F68D determine which CBFβ interctions re essentil for function y performing lnine scnning mutgenesis of the first 6 mino cids of. Our results show tht residues 5 WQVMIVW 11 ply criticl role in inding to CBFβ; specificlly, our results indicte tht W5 of forms criticl triprtite interction with I55 nd F68 of CBFβ. Sustitution of ny one of these three residues lmost completely olished the inding of to CBFβ, s determined in Co-IP ssys. Muttion of W5 of olished s ility to induce degrdtion of A3G, nd mutnts of CBFβ with sustitutions t the I55 nd F68 positions filed to rescue s function when the endogenous CBFβ ws depleted y sirna knockdown. Our results confirm previous study indicting tht F68D muttion of CBFβ rogted CBFβ interction without ffecting the formtion of the CBFβ-RUNX1 heterodimer [14], nd other studies [16, 18] which indicted tht mino cids 5 WQVMIVW 11 re importnt for CBFβ inding. Unlike the previous studies, which performed deletion nd sustitution muttion nlysis in the sence of structure, we performed rtionl, structure-sed systemtic nlysis to mp the mino cid determinnts tht re functionlly criticl for CBFβ interction. Importntly, our results indicte tht the W5 of the 5 WQVMIVW 11 is criticl for CBFβ inding nd prticiptes in triprtite interction with CBFβ residues I55 nd F68, while other mino cids mke minor contriutions to CBFβ inding. We lso showed tht residues of disordered loop of CBFβ (mino cids of loop 3), which re not modeled in the crystl structure due to poorly defined electron density, re dispensle for interctions with.

10 Pge 1 of 13 Flg IP Cell lyste c Endo. CBFβ I55A I55D F68A F68D I55A-F68D I55D-F68D Ctrl sirna IP efficiency (% CBFβ) I55A I55D F68A F68D I55A-F68D I55D-F68D 4.4 Å 3.8 Å 4.4 Å CBFβ Fig. 7 The triprtite interction etween I55 nd F68 of CBFβ nd W5 of is sufficient to stilize CBFβ interction. After tretment of 293T cells with CBFβ-specific sirna, the cells were co-trnsfected with or sustitution mutnts of nd. The cells were treted with MG132 (5 μm) for 16-h efore cell lystes were hrvested. Immunolots of cell lystes (upper three pnels) nd co-ip smples (lower two pnels) proed for, endogenous CBFβ,, nd tuulin (loding control) re shown. Averge immunoprecipittion efficiency of with CBFβ mutnts reltive to CBFβ (set to %). Sttisticl significnce ws determined using t test; P <.5. c The model depicts (red) nd CBFβ (cyn) dpted from PDB: 4N9F. The triprtite hydrophoic interction of I55 (mgent) nd F68 (mgent) of CBFβ nd W5 (lue) of re highlighted in lck frme nd close-up of the highlighted residues is shown with the pproximted distnce etween them in Å We found tht the other mino cids in the 5 WQVMIVW 11 motif did contriute to the interction with CBFβ, ut 3 or 4 lnine sustitution muttions were required to completely olish CBFβ inding. The 5 WQVMIVW 11 forms β strnd tht mkes β rrel-like interctions with the β2 nd β3 strnds of CBFβ. Since ckone hydrogen onds, nd not side-chin onds, re criticl for formtion of β rrels, it is possile tht multiple lnine sustitutions re required to lter the secondry structure of this motif, resulting in loss of interction with CBFβ. Our results indicted tht mino cids E45 S52 plyed role in CBFβ inding. The exct mechnism y which these residues contriute to the interction with CBFβ is not cler. We mutted CBFβ residues W73 R78, which re not resolved in the pentmeric structure [1] nd re prt of the previously reported CBFβ deletion mutnt (mino cids 69 9) tht rogted its inding to HIV-1 [5]. Our doule-lnine sustitution study showed tht these CBFβ residues did not significntly contriute to inding. Our results likely differ ecuse the previous study nlyzed lrger deletion compred to the region nlyzed in our study (mino cids 69 9 vs ). The deletion most likely ffected the overll structure of CBFβ, which resulted in loss of inding; the doule-lnine sustitution mutnts nlyzed in our study did not hve n effect on the structure of CBFβ nd did not ffect inding. Thus, residues

11 Pge 11 of 13 E45 S52 do not compose on fide CBFβ interction site; it is possile tht sustitution muttions of residues E45 S52 my hve indirectly ffected the structure of, resulting in weker interction etween W5 nd I55 or F68. Although it ws not discussed previously, we noted potentil electrosttic interction etween CBFβ E54 nd K5. Interestingly, CBFβ E54 is djcent to I55, which is involved in the triprtite interction; disruption of potentil electrosttic interction etween K5 nd CBFβ E54 my hve indirectly influenced the I55 residue position, leding to weker inding to. The results of these studies indicte tht CBFβ nd -APOBEC3 interction surfces do not overlp nd re mutully exclusive. Muttions in the 14 DRMR 17 nd 4 YRHHY 44 motifs, which were previously shown to e essentil for degrdtion of A3F nd A3G, respectively, did not hve ny effect on the CBFβ interction. We lso found tht severl mino cids, including H27, H28, W38, nd others re likely to e involved in hydrophoic interction tht is essentil for mintining the structure of the α-domin of nd re not directly involved in interction with CBFβ. These results re consistent with prior mutgenesis studies [17]. Results of prior mutgenesis studies [19, 23, 24] nd the current study on the effect of residues H27, H28, W38, T47, N48, P49, K5, I57, P58, nd F112 mutnts of on inding to CBFβ nd APOBEC3 proteins cn e interpreted nd grouped into those tht prticipte in n extensive hydrophoic interction nd those tht mintin the structure of the α-domin of. Previous studies found tht the W21A mutnt ws unle to ind to CBFβ [5, 6, 17, 18], while our studies indicte tht the W21A K22A doule mutnt does not hve ny pprent defect in CBFβ inding. Exmintion of the pentmeric crystl structure shows tht the W21 residue of is uried within the α-domin motif of nd does not interct with CBFβ; SASA for W21 is 6.49 Å 2. Thus, the W21A muttion most likely results in mjor structurl chnge in the α-domin of, resulting in loss of inding to CBFβ. Interestingly, our results suggest tht the lysine residue t the 22 position is required for the structurl chnges induced y the W21A muttion. The pentmeric structure of in complex with CBFβ suggests other potentil interctions etween nd CBFβ. residues W89 is close to CBFβ residue F143, nd Y94 is in close proximity to CBFβ I12, E135, D136, nd Q14. In ddition, C-terminl residues ner F115 re in close proximity to CBFβ residue F153 nd other residues etween 151 nd 156. Although these interctions likely stilize CBFβ inding, they re not sufficient to retin ctivity, since muttions t W5 of, or I55 nd/or F68 of CBFβ were sufficient to lmost completely inhiit s ility to ind to CBFβ nd induce A3G degrdtion. Although these interctions re not sufficient to support function, dditionl studies re needed to determine whether other muttions t these sites cn result in steric hindrnce nd interfere with CBFβ inding. Similrly, the functionl significnce of Cul5 nd EloC interctions descried in the pentmeric structure should e determined to estlish whether these interctions my e vlule trgets for development of smll molecule inhiitors. Conclusions Our results provide detiled insight into the criticl determinnts of the interction interfce etween the N-terminus of nd CBFβ nd identified triprtite interction tht plys mjor role in CBFβ inding. Together with the ville structurl dt on CBFβ E3 uiquitin ligse complex, these results further our understnding of the iology of HIV-1 CBFβ interction nd my id in the development of novel therpeutics tht trget the CBFβ interction nd inhiit -medited degrdtion of A3 proteins. Methods Plsmids nd cell lines Expression plsmids of Flg-A3G, HIV-1 nd its N-terminl doule-lnine sustitution mutnts, HIV-1 vector HDV-eGFP, nd VSV-G expression plsmid phcmv-g were previously descried [19, 25 31]. All mutnts were generted y site-directed mutgenesis using QuickChnge Lightening site-directed mutgenesis kit (Agilent Technologies) nd verified y sequencing. The expression plsmid ws otined from Addgene. HeL-derived reporter TZM-l nd 293T cell lines were mintined in DMEM (Corning Cellgro) supplemented with 1% fetl ovine serum (HyClone) nd 1% penicillin streptomycin (GIBCO). Co immunoprecipittion ssys nd western lotting Flg Co-IP ssys were crried out s previously descried [19, 25]. Briefly, 293T cells were seeded t cells per 1-cm dish nd trnsfected the next dy y the polyethylenimine PEI method [19, 32]. The following DNA mounts were used to trnsfect 293T cells: 4 μg, 4 μg, nd 1.8 μg pgreen Lntern-1 (pgl) (GIBCO; control for trnsfections) expression plsmids. To mintin equivlent DNA mounts, pcdna3.1 ws used when needed. After 48 h, totl cell lystes were hrvested in 1 ml of lysis uffer nd Co-IP ws performed s previously descried [19, 25]. To detect eluted complexes s well s the input cell lystes, western lotting ws performed. CBFβ ws detected using rit nti- Flg polyclonl ntiody (Sigm) t 1:2 dilution,

12 Pge 12 of 13 ws detected using mouse nti- monoclonl ntiody [Clone 319] (66643; Acm) t 1:2 dilution, nd tuulin s loding control ws detected using mouse nti-tuulin ntiody (Sigm) t 1:2, dilution. Rit nd mouse primry ntiodies were detected using 1:5 dilutions of n IRDye 8CW-leled got nti-rit secondry ntiody (Licor) or n IRDye 68-leled got nti-mouse secondry ntiody (Licor). Protein nds were visulized nd quntified using n Odyssey Infrred Imging System (Licor). A3G degrdtion ssys Humn 293T cells were seeded t cells per well in six-well pltes nd trnsfections were crried out using the PEI method. To ssy for -medited degrdtion of A3G, the following plsmids nd mounts were used:.34 nd.67 μg of pflg-a3g, 2.5 μg of, nd.2 μg pgl (used s positive control for trnsfection). To mintin equivlent DNA mount, pcdna3.1 ws dded s needed. After 48 h, cell lystes were hrvested nd immunolotting nlyses were performed to detect stedy-stte expression levels of A3G in the sence or presence of. Flg-A3G ws detected using rit nti-flg polyclonl ntiody (Sigm). HIV-1, endogenous CBFβ nd tuulin were proed s descried ove. Virus production To produce virus, 293T cells were seeded t cells per well in six-well pltes, nd trnsfected using the PEI method with the following plsmids nd mounts: 1 μg of phdv-egfp,.25 μg of phcmv-g,.34 μg or.67 μg of pflg-a3g, nd 2.5 μg of. To produce virus with or without CBFβ depletion, 293T cells were seeded t cells per well in six-well pltes dy efore trnsfection nd the Lipofectmine2 trnsfection method (Invitrogen) ws used with the following plsmids nd mounts: 1 μg of phdv-egfp,.25 μg of phcmv-g,.34 μg or.67 μg of pflg-a3g, nd 2.5 μg of in comintion with the specific sirna s descried elow. After 48 h, the virus-contining superntnts were filtered through.45-μm filter nd kept t 8 C until use. Virus infectivity TZM-l cells were seeded in 96-well pltes (4 1 3 cells per well) nd were infected the next dy in triplicte with viruses normlized for p24 s determined y p24 ELISA (XpressBio). Forty-eight hours lter, luciferse ctivity ws determined y ritelite plus kit (PerkinElmer) following the instructions of the mnufcturer using LUMIstr Glxy luminometer or 145 MicroBet JET (PerkinElmer). CBFβ knockdown For CBFβ knockdown experiments, 293T cells were seeded t cells per well in six-well pltes dy efore trnsfection. Smll interfering RNA (sirna)- medited trnsient knockdown of CBFβ ws performed using Silencer select trgeting coding region (Amion; S247) or sirna-b7 trgeting 3 -UTR region (Invitrogen; NM_ _stelth_865), which ws designed sed on n shrna descried previously [4] nd control sirna t 25 nm using Lipofectmine RNAiMx regent (Invitrogen) following the mnufcturer instructions. Twenty-four hours lter the cells were co-trnsfected with the respective sirna (25 nm) nd expression plsmids of Flg-A3G,, VSV-G, nd HDVeGFP vector using Lipofectmine2 regent in order to produce virus for infectivity (see ove for detils of the mounts of plsmids used) nd mintin efficient CBFβ knockdown. In some CBFβ overexpression rescue experiments, in ddition to the ove co-trnsfection cocktils of plsmids, we dded either wild-type or different mutnts (I55A, I55D, F68A, F68D, I55A-F68D, or I55D-F68D) of CBFβ expression plsmids nd crried out infectivity nd A3G degrdtion ssys s descried ove. After 48 h, producer cell lystes nd superntnts were collected for western lotting nd infectivity ssys, respectively. Additionl files Additionl file 1: Supplementry Fig. S1. W38 nd I57 re involved in mintining the structurl orgniztion of the α-domin of. A rion digrm of HIV-1 highlighting residues W38, I57, I17, Y111, nd F112 in lue. is shown in red nd CBFβ is shown in cyn (PDB: 4N9F). W38, I57, nd I17 re not surfce exposed (SASA. Å 2 ); Y111 nd F112 re only prtilly surfce exposed (SASAs of nd 6.5 Å 2, respectively). Additionl file 2: Supplementry Fig. S2. Effect of single-lnine sustitution in the 5 WQVMVIW 11 motif of on A3G degrdtion nd infectivity. (A) Representtive western lots showing levels of A3G,, nd tuulin in cells co-trnsfected with Flg-A3G nd vrints. Lystes of virus producer cells were collected 48 h post-trnsfection nd nlyzed y western lotting. ws used s loding control for quntittion A3G levels. (B) Quntittion of -induced A3G degrdtion reltive to the no control set to % from two independent experiments. (C) Infectivity of viruses produced in experiments shown in pnel A is plotted reltive to virus produced in the presence of set to %. Infectivity ws determined y mesuring luciferse ctivity in TZM-l cells. Sttisticl significnce ws determined using t test; P <.5. Authors contriutions BAD nd VKP designed experiments; BAD nd JLS performed experiments; BAD, JLS, HM, WSH, nd VKP discussed dt; nd BAD nd VKP wrote the mnuscript. All uthors red nd pproved the finl mnuscript. Author detils 1 Virl Muttion Section, HIV Dynmics nd Repliction Progrm, Center for Cncer Reserch, Ntionl Cncer Institute, Frederick, MD 2172, USA. 2 Bsic Reserch Lortory, Leidos Biomedicl Reserch, Inc., Frederick Ntionl Lortory, Frederick, MD, USA. 3 Virl Recomintion Section, HIV

13 Pge 13 of 13 Dynmics nd Repliction Progrm, Center for Cncer Reserch, Ntionl Cncer Institute, Frederick, MD, USA. Acknowledgements We sincerely thnk Stephen Hughes, Eric Freed, Krist Delviks-Frnkenerry, Ryn Burdick, nd Jonthn Rwson for criticl comments during mnuscript preprtion. Competing interests The uthors declre tht they hve no competing interests. Funding This reserch ws supported in prt y the Intrmurl Reserch Progrm of the NIH, Ntionl Cncer Institute, Center for Cncer Reserch. The content of this puliction does not necessrily reflect the views or policies of the U.S. Deprtment of Helth nd Humn Services, nor does mention of trde nmes, commercil products, or orgniztions imply endorsement y the U.S. Government. Received: 31 Jnury 217 Accepted: 8 Mrch 217 References 1. Desimmie BA, Delviks-Frnkenerrry KA, Burdick RC, Qi D, Izumi T, Pthk VK. Multiple APOBEC3 restriction fctors for HIV-1 nd one to rule them ll. J Mol Biol. 214;426(6): Stvrou S, Ross SR. 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