Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

27 Pages·2012·4.55 MB·English

Nangolaetal.Retrovirology2012,9:17
http://www.retrovirology.com/content/9/1/17
RESEARCH Open Access
Antiviral activity of recombinant ankyrin targeted
to the capsid domain of HIV-1 Gag polyprotein
Sawitree Nangola1,2,3, Agathe Urvoas3, Marie Valerio-Lepiniec3, Wannisa Khamaikawin1,2,
Supachai Sakkhachornphop1,2, Saw-See Hong4,5, Pierre Boulanger4,5*, Philippe Minard3* and
Chatchai Tayapiwatana1,2*
Abstract
Background: Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike
intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-
independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular
antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1.
Results: A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of
the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named
AnkGAG1D4 (16.5 kDa) was isolated. AnkGAG1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1
and a dissociation constant of K ~ 1μ M, and the AnkGAG1D4 binding site was mapped to the N-terminal domain
d
of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing AnkGAG1D4 in both N-
myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. AnkGAG1D4
expression also reduced the production of MLV, a phylogenetically distant retrovirus. The AnkGAG1D4-mediated
antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor
processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the
negative interference of AnkGAG1D4-CA with the Gag assembly and budding pathway.
Conclusions: The resistance of AnkGAG1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin
AnkGAG1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1
assembly based on ankyrin-repeat modules.
Keywords: HIV-1, HIV-1 assembly, Gag polyprotein, CA domain, virus assembly inhibitor, ankyrins, artificial ankyrin
library, intracellular antiviral agent
Background Several strategies for anti-HIV gene therapy are cur-
In recent years, significant progress has been made in rently under development, and certain ones have been
the control of HIV-1 infections using highly active anti- tested in hematopoietic cells [6-8]. They can be classi-
retroviral therapy (HAART). Nevertheless, the occur- fied into two major categories: (i) RNA-based agents
rence of multi-drug resistant mutants and the side including antisense, ribozymes, aptamers and RNA
effects of HAART justify the exploration of alternative interference [9]; (ii) protein-based agents including
therapeutic approaches, such as gene therapy [1-5]. dominant-negative mutant proteins, intrabodies, intra-
kines, fusion inhibitors and zinc-finger nucleases [10,11].
The most commonly transduced genes with antiviral
*Correspondence:[email protected];[email protected];
potential consist of those encoding derivatives of immu-
[email protected]
1DivisionofClinicalImmunology,DepartmentofMedicalTechnology, noglobulins. However, the complex structure of these
FacultyofAssociatedMedicalSciences,ChiangMaiUniversity,ChiangMai, molecules limits their antiviral function within cells,
Thailand50200
since their stability relies on disulfide bond(s) which
3InstitutdeBiochimieetdeBiophysiqueMoléculaireetCellulaire(IBBMC)
UMR-8619,UniversitédeParis-SudetCNRS,OrsayCedex91405,France
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Nangolaetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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rarely occur(s) in the reducing conditions of the intra- was mapped to the N-terminal domain of the CA, and
cellular milieu [12-16]. SupT1 cells that stably expressed AnkGAG1D4 showed a
Several methods and novel molecules have been devel- reduced permissiveness to HIV-1 infection. The
oped to overcome the limitations of antibodies and their AnkGAG1D4-mediated antiviral effect was found to
derivatives (e.g. scFv), in terms of stability, facility of occur at post-integration steps of the HIV-1 life cycle
modifications, robustness, and cost-efficient production involving the Gag protein assembly and budding
[13,17-19]. This is the case for molecules based on pro- machinery. This study demonstrated the potential of
tein frameworks or scaffolds which interact with poten- ankyrin-repeat proteins as a novel class of intracellular
tial therapeutic targets by mimicking the binding antivirals and suggested that AnkGAG1D4 could serve as
process of immunoglobulins to their specific antigens. a protein platform for the design of efficient intracellular
The ankyrin-repeat proteins represent an attractive scaf- inhibitors of HIV-1 assembly.
fold to generate this type of specific binders [20,21].
Analysis of the protein sequence-structure relationship Results
in natural ankyrins has defined consensus ankyrin motifs Design of artificial ankyrin-repeat motifs and construction
(or modules), and the results have been used to generate of an ankyrin library
large libraries of artificial proteins, called ‘Designed The construction of an artificial ankyrin library implies
Ankyrin-Repeat Proteins’ or DARPins. Several DARPins the randomization of amino acid residues localized on
with desired binding specificity to various target mole- the accessible surface of ankyrin which has a potential
cules have successfully been isolated from such libraries interaction with the desired target, while maintaining
[12,21-27], including competitors of HIV-1 binding to intact the conserved residues of the consensus repeat
the viral receptor CD4 [28]. modules which form the rigid framework of ankyrin.
Ankyrinsmediatemanyimportantprotein-proteininter- The consensus sequence of the ankyrin-repeat modules
actionsinvirtuallyallspeciesandarefoundinallcellular generated in this study was based on previous DARPins
compartments, indicating that these proteins can be libraries [12-14,23,29,30,32], with minor modifications,
adaptedtofunctioninavarietyofenvironments,intracel- as described in the Materials and Methods section. For
lular as well as extracellular [12,20,21,23,25,29,30]. For example, the lysine residue (K) at position 25 was sub-
example, lentiviral vectors pseudotyped withHER2/neu- stituted for glutamate (E) to prevent a possible repulsion
specificDARPinshavebeenfoundtoefficientlytransduce with the positively charged arginine (R) at position 21
their specific targets, HER2/neu-positive cells [31]. The (Tables 1 and Figure 1). Our ankyrin library was gener-
major advantages of ankyrin-repeat proteins reside in (i) ated by randomization of amino acids at positions 2, 3,
theirbindingspecificityandaffinity,asobservedinDAR- 5, 10, 13, and 14 (Figure 2 and 3). The amino acid side
Pins selected from large libraries; (ii) their solubility and chains at these positions were all oriented outwards and
stability,eveninthereducingconditionsoftheintracellu- belonged to the same surface-exposed surface of the
larmilieu;(iii)theirsequencefeaturespresentinDARPins, ankyrin-repeat module (Figure 3B).
whicharenaturallyexpressedinhumancells:asaconse- The artificial ankyrin-repeat proteins obtained were
quence,ankyrin-repeatproteinsareexpectednottobeas made of a variable number of ankyrin modules flanked
immunogenic as foreign proteins. Artificial ankyrins are by N- and C-capping sequences (N-cap and C-cap;
therefore promising candidates as protein interfering Figure 2B). The library was generated using the direc-
reagentscapableofactingbothextra-andintra-cellularly tional polymerization of one ankyrin microgene, each
[24]. microgene corresponding to one single repeat motif.
In the present study, we designed artificial ankyrin Polymerizationwasrealizeddirectlyintoaphagemidvec-
molecules targeted to the HIV-1 Gag polyprotein and tor[33].Thisresulted inproteins withvariablenumbers
evaluated their potential as intracellular therapeutic ofrepeatsandsequence lengths.Thelengthdistribution
agents which would negatively interfere with HIV-1 inthelibrarywasdeterminedby digestingthephagemid
replication, and more specifically with the virus particle pool with restriction enzymes whose sites were located
assembly machinery. To this aim, we constructed a on both sides of the ankyrin coding sequence, followed
library of ankyrin-repeat protein library expressed at the byanalysisoftheDNAfragmentsbygelelectrophoresis.
surface of recombinant filamentous bacteriophages. This Amaximumnumberof15ankyrinrepeatswasobtained,
phage-displayed library was screened on immobilized andthemostfrequentnumbersrangedfrom1to6.
matrix (MA)-capsid (CA) domain (MA-CA) of the HIV- Our final phage-displayed ankyrin library represented
1 Gag precursor (Pr55Gag, or more simply Gag), and a as many as 1.9× 108 independent clones. The quality of
panel of Gag-specific artificial ankyrins were isolated. our library was first evaluated by sequencing proteins
One particular Gag binder, AnkGAG1D4, was selected from randomly picked clones. Nine outof fifteen clones
for further characterization. AnkGAG1D4 binding site (60%)presenteddiscontinuoussequences,withsequence
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Table 1Randomization schemes used to introduce variability at specific positions ofankyrin repeats (a)
Repeatposition Degeneratedcodons Subsetofencodedaminoacid
2 VDK,DMY,RAA A,D,E,G,H,I,K,L,M,N,Q,R,S,T,V,Y
3 VDK,DMY,VAN A,D,E,G,H,I,K,L,M,N,Q,R,S,T,V,Y
5 VDK,DMY,VAN,TGG A,D,E,G,H,I,K,L,M,N,Q,R,S,T,V,W,Y
10 CTG,TGG,TAC,RTC I,L,V,W,Y
13 KCK,TAC,CGY,VAR A,E,F,I,K,L,M,Q,R,S,Y
14 KCK,VAR,AAC,SGY,YAY,NTG A,E,G,H,K,L,M,N,Q,R,S,V,Y
(a)Foreachpositionindicatedontheleftmostcolumn,thedegeneratedcodonsusedforthemicrogenesynthesisandthecorrespondingencodedamino-acids
areindicatedinthemiddleandrightmostcolumns,respectively.Standardletterformixedbasesreferto:N=A/T/G/C,V=A/C/G,D=A/G/T,K=G/T,M=A/C,Y
=C/T,R=A/G.
frameshifts and stop codons, which likely resulted from tag, as monitored by colony filtration blotting (COFI
errorsinoligonucleotidesynthesisand/orassembly.Dis- blot; data not shown). We therefore estimated the real
continuous sequences occurred with a higher frequency diversity of our library to one third of the total number
inankyrinswithnumerous modules,whilemostankyrin of independent clones, i.e. 6 × 107 independent ankyrin
moleculeswithfewerrepeatsshowedcorrect,openread- codingsequences.
ing frames. The proportionof clones inour library with
readthroughankyrin sequences was also evaluated from Production and purification of the viral protein target:
theproportionofcolonieswhichexpressedC-terminally HIV-1 Gag precursor H MA-CA
6
His-tagged soluble ankyrin protein: 34% (24 out of 72 The viral target used for screening our phage-displayed
clones)werefoundtobepositivefortheC-terminalHis- ankyrins consisted of the His-tagged recombinant
Repeat 1 5 10 15 20 25 30
position
A D X X G X T P L H L A A X X G H L E I E V L L L K X G A D V N A X
B D X X G X T P L H X A A X X G H L E I V R L L L E H G A D V N A R
C gac xxx xxx ggt xxx acc ccg ctg cac xxx gct gcg xxx xxx ggt cat ctg gaa atc gtt cgt ctc ctg ctg gaa cac ggc gca gac gta aac gcg cgt

D* vdk vdk vdk ctg kck kck
dmy dmy dmy tgg tac var

raa van van tac cgy aac
tgg rtc var sgy
yay
ntg
E ------------Va --------(cid:198)(cid:197)----Vb----(cid:198)(cid:197)----------- Vc ------------(cid:198)(cid:197)----------------------- C1 ---------------------(cid:198)(cid:197)-Va---
- C3-> (cid:197)-------- Vb rev -------(cid:198) (cid:197)--------------- C2 ------------(cid:198)(cid:197)---------------- C3 -----------------
Figure1Aminoacidandnucleotidessequencesoftheankyrin-repeatmicrogenes.(A),Amino-acidsequenceofthepreviouslydescribed
ankyrin-repeatmodule[23].(B),Amino-acidsequenceoftheankyrin-repeatusedinthepresentstudy.(C),Nucleotidesequenceoftheankyrin
microgenes.(D),Setofpartiallyrandomizedcodonsusedateachvariegatedpositionisindicated.(*),thefullsequenceofthesetof
oligonucleotideswillbecommunicateduponrequest.(E),Positionofthedifferenttypesofoligonucleotidesusedtosynthetizedtheankyrins
microgenesalongthenucleotidesequence.
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A
( ) n
Ank Repeat
Not I Bsm BI Bsp MI Hind III

T7p pLac/RBS DsbA ss St2 N-cap Rep cloning sites C-cap H6 p3
B
Ankyrin Repeat
23 25 30 33 1 5 10 15 20 22
L L E H G A D V N A R D X X G X T P L H X A A X X G H L E I V R L

(cid:5)(cid:4)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:4)(cid:2)(cid:2)(cid:1)(cid:3)(cid:2)(cid:3)(cid:1)(cid:4)(cid:4)(cid:3)(cid:1)(cid:4)(cid:3)(cid:2)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:4)(cid:5)(cid:2)(cid:1)(cid:2)(cid:2)(cid:3)(cid:1)(cid:4)(cid:3)(cid:4)(cid:1)(cid:3)(cid:4)(cid:5)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:7)(cid:7)(cid:7)(cid:1)(cid:1)(cid:1)(cid:7)(cid:7)(cid:7)(cid:1)(cid:1)(cid:4)(cid:4)(cid:5)(cid:1)(cid:1)(cid:7)(cid:7)(cid:7)(cid:1)(cid:1)(cid:2)(cid:3)(cid:3)(cid:1)(cid:3)(cid:3)(cid:4)(cid:1)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:1)(cid:3)(cid:2)(cid:3)(cid:1)(cid:1)(cid:7)(cid:7)(cid:7)(cid:1)(cid:1)(cid:4)(cid:3)(cid:5)(cid:1)(cid:1)(cid:4)(cid:3)(cid:4)(cid:1)(cid:1)(cid:7)(cid:7)(cid:7)(cid:1)(cid:1)(cid:7)(cid:7)(cid:7)(cid:1)(cid:1)(cid:1)(cid:4)(cid:4)(cid:5)(cid:1)(cid:1)(cid:3)(cid:2)(cid:5)(cid:1)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:1)(cid:4)(cid:2)(cid:2)(cid:1)(cid:1)(cid:2)(cid:5)(cid:3)(cid:1)(cid:1)(cid:4)(cid:6)(cid:1)(cid:1)(cid:3)(cid:4)(cid:5)(cid:1)(cid:1)(cid:3)(cid:5)(cid:3)(cid:1)(cid:1)(cid:3)(cid:1)
(cid:3)(cid:1)(cid:3)(cid:6)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:4)(cid:2)(cid:1)(cid:1)
N-cap Not I B sm BI
A A A D L G K K L L E A A R A G Q D D E V R L
(cid:11)(cid:9)(cid:1)(cid:1)(cid:4)(cid:3)(cid:4)(cid:1)(cid:1)(cid:4)(cid:3)(cid:3)(cid:1)(cid:1)(cid:1)(cid:4)(cid:3)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:4)(cid:5)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:2)(cid:2)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:2)(cid:2)(cid:2)(cid:1)(cid:1)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:3)(cid:5)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:3)(cid:5)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:4)(cid:5)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:3)(cid:5)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:4)(cid:5)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:2)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:6)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:2)(cid:3)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:2)(cid:4)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:1)(cid:1)(cid:1)
(cid:10)(cid:9)(cid:1)(cid:1)(cid:3)(cid:4)(cid:3)(cid:1)(cid:1)(cid:3)(cid:4)(cid:4)(cid:1)(cid:1)(cid:1)(cid:3)(cid:4)(cid:5)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:3)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:6)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:6)(cid:5)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:6)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:4)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:4)(cid:2)(cid:1)(cid:1)(cid:1)(cid:4)(cid:3)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:4)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:3)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:5)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:6)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:2)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:3)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:4)(cid:2)(cid:1)(cid:1)
Rep Cloning sites
Kpn I Bsp MI
(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:5)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:3)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:2)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:2)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:3)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:5)(cid:2)(cid:2)(cid:1)(cid:1)(cid:1)(cid:5)(cid:1)
(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:5)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:5)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:3)(cid:2)(cid:1)(cid:1)(cid:1)(cid:1)(cid:5)(cid:4)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:5)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:5)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:5)(cid:4)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:2)(cid:3)(cid:4)(cid:1)(cid:1)(cid:1)(cid:1)(cid:2)(cid:6)(cid:1)(cid:1)(cid:1)(cid:1)(cid:2)(cid:4)(cid:3)(cid:1)(cid:1)(cid:1)(cid:1)(cid:4)(cid:2)(cid:1)(cid:1)
C-cap
L L K H G A D V N A N D H F G K T A F D I S I D N G N E D L A E I L
(cid:5)(cid:4)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:2)(cid:2)(cid:4)(cid:1)(cid:3)(cid:2)(cid:3)(cid:1)(cid:4)(cid:4)(cid:5)(cid:1)(cid:4)(cid:3)(cid:5)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:4)(cid:6)(cid:1)(cid:2)(cid:2)(cid:3)(cid:1)(cid:4)(cid:3)(cid:5)(cid:1)(cid:2)(cid:2)(cid:5)(cid:1)(cid:1)(cid:4)(cid:2)(cid:5)(cid:1)(cid:3)(cid:2)(cid:3)(cid:1)(cid:1)(cid:6)(cid:3)(cid:1)(cid:1)(cid:4)(cid:4)(cid:5)(cid:1)(cid:1)(cid:2)(cid:2)(cid:4)(cid:1)(cid:1)(cid:2)(cid:3)(cid:3)(cid:1)(cid:4)(cid:3)(cid:5)(cid:1)(cid:6)(cid:3)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:2)(cid:5)(cid:3)(cid:1)(cid:1)(cid:5)(cid:3)(cid:3)(cid:1)(cid:1)(cid:2)(cid:5)(cid:3)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:2)(cid:2)(cid:3)(cid:1)(cid:1)(cid:4)(cid:4)(cid:5)(cid:1)(cid:1)(cid:2)(cid:2)(cid:3)(cid:1)(cid:1)(cid:4)(cid:2)(cid:4)(cid:1)(cid:1)(cid:4)(cid:2)(cid:3)(cid:1)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)(cid:1)(cid:4)(cid:3)(cid:5)(cid:1)(cid:1)(cid:4)(cid:2)(cid:2)(cid:1)(cid:1)(cid:2)(cid:5)(cid:3)(cid:1)(cid:1)(cid:3)(cid:5)(cid:4)(cid:1)
(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)(cid:3)(cid:1)(cid:1)(cid:6)(cid:3)(cid:1)(cid:8)(cid:1)(cid:1)(cid:1)(cid:1)
Figure2SchematicconstructionoftheankyrinrepeatlibraryusingthepHDiExacceptorvector.(A),Themono-ankyrinmicrogeneswere
polymerizedbyinsertion/ligationtopHDiExdoubledigestedbyBsmBIandKpnI.TheconstructionwassubsequentlydigestedbyBspMIand
recircularisedbyintramolecularligation.ThisresultedinthesubstitutionoftheregioncontainingtheRepcloningsitesbytheankyrinrepeats.
Thenumberofrepeatsdifferedfromoneclonetoanother,andusuallyrangedfrom2to6repeats.(B),DetailedsequencesofthedifferentDNA
regions.Abbreviations:T7p,T7promoter;pLac/RBS,lactosepromoterandRibosomebindingsite;DsbAss,DsbAsignalsequence;St2,Strep-Tag2
tag;H6,hexa-histidinetag.
polyprotein H MA-CA, corresponding to the MAp17 H MA-CA or BV-H CA, and the recombinant Gag pro-
6 6 6
and CAp24 domains of the HIV-1 Gag precursor. The teins purified by affinity chromatography on nickel-
rationale for screening our ankyrin library on the MA- sepharose column.
CA target was not only to search for MA- or/and CA-
binders, but also for ankyrin(s) which recognize(s) the Screening of Gag-binding ankyrins using the phage-
MAp17-CAp24 hinge region, which contains the clea- display method
vage site of the viral protease (PR). His-tagged recombi- The phage-displayed library of ankyrins was amplified
nant protein H CA, which corresponded to the mature using a conventional protocol [34,35], and Gag-binders
6
capsid protein CAp24, was used to identify the struc- were isolated by three rounds of selection/elution from
tural domains of the Gag precursor which contained the surface-immobilized H MA-CA protein. Elution of
6
ankyrin-binding site. Large amounts of recombinant H MA-CA-bound phages was performed using acidic
6
H MA-CA and H CA proteins were produced in Sf9 bufferforthefirsttworounds,followedbyspecificligand
6 6
cells infected with recombinant baculoviruses BV- elutionusingexcessof soluble H MA-CAproteinasthe
6
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A
1--- - - - ---10----------20----------30-33
DARPin : DXXGXTPLHLAAXXGHLEIVEVLLKZGADVNAX
Library: DXXGXTPLHXAAXXGHLEIVRLLLEHGADVNAR
B
C
Figure3Consensussequenceandthree-dimensionalmodelofankyrinmodule.(A),SequencecomparisonbetweentheconsensusDARPin
repeatmotifandtherepeatmotifofourankyrinlibrary.Theredlettersrefertothepositionsofrandomaminoacids,andbluelettersrepresent
theresidueswhichdifferfromtheconsensusDARPinsequence.ThepositionoftherecognitionsitefortherestrictionnucleaseBsmBIis
underlined.(B),Structuralmodelofonesingleankyrinrepeatmotif(ormodule).(C),Spatialarrangementofthreemodulesbelongingtothe
sameankyrinlinearsequence(triple-repeatmotifankyrinmolecule).Thefixedstructureoftherepeatmotifispresentedasayellowribbon.The
variableaminoacidsonthesolvent-exposedsurfaceareshownasstickpattern;theirrespectivenumberinthelinearsequenceisindicatedin
panelB.
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competititor in the third round [34,35]. Phage clones Identification of the ankyrin binding domain on HIV-1
were picked at random in each eluate, and tested by Gag precursor
ELISA for binding to H MA-CA. Only 20% of Gag-bin- ThestructuraldomainofPr55Gagrecognizedbyeachof
6
ders were foundin the first eluate, whereas a significant the three Gag-binders AnkGAG1B8, AnkGAG1D4 and
enrichmentwasobservedinthesecondandthirdeluates, AnkGAG6B4wasdeterminedbyFarWesternblotanalysis
with 70% of Gag-binders in both. Clones which gave a and ELISA. Lysates of H MA-CA-expressing Sf9 were
6
signal 5-fold over the background signal were picked in analyzed by SDS-PAGE, and proteins transferred to
alleluatesandsequenced.Allpositiveclonesshowedtwo PVDF membranes. Spontaneous cleavage of H MA-CA
6
or three ankyrin repeats flanked by N-cap and C-cap. by insect cell proteases resulted in the occurrence of
Three different clones, referred to as AnkGAG1B8, His-tagged N-terminal domain, H MA, migratingas the
6
AnkGAG1D4 and AnkGAG6B4 and containing three mature matrix protein of the virion, MAp17 (Figure 4B;
ankyrinmoduleseach,wereidentifiedseveraltimes;they control, rightmost lane). All three Gag-binders,
werethereforeselectedforfurtherstudies. AnkGAG1B8, AnkGAG1D4and AnkGAG6B4, reactedwith
To evaluate the specificity of our Gag-binders, an irre- H MA-CAonblot,butnotwithH MA(Figure4B).This
6 6
levant target protein, aRep-A3, was used in lieu of indicatedthattheankyrinbindingsitewasnotlocatedin
H MA-CA. Protein aRep-A3, previously described theMAdomain,butintheCAdomain.Asexpected,no
6
under the acronym aRep-n4-a in our previous study reaction was obtained with the control aRep-A3-binder
[33], is an artificial alpha-helicoidal repeat protein AnkA32D3 (Figure 4B). The reactivity towards the CA
(aRep) based on thermostable HEAT-like repeats, which domainwasconfirmedbyindirectELISA,usingrecombi-
folds cooperatively and shows a high stability [33]. Our nant H CA protein immobilized on nickel-coated wells.
6
phage-displayed ankyrin library was screened on immo- Positive signals with the CA protein were detected
bilized aRep-A3 protein, and aRep-A3-bound clones with all three Gag-binders, but not with AnkA32D3
were checked for binding specificity and sequenced. (Figure 4C). This indicated that the binding sites of
One ankyrin clone with a high affinity and specificity for AnkGAG1B8,AnkGAG1D4andAnkGAG6B4onH MA-CA
6
the aRep-A3 target, referred to as AnkA32D3, was used proteinwereallsituatedintheCAdomain.
as the irrelevant control of H MA-CA binders in the
6
rest of the present study. Biochemical characterization of Gag-binding ankyrins
As shown in Figure 4C, AnkGAG1B8 reacted with H CA
6
Gag-ankyrin interaction withthehighestapparentaffinity.However,DNAsequen-
Gag- and aRep-A3-bindingankyrinswere purified,che- cingshowedseveralnonconservativeaminoacidsubstitu-
mically biotinylated, and assayed for their capacity of tionswithinthe highly structured scaffolddomain ofthe
binding to their specific target in vitro. Importantly, no AnkGAG1B8 modules, as well as in AnkGAG6B4. Since
change wasdetectedintheinteractionofthe threeGag- thesemutationscouldadverselyaffecttheankyrin-repeat
binders AnkGAG1B8, AnkGAG1D4 and AnkGAG6B4, and motifs,AnkGAG1B8andAnkGAG6B4wereexcludedfrom
ofcontrolaRep-A3-binderAnkA32D3,withtheirrespec- our nextanalyses,andonlyAnkGAG1D4wasselectedfor
tive substrates, as determined by ELISA (data not further characterization. DNA sequencing revealed that
shown). This indicated that biotinylation did not alter AnkGAG1D4proteincomprisedofthreeankyrinmodules,
theirGag-oraRep-A3-specificbindingactivity. each containing different types of amino acids at the six
The degree of Gag-specificity of biotinylated assigned positions for variable residues (Figure 5A). The
AnkGAG1B8,AnkGAG1D4andAnkGAG6B4wasevaluated oligo-histidine tag allowedus to purify AnkGAG1D4pro-
inthepresenceofspecificornonspecificcompetitors,and teintohomogeneitybyusingatwo-stepchromatographic
tested in ELISA using H MA-CA-coated wells. Controls procedure,(i)affinitychromatography(Figure5B,lane3),
6
consisted of AnkA32D3 and aRep-A3-coated wells. and(ii)gelfiltration(Figure5B,lane2).SDS-PAGEanaly-
Competitors were (i) the same ankyrin protein in its sis showed that AnkGAG1D4 migrated with an apparent
non-biotinylatedformand(ii)non-biotinylatedaRep-A3 molecular mass of 16.5 kDa (Figure 5B), consistent with
protein. AnkGAG1B8, AnkGAG1D4, AnkGAG6B4 and thetheoretical mass 17.9kDaforaproteinof163amino
AnkA32D3 were all competed with their respective non- acidresidues.
biotinylatedversions,whilenosignificantcompetitionwas
observed between ankyrins AnkGAG1B8, AnkGAG1D4, Mapping of the AnkGAG1D4 binding site on the CA
AnkGAG6B4 on one hand, and aRep-A3 protein on the domain
otherhand(Figure4A).Interestingly,AnkGAG1D4showed A more refined mapping of the ankyrin binding site on
thehighestsignalofbindingtotheH MA-CAtarget,and the CA domain was performed using carboxyterminal
6
the highest competition effect was observed with non- deletion mutants of Gag expressed as recombinant pro-
biotinylatedAnkGAG1D4(Figure4A). teins in baculovirus-infected cells. Gagamb276 and
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A
No inhibitor Non-biotinylated Gag binder
2.5
Biotinylated (cid:1)Rep-A3 binder Non-biotinylated (cid:1)Rep-A3 binder
2.0
m
n
1.5
0
5
4
1.0
D
O
0.5
0
AnkGAG1B8 AnkGAG1D4 AnkGAG6B4 AnkA32D3
H MA-CA (cid:1)Rep-A3
6
B C
2.5
HCA
6
2.0
BG

m
n 1.5

0
5
< HMA-CA 4
6 D 1.0
O

0.5
< HMA 0
6
Figure4Gag-bindingactivityofartificialankyrins.(A),CompetitionELISA.SamplesofbiotinylatedGag-bindersAnkGAG1B8,AnkGAG1D4and
AnkGAG6B4,andofcontrolbiotinylatedaRep-A3-binderAnkA32D3weremixedwiththeircorrespondingnon-biotinylatedform(blackbars),or
mixedwithirrelevantsolubletarget(greybars),ormixedwithbuffercontainingnoinhibitor(whitebars).MixtureswereaddedtoHMA-CA-or
6
aRep-A3-coatedwells,asindicatedatthebottomofthepanel.Bound-ankyrinsweredetectedbyadditionofHRP-conjugatedextravidin,
followedbytheTMBsubstrate.(B),FarWesternblotting.LysatesofBV-HMA-CA-infectedSf9cellswereelectrophoresedinSDS-gel,proteins
6
transferredtoaPVDFmembrane,andmembranecutintostrips.Gag-bindingactivitywasdeterminedbyincubationofthestripswiththe
differentbiotinylatedankyrinsAnkGAG1B8,AnkGAG1D4,AnkGAG6B4,andAnkA32D3,asindicatedontopofthestrips.Ontherightmoststrip,the
respectivepositionsoftheGagproteinsHMA-CAandHMAweredeterminedusinganti-histidinetagantibody(arrowheads).(C),Indirect
6 6
ELISA.HCAwascapturedonnickel-coatedplate,andusedassubstrateforbindingassayofbiotinylatedankyrinsAnkGAG1B8,AnkGAG1D4,
6
AnkGAG6B4,andAnkA32D3.Bound-ankyrinswerequantitatedasin(A).BG,backgroundsignal.
Gagamb241 mutants carried an amber stop codon in 110, corresponding to positions 132-241 in the Pr55Gag
the Pr55Gag sequence at positions 276 and 241, respec- sequence of 500 amino acids.
tively [36]. Both recombinant Gag proteins had in com-
mon the MA domain, plus 110 residues of the CA Gag-binding parameters of AnkGAG1D4
domain for Gagamb241, and 145 residues of the CA ThespecificityandbindingparametersofAnkGAG1D4to
domain for Gagamb276 [36]. AnkGAG1D4 was found to itsH MA-CAsubstrateweredeterminedbymicrocalori-
6
bind to both C-truncated Gag proteins (Figure 6). This metry (ITC). Titration of increasing amounts of
restricted the AnkGAG1D4 binding site to the N-term- AnkGAG1D4proteinintosample cellcontaining purified
inal region of the CA domain spanning residues 1 to H MA-CA protein gave the approximate value of 1 μM
6
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A B

Lane : 1 2 3
20 kDa >
AnkGAG1D4
15 kDa >
Figure5CharacterizationofAnkGAG1D4.(A),AminoacidsequenceoftheAnkGAG1D4protein,representedbyusingthesinglelettercode.
Thevariableresiduesatthesixpredefinedpositionsontheankyrinsolvent-exposedsurfacewerehighlightedinred.(B),SDS-PAGEanalysisof
AnkGAG1D4.TheAnkGAG1D4proteinwasfirstisolatedbyaffinitychromatographyonnickel-column(HisTrapcolumn;lane3),andfurtherpurified
bygelfiltrationchromatography(SephadexG-75column;lane2).Lane1,molecularmassmarkers.
2.0
1.5

m
n

0
5 1.0
4

D
O

0.5
0
Figure6MappingoftheAnkGAG-1D4bindingsiteonHIV-1GagCAdomain.ThebindingactivityofbiotinylatedAnkGAG1D4proteinwas
testedinELISAtowardssurface-immobilizedlysatesofmock-infectedSf9cells,orbaculovirus-infectedSf9cellsexpressingrecombinantHMA-
6
CA,HCA,Gagamb241,orGagamb276protein.ThequantityofboundAnkGAG1D4wasassayedbyreactionwithstreptavidin-HRP.Datapresented
6
arefromtriplicateexperiments(m±SEM).
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forthedissociationconstant(K )ofthespecificreaction respectively,andfusedtoHis-taggedGFPattheN-termi-
d
of the binder with its target protein (Figure 7; leftmost nus(Figure8A)weretransfectedintotheSupT1cellline.
top panel). In control experiments, no interaction was Clones that stably expressed AnkGAG1D4-GFP protein
detected between AnkGAG1D4 and aRep-A3 (Figure 7; (SupT1/Myr+AnkGAG1D4-GFP and SupT1/Myr0Ank-
middle toppanel). Bycomparison,AnkA32D3interacted GAG1D4-GFP)wereidentified byfluorescentmicroscopy,
withitssubstrateaRep-A3withaK =18nM(Figure7; isolatedandexpandedunderthehygromycin-Bselection.
d
rightmosttoppanel). Two control SupT1cell lines harboring the pCEP4 plas-
The stoichiometry (N) of the interacting molecules in midsencodingtheMyr+andMyr0versionsofGag-irrele-
the protein complexes and the number of binding sites vant AnkA32D3-GFP (SupT1/Myr+AnkA32D3-GFP and
were calculated from the fitting curves of ITC data. The SupT1/Myr0AnkA32D3-GFP) were generated in parallel.
stoichiometry of protein monomers was found to be N ConfocalmicroscopyshowedthatMyr+AnkGAG1D4-GFP
= 0.91 for the pair AnkGAG1D4/H MA-CA, and N = andMyr+AnkA32D3-GFPlocalizedinboththecytoplasm
6
0.62 for the control pair AnkA32D3/aRep-A3 (Figure 7). and the plasma membrane, as expected for N-myristoy-
The aRep-A3 protein is known to occur as a homodi- lated proteins, whereas Myr0AnkGAG1D4-GFP and
mer [33], and the experimental value of 0.62 was close Myr0AnkA32D3-GFPshowedadiffusecytoplasmicfluor-
to the theoretical ratio of 0.5. The data therefore sug- escence.Flowcytometryanalysisshowedthatalmost80%
gested that one molecule of AnkA32D3 bound to an of ankyrin-expressing cells were GFP-positive, and that
aRep-A3 homodimer to form a ternary AnkA32D3/ Myr+AnkGAG1D4-GFPorMyr0AnkGAG1D4-GFPdidnot
(aRep-A3) complex. By contrast, AnkGAG1D4 bound to negatively interfere with the surface expression of CD4
2
the H MA-CA monomer in a 1-to-1 binary complex. (Figure 8B). The status of CD4 molecules, the primary
6
receptors of HIV-1, was important to assess in ankyrin-
Construction of SupT1 cell lines stably expressing Gag- expressing cells prior to HIV-1 infection, in order to
binding ankyrin proteins ensure that SupT1/Myr+AnkGAG1D4-GFP and SupT1/
Two pCEP4-based episomal plasmids encoding the Myr0AnkGAG1D4-GFP cells could serve as host cells for
AnkGAG1D4 protein in its Myr+ and Myr0 versions, testingthefunctionalityofAnkGAG1D4asantiviralagent.
(cid:2)(cid:17)(cid:15)(cid:5)(cid:2)(cid:5)(cid:31)(cid:4)(cid:34)(cid:1)(cid:39)(cid:1)(cid:6) (cid:7)(cid:2)(cid:25)(cid:3)(cid:2) (cid:2)(cid:17)(cid:15)(cid:5)(cid:2)(cid:5)(cid:31)(cid:4)(cid:34)(cid:1)(cid:39)(cid:1)(cid:1)(cid:9)(cid:12)(cid:19)(cid:25)(cid:2)(cid:33) (cid:2)(cid:17)(cid:15)(cid:2)(cid:33)(cid:32)(cid:4)(cid:33)(cid:1)(cid:39)(cid:1)(cid:1)(cid:9)(cid:12)(cid:19)(cid:25)(cid:2)(cid:33)
(cid:36)
K (cid:1)(cid:26)μ(cid:7)(cid:27)(cid:23)(cid:1)(cid:1)(cid:1)(cid:1)(cid:31)(cid:24)(cid:30)(cid:38)(cid:1)(cid:40)(cid:1)(cid:30)(cid:24)(cid:31)(cid:32) (cid:8)(cid:2) (cid:30)(cid:24)(cid:30)(cid:31)(cid:37)(cid:1)(cid:40)(cid:1)(cid:30)(cid:24)(cid:30)(cid:32)(cid:31)
d
(cid:10)(cid:21)(cid:18)(cid:14)(cid:11)(cid:13)(cid:14)(cid:18)(cid:16)(cid:12)(cid:21)(cid:20)(cid:22)(cid:1)(cid:26)(cid:8)(cid:27)(cid:23)(cid:1)(cid:1)(cid:1)(cid:30)(cid:24)(cid:38)(cid:31)(cid:1)(cid:40)(cid:1)(cid:30)(cid:24)(cid:30)(cid:31) (cid:8)(cid:2) (cid:30)(cid:24)(cid:36)(cid:32)(cid:35)(cid:1)(cid:40)(cid:1)(cid:30)(cid:24)(cid:30)(cid:31)(cid:34)
Figure 7 Gag-binding activityof AnkGAG1D4 analyzed bymicrocalorimetry (ITC):affinity andspecificity. The affinity of AnkGAG1D4
towardsitsGagsubstrateH6MA-CA(leftmostpanel)andtheaffinityofAnkA32D3towardsitsaRep-A3substrate(rightmostpanel)were
determinedbyITCmeasurementsofankyrin/substratemixturesatvariousproteinratios.TheGag-bindingspecificitywasevaluatedbyITC
analysisofthemixtureofAnkGAG1D4andirrelevantsubstrateaRep-A3atvariousproteinratios(middlepanel).Theparametersofeachbinding
reaction,K andstoichiometry(N),areshownunderthecorrespondingpanel.NA,calculationnotapplicable.
d
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A
(a) pCEP4-Myr+AnkGAG1D4-GFP
Myr
I GFP
MGSSKSK- Ankyrin - His6
(b) pCEP4-Myr0AnkGAG1D4-GFP
GFP
M- Ankyrin - His6
B

4
D
C
GFP
Figure8CharacterizationofSupT1cellsstablyexpressingartificialankyrins.(A),Ankyrinconstructs.Schematicrepresentationofthe
artificialankyrinconstructsdesignedforstableexpressioninSupT1cells,usingpCEP4-basedvector.Histidine-tagandgreenfluorescenceprotein
(GFP;greenbox)wereinsertedattheC-terminusandin-phasewiththeankyrinsequence(whitebox).AdditionofaN-myristoylationsignal(in
purpleredletters)totheMyr+AnkGAG1D4-GFPcloneresultedintheremovaloftheN-terminalmethionine(M)andcovalentlinkageofmyristic
acidchain(Myr;inblueletters)toglycine-2(G).(B),Flowcytometry.ExpressionofCD4moleculesatthesurfaceofcontrolSupT1cells,SupT1/
Myr+AnkGAG1D4-GFPandSupT1/Myr0AnkGAG1D4-GFPcells.Flowcytometryanalysiswasperformedonnonpermeabilizedcells,usingmonoclonal
antibodyagainstCD4,followedbyPE-conjugatedgoatanti-mouseIgG.