Vol. 180, No. 2, 1991 October 31, 1991
81OCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages l O l O - l O l 8
FIRST LARGE SCALE CHEMICAL SYNTHESIS OF THE 7 2 AMINO ACID HIV-I NUCLEOCAPSID PROTEIN NCp7 IN AN ACTIVE FORM H. de R o c q u l g n y , D. F i c h e u x , C. Gabus*, M.-C. Fourni6-Zaluski, J.-L. Dariix* a n d B.P. R o q u e s ÷
D6partement de Chimie Organique, U266 INSERM-UA498 CNRS UFR des Sciences Pharmaceutiques et Biologiques 4, avenue de rObservatoire, 75270 Paris C6dex 06, France * Labo R6tro C J F INSERM, ENS 46, all6e d'Italie, 69364 Lyon Cedex 07, France Received September 12, 1991
The n u c l e o c a p s i d p r o t e i n (NC) of t h e h u m a n i m m u n o d e f l c i e n c y virus t y p e I p l a y s a c r u c i a l role in t h e f o r m a t i o n o f i n f e c t i o u s vlral p a r t i c l e s a n d t h e r e f o r e should be a major t a r g e t for t h e d e v e l o p m e n t of antiviral agents. T h i s r e q u i r e s an i n v e s t i g a t i o n o f NC p r o t e i n s t r u c t u r e a n d o f i t s i n t e r a c t i o n s w i t h b o t h p r i m e r tRNALys, 3 and g e n o m i c RNA. Nucleocapsid p r o t e i n NCp7, w h i c h r e s u l t s f r o m t h e m a t u r a t i o n of NCp15, c o n t a i n s two zinc fingers flanked b y s e q u e n c e s rich in basic a n d prollne residues. H e r e we r e p o r t t h e first s y n t h e s i s of large q u a n t i t i e s of NCp7 able t o a c t i v a t e HIV-1 RNA d i m e r i z a t i o n a n d r e p l i c a t i o n p r i m e r tRNALyS, z a n n e a l i n g t o t h e i n i t i a t i o n site o f r e v e r s e t r a n s c r i p t i o n . In a d d i t i o n UV s p e c t r o s c o p i c a n a l y s e s p e r f o r m e d t o c h a r a c t e r i z e t h e Co 2+ b i n d i n g p r o p e r t i e s of e a c h zinc f n g e r suggest t h a t t h e two fingers probably i n t e r a c t in NCp7. ~ ~991 Academic Press, Inc.
The GAG open reading frame of the h u m a n immunodeficiency virus type 1 (HIV-1) codes for the m a j o r core s t r u c t u r a l p r o t e i n s MApl7, CAp24 and N C p l 5 (1,2). Veronese et al. (3) have reported t h a t N C p l 5 is processed into two smaller proteins that are NCp7 and NCp6. Interactions of the NC protein with the viral RNA and replication primer tRNALys, 3 appear to be critical for the production of infectious viral particles (4). In fact, interactions of N C p l 5 with HIV-1 RNA promoted dimerization of the viral RNA, a process which a p p e a r s closely linked to genomic RNA encapsidation. In addition, interactions of N C p l 5 with b o t h primer tRNALys, 3 (5) and HIV-1 RNA resulted in the annealing of primer tRNA to the initiation site of reverse transcription (PBS). NC proteins also +To whom correspondence should be addressed.
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1010
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
participate in s p h e r i c a l capsid formation, p r o b a b l y t h r o u g h protein aggregation and tight interactions with the genomic RNA dimer (4-10). The NH2 t e r m i n u s of NCp15 c o r r e s p o n d i n g to NCp7 (72 amino acids) is a s s u m e d to be the functional domain in RNA recognition and b i n d i n g since it c o n t a i n s two s u c c e s s i v e zinc fingers of t h e type CX2CX4HX4C. At least one zinc finger of this type is found in all the highly conserved sequences of NC proteins of retroviruses (12). In fact genetic data have indicated that the zinc fingers play a crucial role in the specific encapsidation of genomic RNA (9). The zinc fingers bind Zn 2+ with high affinity (-1012 M -I) (13-15) and the binding of zinc was shown by NMR spectroscopy to induce folding of the peptide backbone {16-19). Although deletions or m u t a t i o n s of residues in the zinc fingers were shown to lower or abolish genomic RNA packaging (20), the precise role of the zinc fingers is still unclear (21), Moreover, the peptide sequences directly involved in the various functions of the NC protein are unknown, t h u s preventing a rational a p p r o a c h in the design of anti-viral agents aimed at inhibiting the NC protein functions. Hence structural analyses of the NC protein, either alone or in tight interactions with the viral RNA or primer tRNA, should be of great interest and well facilitated by the use of NCp7 provided that this small NC protein has all the biological activities of NCp15. Here we report the first synthesis of HIV-1 NCp7 protein in large quantities. The proximal (13-30) a n d the distal (34-51) zinc fingers of NCp7 (Fig. I) were also synthesized for s t r u c t u r a l and biological studies, Only NCp7 is shown to possess a high biological activity in vitro. Moreover, preliminary UV spectroscopic analyses of the Co 2+ binding properties of NCp7 and the zinc fingers suggest that the two fingers probably interact in the NCp7 protein. MATERIALS AND METHODS
Peptide synthesis. Fmoc p r o t e c t e d a m i n o acids ,HMP resin, p i p e r i d i n e , N - m e t h y l p y r r o l i d o n e (NMP), d i c h l o r o m e t h a n e (DCM), dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) were p u r c h a s e d from Applied Biosystems. Trifluoroacetic acid (TFA) was from Neosystem Laboratory (Strasbourg, France) and 1,2 ethanedithiol, phenol and CoCl 2 from Aldrich Chemie (Paris). Assembly of the protected peptide c h a i n s was carried out u s i n g the stepwise solid p h a s e m e t h o d of Merrifield (22) on a n Applied Biosystems 431A peptide synthetizer, with H O B t / D C C as coupling reagents. Fmoc amino acids were u s e d with the following side chains protections : t-butyl ether (Ser, Thr, Tyr), t-butyl ester (Glu, Asp), trityl (Cys, His, Asn, Gin), pmc (Arg) and boc (Lys). D e p r o t e c t i o n of the Fmoc group was o b t a i n e d b y two s u c c e s s i v e treatments of 3 min and 15 min with 20% piperidine in NMP. At the end of the synthesis, the peptide resin was treated for two h o u r s with 50 ml of TFA in the presence of phenol (3.75g), EDT (1.25ml), thioanisole (2.25 1011
Vol. 18o, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NCp7
6 Kr6N.._
GKE6HQ
NC~
z~, IA R
~C ~
"c ~s-M~RGNFRN~RKNV K 1
z~,.-" M K
c
c,
RAPRKKG
RR~ANFLGKIWP8YKGRP GNFL-C00fl 72 GKE611Q C -- M K ~ -: K
w c~
~=-x G 34
13-30 NCp7 cGKE611-T N ~Za
~-v x 13 Figure
1,
="
o %c~r
34-51
NCp7
~-cooa 51
AR
%-cooe 3O
Primary sequence of NCp7, (13-30)NCp7 and (34-51)NCp7.
ml), a n d H 2 0 (2.25 ml) to remove p r o t e c t i n g g r o u p s a n d to cleave the peptide from t h e resin. The entire r e a c t i o n m i x t u r e w a s applied to a C4 Vydac 5 g m (220x10 ram) a n d eluted w i t h a linear g r a d i e n t of 10-90% B in 30 m i n (A : H 2 0 , 0,1% TFA, B : CH3CN, H 2 0 , 70%, 30%, 0,09% TFA) at a flow r a t e of 3 m l / m i n with d e t e c t i o n at 214 n m u s i n g a n Applied B i o s y s t e m s 151A S e p a r a t i o n System. All the purifications were carried out u n d e r Argon (fig.2).
Biological activity of NCp7 and related zinc finger peptides. To s t u d y t h e biological activity of t h e s y n t h e t i c p e p t i d e s we u s e d t h e biological s u b s t r a t e s HIV-1 RNA a n d p r i m e r 32P-tRNALy s,3. HIV-1 RNA w a s s y n t h e s i z e d in vitro u s i n g T7 RNA p o l y m e r a s e a n d c o n t a i n s R, U5, PBS, the d i m e r i s a t i o n e n c a p s i d a t i o n d o m a i n a n d AUG of gag (positions 1 to 415) (4,6). HIV-1 RNA (1-415) a n d 32P-tRNALy s,3 were i n c u b a t e d in t h e a b s e n c e or p r e s e n c e of i n c r e a s i n g c o n c e n t r a t i o n s of NCp7 a n d r e l a t e d zinc fingers. After 30 m i n u t e s at 37°C the RNAs were p h e n o l e x t r a c t e d a n d a n a l y s e d by a g a r o s e gel e l e c t r o p h o r e s i s u n d e r native conditions. As reported in figure 3A l a n e s 1-3, NCp7 can strongly activate dimerization of HIV- 1 RNA (1-415). L a n e s 4-7 (fig. 3B) s h o w t h a t a n n e a l i n g of replication p r i m e r tRNALys,3 to the PBS was also activated b y NCp7. U n d e r the s a m e e x p e r i m e n t a l c o n d i t i o n s b o t h zinc finger p e p t i d e s were inactive (data n o t shown). Analysis of Co2+ binding properties of NCp7 and its zinc fingers by UV-visible spectroscopy. Visible absorption s p e c t r a were carried o u t on a Pye U n i c a m SP8-100 ultraviolet s p e c t r o m e t e r . A b o u t 0.5 mM of peptide u n d e r r e d u c e d form w a s dissolved in 50 mM Hepes, 100 mM KC1 buffer, pH 7.5, c o n t a i n i n g 1.5 eq. of COC12. In c o m p e t i t i o n e x p e r i m e n t s , a s o l u t i o n of EGTA (10 eq.) in Tris 1M w a s r a p i d l y a d d e d to t h e peptide solution in Hepes buffer. E x p e r i m e n t a l dissociation curves were t r e a t e d b y t h e c o m p u t e r p r o g r a m m LIGAND (Elsevier Biosoft). B i p h a s i c d i s s o c i a t i o n c u r v e s gave a good fit b e t w e e n e x p e r i m e n t a l a n d c a l c u l a t e d d a t a . 1012
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
F o r m a t i o n of t h e c o m p l e x b e t w e e n t h e s y n t h e s i z e d p e p t i d e s in H e p e s buffer pH 7.5 solutions a n d Co 2+ ions w a s evidenced by the a p p e a r a n c e of a blue color a n d of large absorption b a n d s with m a x i m a (~, expressed in Mlcm-1) a r o u n d 695 (a = 473), 642(c = 550), 615 n m (e = 348) c o r r e s p o n d i n g to the d-d electronic t r a n s i t i o n s of t h e m e t a l (fig.4) a n d in t h e 350 (a = 1374), 310 n m (a = 2560} r a n g e c o r r e s p o n d i n g to S-Co 2+ c h a r g e t r a n s f e r b a n d s . E x p e r i m e n t s p e r f o r m e d w i t h c o n c e n t r a t i o n s of Co 2+ g r e a t e r t h a n a 1:1 m o l a r ratio s h o w e d no f u r t h e r i n c r e a s e in absorption. Addition of ZnC12 to the Co 2+ complex abolishes the Co 2+ d-d t r a n s i t i o n b a n d at 600-700 nm. A t t e m p t s to m e a s u r e t h e E G T A - i n d u c e d d i s s o c i a t i o n c o n s t a n t s of t h e Co 2+ ions from the zinc finger(s) in p s e u d o first o r d e r c o n d i t i o n s (excess of EGTA>50 eq.) w a s h i n d e r e d by t h e complete d i s a p p e a r a n c e of t h e a b s o r p t i o n b a n d at 642 n m w h i c h o c c u r r e d too r a p i d l y to be m o n i t o r e d w i t h a c l a s s i c a l UV s p e c t r o m e t e r . T h e r e f o r e a q u a l i t a t i v e e v a l u a t i o n of t h e s t a b i l i t y of t h e Co 2+ c o m p l e x e s w a s a c h i e v e d b y competition s t u d i e s with 10 eq. of EGTA, l e a d i n g to a m e a s u r a b l e biphasic t i m e - d e p e n d e n t r e d u c t i o n of the a b s o r p t i o n at 642 n m with the following a p p a r e n t d i s s o c i a t i o n r a t e s (fig.5) : 1.04x10 -1 a n d 1.17x10 -3 sec -1 for NCp7, 3 . 0 9 x 1 0 -2 a n d 5.25x10 -4 sec -1 for (13-301NCp7, 4 . 4 0 x 1 0 -2 a n d 2 . 4 1 x 1 0 -3 sec -1 for (34-51)NCp7.
RESULTS AND DISCUSSION
Until now, the s t u d i e s devoted to N C p l 5 or its f r a g m e n t s have b e e n done with p r o t e i n s purified from virions or infected cells o b t a i n e d from o v e r p r o d u c t i o n of a r e c o m b i n a n t NCp15 e x p r e s s e d in E.coli. This s t u d y r e p o r t s t h e first s y n t h e s i s of NCp7 u n d e r c o n d i t i o n s w h i c h allow t h i s m i n i p r o t e i n to be o b t a i n e d in large q u a n t i t e s a n d w i t h a h i g h degree of p u r i t y . The c r u c i a l p a r a m e t e r s for s u c c e s s f u l s y n t h e s i s of NCp7 were d e t e r m i n e d by modifying the s t a n d a r d p r o g r a m of the synthetizer. T h u s , all p e p t i d e s were p r e p a r e d u s i n g F m o c c h e m i s t r y on HMP r e s i n (110 mg, 0,9 m m o l / g ) b u t c o u p l i n g t i m e s w e r e i n c r e a s e d progressively from 20 to 60 m i n as a f u n c t i o n of t h e growing size of t h e peptide. In the case of NCp7, double coupling (1 m m o l p e r coupling) was s y s t e m a t i c a l l y achieved from the first to the 22 n d a m i n o acid. Then, after the 22 n d , 44 t h a n d 60 t h c o u p l i n g steps, h a l f of t h e r e s i n w a s r e m o v e d b o t h to r e d u c e t h e p e p t i d e resin v o l u m e a n d to i n c r e a s e t h e a m o u n t of Fmoc a m i n o acid a d d e d to the resin from 10 to 80 eq. After TFA t r e a t m e n t , t h e c r u d e p r o d u c t w a s p u r i f i e d b y r e v e r s e p h a s e HPLC (fig.2) yielding to 124.6, 117.7 a n d 173 m g of p u r e NCp7, (13-30)NCp7 a n d (34-51)NCp7, respectively. The p e p t i d e s were j u d g e d to be >99% p u r e (yield a r o u n d 60%). Amino acid a n a l y s i s a n d s e q u e n c i n g s h o w e d a good a g r e e m e n t b e t w e e n p r e d i c t e d a n d e x p e r i m e n t a l c o m p o s i t i o n of t h e p e p t i d e s (table 1). 1013
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NCp7 8.94 min.
13-30 NCD 7 '89 min.
34-51 ~c~7 8.27 rain. 10
lb g I~tu'e 2.
~b
HPLC of purified NCp7 and related fragments. C o l u m n C4 Vydac 5 ~m (220 x 10 mm}. Gradient 10%-90% B. Solvent A 0. I% TFA in H 2 0 . Solvent B 0.09% TFA in C H 3 C N / H 2 0 {70/30,v/v), Flow rate 3 m l / m i n .
To p r e s e r v e t h e h i g h l y o x i d i z a b l e c y s t e i n e s u n d e r r e d u c e d f o r m s , all manipulations
were carried
out under
argon,
In these
conditions,
the
t i t r a t i o n of f r e e t h i o l s b y 5,5' d i t h i o b i s ( 2 - n i t r o b e n z o i e aeid) (24) g a v e 5 . 8 5
TABLE 1 . Amino acid c o n t e n t (uncorrected values) of purified peptides
AMINO ACID
NCp7
Asx Thr Ser Glx Pro Gly Ala Val Leu Tyr Lys His Arg Cys Phe Met Trp Ile
8.24 2.00 0.91 6.77 2.90 8.80 2.87 1.00 2.20 0.96 9.30 1.96 7.43 5.85 3.90 1.50 2.00 1.00
(13-30)NCp7
(34-5 I ) N C p 7
1.98 {2) 1.20 (1}
1.00 {1) 1.30 (1}
0,97 (i}
2,78 {3)
1.78 (2) 1.71 {2) 1.00 (I}
2.73 (3}
1.84 0.99 1.99 2.90 0.95
3.42 (4) 0.95 (i)
{8) {2) (I) (7) (3) (9) (3) (I} (2} (I) (I0} (2} (8) (6} (4) {2) (2} (1)
(2} (i) (2) (3) (I)
2.90 (3) 0.50 (I} 1.00 (I}
The theoretical amino acid c o m p o s i t i o n of each peptide is given in parentheses.
1014
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NCp7 j c
NCp7
i 12
3 I
2
s
x
T
E 0
7
B
R
~.
4-
i 600
9
M
®
Q
700 rim
Agarose gel e l e c t r o p h o r e s i s showing HIV-1 RNA dimerization (gel A) and primer tRNA annealing onto genomic HIV-1 RNA (gel B). Left lanes c o r r e s p o n d to the control with 32p tRNALys.3 (3x106 cpm/~tg) alone or in the presence of HIV-1 RNA. Lanes 1, 4 : 25 ng NCp7 ; lanes 2, 5 : 50 ng NCp7 ; lanes 3, 6 : 75 ng NCp7 showing a c o n c e n t r a t i o n d e p e n d e n t i n d u c t i o n of HIV-1 RNA dimerization and tRNA a n n e a l i n g onto m o n o m e r i c and dimeric HIV-1 RNA. Flgt~e 4 .
Visible a b s o r p t i o n s p e c t r u m of C o 2 + - N C p 7 (-), C o 2 + - ( 1 3 30)NCp7 (. . . . } and Co2+-(34-511NCp7 (.... ) between 700 and 600 n m corresponding to the d-d electronic transition.
e q u i v a l e n t s for N C p 7 a n d 2 . 9 for ( 1 3 - 3 0 ) N C p 7 a n d ( 3 4 - 5 1 ) N C p T , in good agreement with the expected number of SH groups. The
involvement
of the
nucleocapsid
protein
in retroviral
RNA
d i m e r i z a t i o n , e n c a p s i d a t i o n a n d a n n e a l i n g of t h e r e p l i c a t i o n p r i m e r tRNA o n t o t h e g e n o m i c RNA is n o w well d o c u m e n t e d
(4,8). In t h i s a r t i c l e w e
s h o w for t h e f i r s t t i m e t h a t N C p 7 c o n t a i n s all t h e s t r u c t u r a l e l e m e n t s r e q u i r e d to p r o m o t e HIV-1 RNA d i m e r i z a t i o n a n d a n n e a l i n g o f p r i m e r tRNA to t h e P B S (fig.3) w h i c h is c r i t i c a l for t h e i n i t i a t i o n
of reverse
t r a n s c r i p t i o n (8}. In c o n t r a s t , n e i t h e r of t h e two z i n c f i n g e r s w a s f o u n d to b e a c t i v e in v i t r o u s i n g t h e biological a s s a y s . T h e s e d a t a s u g g e s t t h a t b o t h N and C terminal elements surrounding the zinc fingers play an important role in RNA r e c o g n i t i o n a n d a n n e a l i n g . T h u s t h e z i n c f i n g e r s w o u l d n o t b e t h e o n l y e s s e n t i a l e l e m e n t s for t h e specific p a c k a g i n g o f t h e g e n o m i c RNA dimer. NCp7 and the finger domains (13-30)NCp7 and (34-51)NCp7 bind t h e Co 2 + i o n s in 2 / 1 a n d 1 / 1 r a t i o r e s p e c t i v e l y . T h e p o s i t i o n a n d t h e intensity of the bands
a t 6 9 5 , 6 4 2 a n d 6 1 5 n m (fig.4} a r e t y p i c a l o f a n
almost symmetrical tetrahedral arrangement intense
bands
at 349 and
314 nm
of t h e m e t a l w h i l e t h e t w o
a r e i n d i c a t i v e o f Co(II)-S- c h a r g e
t r a n s f e r (16). I n t e r e s t i n g l y t h e a b s o r p t i o n s p e c t r a of N C p 7 a n d t h e f i n g e r 1015
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
," 6[ o
5
×
4
T
2
T
time, s e c
leigttre 5 .
Time-dependent dissociation curves, followed at 642 nm, of the complexes formed between CO 2+ and (13-30)NCp7, (B} (3451)NCp7 ($} or NCp7 {-) in the presence of 10 eq. of EGTA.
d o m a i n s are very similar s u g g e s t i n g t h e l a c k of large differences in t h e g e o m e t r y of t h e complexed m e t a l for t h e t h r e e peptides. Similar r e s u l t s h a v e b e e n r e p o r t e d for t h e N C p l 0 of MoMuLV (24, 25) a n d for a r e c o m b i n a n t N C p l 5 from H1V (16). A d d i t i o n of a small excess of EGTA to NCp7 or its finger d o m a i n s allowed for t h e first time the stability of t h e complexes f o r m e d b e t w e e n Co 2+, a n d by e x t e n s i o n Zn 2+, w i t h NCp7 (fig. 5} to be e x p e r i m e n t a l l y evaluated. The affinity of Co 2+ ions is clearly higher for the proximal (1330)NCp7 t h a n for the distal finger d o m a i n (34-51)NCp7. T h u s , after 150 sec. a b o u t 80% of the complexed form of t h e distal finger h a s d i s a p p e a r e d as c o m p a r e d to only 50% of the proximal d o m a i n . In t h e case of NCp7, the dissociation rate of the Co 2+ complex is f a s t e r t h a n t h o s e f o u n d with b o t h fingers alone a n d after 150 sec the p e r c e n t a g e of b o u n d form is a r o u n d 40%. Differences in the s t a b i l i t y of t h e c o m p l e x e d zinc fingers a n d t h e negative c o o p e r a t i v i t y b e t w e e n t h e m e v i d e n c e d here, could i n d i c a t e t h e o c c u r r e n c e of spatial i n t e r a c t i o n s between b o t h finger d o m a i n s in NCp7. A similar h y p o t h e s i s h a s b e e n proposed from 1H NMR s t u d y of a s h o r t e r 55 a m i n o acid NCp7 e x t r a c t e d from a HIV-1 m u t a n t (17) a n d from l l 5 c d NMR s p e c t r o s c o p y of a r e c o m b i n a n t H1V-1 NCp15 (16). A l t h o u g h in m a n y retroviruses, t h e NC proteins have only one zinc finger, it is interesting to observe t h a t in t h e case of RSV, w h i c h c o n t a i n s two fingers, deletion of t h e p r o x i m a l d o m a i n s u p p r e s s e s a n y detectable infectivity, while deletion of t h e distal finger one is less deleterious (10,11}. The s t r u c t u r a l origin of t h e s e differences r e m a i n s u n k n o w n . In conclusion, the NCp7, b u t n o t its zinc fingers alone, w a s s h o w n to e x h i b i t v a r i o u s b i o l o g i c a l a c t i v i t i e s m i m i c k i n g t h o s e of N C p l 5 . E x p e r i m e n t s are n o w in p r o g r e s s to d e t e r m i n e t h e m i n i m a l s e q u e n c e 1016
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
required for full retention of biological activities with the aim to u s e this data in designing antiviral agents.
Acknowledgments We a c k n o w l e d g e Mary LAPADAT for a critical r e a d i n g of the m a n u s c r i p t and A. B O U J U and C. DUPUIS for typing. This work w a s supported b y the French Program (ANRS) against AIDS.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Barrd-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L. (1983) Science, 220, 868-870. Ratner, L., Haseltine, W., Patarca, R., Livak, K.J., Starcich, B., J o s e p h s , S.F., Doran, E.R., Rafalski, J.A., Whitehorn, E.A., B a u m e i s t e r , K., Ivanoff, L., Petteway Jr, S.R., Pearson, M.L., Lautenberger, J.A., Papas, T.S., Ghrayeb, J., Chang, N.T., Gallo, R.C. and Wong-Staal, F. (1985) Nature, 313, 277-284. Di Marzo Veronese, F., Rahman, R., Copeland, T.D., Oroszlan, S., Gallo, R.C. and Sarngadharan, M.G. (1987) AIDS IRes. H u m a n Retroviruses, 3, 253-264. Darlix, J.L., Gabus, C., Nugeyre, M.T., Clavel, F. and Barr6-Sinoussi, F. (1990) J. Mol. Biol. 216, 689-699. Barat, C., Lullien, V. Schatz, O., Keith, G., Nugeyre, M.T., GrfiningerLeitch, F., Barrd-Sinoussi, F., Le Griee, S.F.J. and Darlix J.L. {1989) EMBO J., 8, 3279-3285. Bieth, E., Gabus, C. and Darlix, J.C. (1990) Nuc. Acids Res. 18, 119127, Prats, A.C., Roy, C., Wang, P., Erard, M., H o u s s e t , V., G a b u s , C., Paoletti, C. and Darlix, J.L. {1990) J. Virol. , 64, 774-783. Prats, A.C., Sarih, L., Gabus, C., Litvak, S., Keith, G. and Darlix, J.L. (1988) The EMBO J., 7, 1777-1783. Aldovini, A. and Young, R.A. (1990) J. Virol., 64, 1920-1926. Meric, C. and Spahr, P.F. (1986) J. Virol. 60, 450-459. Merit, C., Gouilloud, E. and Spahr, P.F. (1988) J. Virol., 62, 33283333. Berg, J.M. (1986) Reports, 232, 485-487. Cornille, F., Mely, Y., Ficheux, D., Savignol, I., G6rard, D., Darlix, J.L., Fournid-Zaluski, M.C. and Roques, B.P. (1990) Int. J. Pept. Prot. Res. , 36, 551-558. Green, L.M. and Berg, J.M. (1990) Proc. Natl. Acad. Sci., 87, 64036407. Mely, Y., Cornille, F., Fourni6-Zaluski, M.C., Darlix, J.L., Roques, B.P. and G6rard, D. (1991) Biopolymers (in press). Fitzgerald, D.W. and Coleman, J.E. (1991) Biochemistry, 30, 51915201. South, T.L., Blake, P.R., Hare, D.R. a n d S u m m e r s , M.F. (1991) Biochemistry, 30, 6342-6349. Summers, M.F. (1991) J. Cell. Biochem. , 45, 41-48. S u m m e r s , M.F., S o u t h , T.L., Kim, B. a n d Hare, D.R. (1990) Biochemistry, 29, 329-340. Gorelick, R.J., Henderson, L.E., Hanser, J.P. and Rein, A. (1988) Proc. Natl. Acad. Sci. USA, 85, 8420-8424. 1017
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
21. Jentoft, J.E., Smith, L.M., Fu, X., J o h n s o n , M, and Leis, J. (1988} Proc. Natl. Acad. Sci., USA, 85, 7094-7098. 22. Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and Meirnhofer, J., Eds) Vol. 2, pp. 1-224, Academic Press, New-York. 23. Ellman, G.L. (1959) Arch. Biochem. Biophys. , 82, 70-77. 24. Roberts, W.J., Pan, T., Elliot, J.I., Coleman, J.E. and Williams, K.R. (1989) Biochemistry, 28, 10043-10047. 25. Green, L.M. and Berg, J.M. (1989) Proc. Natl. Acad. Sci. USA, 86, 4047-4051.
1018
81OCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages l O l O - l O l 8
FIRST LARGE SCALE CHEMICAL SYNTHESIS OF THE 7 2 AMINO ACID HIV-I NUCLEOCAPSID PROTEIN NCp7 IN AN ACTIVE FORM H. de R o c q u l g n y , D. F i c h e u x , C. Gabus*, M.-C. Fourni6-Zaluski, J.-L. Dariix* a n d B.P. R o q u e s ÷
D6partement de Chimie Organique, U266 INSERM-UA498 CNRS UFR des Sciences Pharmaceutiques et Biologiques 4, avenue de rObservatoire, 75270 Paris C6dex 06, France * Labo R6tro C J F INSERM, ENS 46, all6e d'Italie, 69364 Lyon Cedex 07, France Received September 12, 1991
The n u c l e o c a p s i d p r o t e i n (NC) of t h e h u m a n i m m u n o d e f l c i e n c y virus t y p e I p l a y s a c r u c i a l role in t h e f o r m a t i o n o f i n f e c t i o u s vlral p a r t i c l e s a n d t h e r e f o r e should be a major t a r g e t for t h e d e v e l o p m e n t of antiviral agents. T h i s r e q u i r e s an i n v e s t i g a t i o n o f NC p r o t e i n s t r u c t u r e a n d o f i t s i n t e r a c t i o n s w i t h b o t h p r i m e r tRNALys, 3 and g e n o m i c RNA. Nucleocapsid p r o t e i n NCp7, w h i c h r e s u l t s f r o m t h e m a t u r a t i o n of NCp15, c o n t a i n s two zinc fingers flanked b y s e q u e n c e s rich in basic a n d prollne residues. H e r e we r e p o r t t h e first s y n t h e s i s of large q u a n t i t i e s of NCp7 able t o a c t i v a t e HIV-1 RNA d i m e r i z a t i o n a n d r e p l i c a t i o n p r i m e r tRNALyS, z a n n e a l i n g t o t h e i n i t i a t i o n site o f r e v e r s e t r a n s c r i p t i o n . In a d d i t i o n UV s p e c t r o s c o p i c a n a l y s e s p e r f o r m e d t o c h a r a c t e r i z e t h e Co 2+ b i n d i n g p r o p e r t i e s of e a c h zinc f n g e r suggest t h a t t h e two fingers probably i n t e r a c t in NCp7. ~ ~991 Academic Press, Inc.
The GAG open reading frame of the h u m a n immunodeficiency virus type 1 (HIV-1) codes for the m a j o r core s t r u c t u r a l p r o t e i n s MApl7, CAp24 and N C p l 5 (1,2). Veronese et al. (3) have reported t h a t N C p l 5 is processed into two smaller proteins that are NCp7 and NCp6. Interactions of the NC protein with the viral RNA and replication primer tRNALys, 3 appear to be critical for the production of infectious viral particles (4). In fact, interactions of N C p l 5 with HIV-1 RNA promoted dimerization of the viral RNA, a process which a p p e a r s closely linked to genomic RNA encapsidation. In addition, interactions of N C p l 5 with b o t h primer tRNALys, 3 (5) and HIV-1 RNA resulted in the annealing of primer tRNA to the initiation site of reverse transcription (PBS). NC proteins also +To whom correspondence should be addressed.
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1010
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
participate in s p h e r i c a l capsid formation, p r o b a b l y t h r o u g h protein aggregation and tight interactions with the genomic RNA dimer (4-10). The NH2 t e r m i n u s of NCp15 c o r r e s p o n d i n g to NCp7 (72 amino acids) is a s s u m e d to be the functional domain in RNA recognition and b i n d i n g since it c o n t a i n s two s u c c e s s i v e zinc fingers of t h e type CX2CX4HX4C. At least one zinc finger of this type is found in all the highly conserved sequences of NC proteins of retroviruses (12). In fact genetic data have indicated that the zinc fingers play a crucial role in the specific encapsidation of genomic RNA (9). The zinc fingers bind Zn 2+ with high affinity (-1012 M -I) (13-15) and the binding of zinc was shown by NMR spectroscopy to induce folding of the peptide backbone {16-19). Although deletions or m u t a t i o n s of residues in the zinc fingers were shown to lower or abolish genomic RNA packaging (20), the precise role of the zinc fingers is still unclear (21), Moreover, the peptide sequences directly involved in the various functions of the NC protein are unknown, t h u s preventing a rational a p p r o a c h in the design of anti-viral agents aimed at inhibiting the NC protein functions. Hence structural analyses of the NC protein, either alone or in tight interactions with the viral RNA or primer tRNA, should be of great interest and well facilitated by the use of NCp7 provided that this small NC protein has all the biological activities of NCp15. Here we report the first synthesis of HIV-1 NCp7 protein in large quantities. The proximal (13-30) a n d the distal (34-51) zinc fingers of NCp7 (Fig. I) were also synthesized for s t r u c t u r a l and biological studies, Only NCp7 is shown to possess a high biological activity in vitro. Moreover, preliminary UV spectroscopic analyses of the Co 2+ binding properties of NCp7 and the zinc fingers suggest that the two fingers probably interact in the NCp7 protein. MATERIALS AND METHODS
Peptide synthesis. Fmoc p r o t e c t e d a m i n o acids ,HMP resin, p i p e r i d i n e , N - m e t h y l p y r r o l i d o n e (NMP), d i c h l o r o m e t h a n e (DCM), dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) were p u r c h a s e d from Applied Biosystems. Trifluoroacetic acid (TFA) was from Neosystem Laboratory (Strasbourg, France) and 1,2 ethanedithiol, phenol and CoCl 2 from Aldrich Chemie (Paris). Assembly of the protected peptide c h a i n s was carried out u s i n g the stepwise solid p h a s e m e t h o d of Merrifield (22) on a n Applied Biosystems 431A peptide synthetizer, with H O B t / D C C as coupling reagents. Fmoc amino acids were u s e d with the following side chains protections : t-butyl ether (Ser, Thr, Tyr), t-butyl ester (Glu, Asp), trityl (Cys, His, Asn, Gin), pmc (Arg) and boc (Lys). D e p r o t e c t i o n of the Fmoc group was o b t a i n e d b y two s u c c e s s i v e treatments of 3 min and 15 min with 20% piperidine in NMP. At the end of the synthesis, the peptide resin was treated for two h o u r s with 50 ml of TFA in the presence of phenol (3.75g), EDT (1.25ml), thioanisole (2.25 1011
Vol. 18o, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NCp7
6 Kr6N.._
GKE6HQ
NC~
z~, IA R
~C ~
"c ~s-M~RGNFRN~RKNV K 1
z~,.-" M K
c
c,
RAPRKKG
RR~ANFLGKIWP8YKGRP GNFL-C00fl 72 GKE611Q C -- M K ~ -: K
w c~
~=-x G 34
13-30 NCp7 cGKE611-T N ~Za
~-v x 13 Figure
1,
="
o %c~r
34-51
NCp7
~-cooa 51
AR
%-cooe 3O
Primary sequence of NCp7, (13-30)NCp7 and (34-51)NCp7.
ml), a n d H 2 0 (2.25 ml) to remove p r o t e c t i n g g r o u p s a n d to cleave the peptide from t h e resin. The entire r e a c t i o n m i x t u r e w a s applied to a C4 Vydac 5 g m (220x10 ram) a n d eluted w i t h a linear g r a d i e n t of 10-90% B in 30 m i n (A : H 2 0 , 0,1% TFA, B : CH3CN, H 2 0 , 70%, 30%, 0,09% TFA) at a flow r a t e of 3 m l / m i n with d e t e c t i o n at 214 n m u s i n g a n Applied B i o s y s t e m s 151A S e p a r a t i o n System. All the purifications were carried out u n d e r Argon (fig.2).
Biological activity of NCp7 and related zinc finger peptides. To s t u d y t h e biological activity of t h e s y n t h e t i c p e p t i d e s we u s e d t h e biological s u b s t r a t e s HIV-1 RNA a n d p r i m e r 32P-tRNALy s,3. HIV-1 RNA w a s s y n t h e s i z e d in vitro u s i n g T7 RNA p o l y m e r a s e a n d c o n t a i n s R, U5, PBS, the d i m e r i s a t i o n e n c a p s i d a t i o n d o m a i n a n d AUG of gag (positions 1 to 415) (4,6). HIV-1 RNA (1-415) a n d 32P-tRNALy s,3 were i n c u b a t e d in t h e a b s e n c e or p r e s e n c e of i n c r e a s i n g c o n c e n t r a t i o n s of NCp7 a n d r e l a t e d zinc fingers. After 30 m i n u t e s at 37°C the RNAs were p h e n o l e x t r a c t e d a n d a n a l y s e d by a g a r o s e gel e l e c t r o p h o r e s i s u n d e r native conditions. As reported in figure 3A l a n e s 1-3, NCp7 can strongly activate dimerization of HIV- 1 RNA (1-415). L a n e s 4-7 (fig. 3B) s h o w t h a t a n n e a l i n g of replication p r i m e r tRNALys,3 to the PBS was also activated b y NCp7. U n d e r the s a m e e x p e r i m e n t a l c o n d i t i o n s b o t h zinc finger p e p t i d e s were inactive (data n o t shown). Analysis of Co2+ binding properties of NCp7 and its zinc fingers by UV-visible spectroscopy. Visible absorption s p e c t r a were carried o u t on a Pye U n i c a m SP8-100 ultraviolet s p e c t r o m e t e r . A b o u t 0.5 mM of peptide u n d e r r e d u c e d form w a s dissolved in 50 mM Hepes, 100 mM KC1 buffer, pH 7.5, c o n t a i n i n g 1.5 eq. of COC12. In c o m p e t i t i o n e x p e r i m e n t s , a s o l u t i o n of EGTA (10 eq.) in Tris 1M w a s r a p i d l y a d d e d to t h e peptide solution in Hepes buffer. E x p e r i m e n t a l dissociation curves were t r e a t e d b y t h e c o m p u t e r p r o g r a m m LIGAND (Elsevier Biosoft). B i p h a s i c d i s s o c i a t i o n c u r v e s gave a good fit b e t w e e n e x p e r i m e n t a l a n d c a l c u l a t e d d a t a . 1012
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
F o r m a t i o n of t h e c o m p l e x b e t w e e n t h e s y n t h e s i z e d p e p t i d e s in H e p e s buffer pH 7.5 solutions a n d Co 2+ ions w a s evidenced by the a p p e a r a n c e of a blue color a n d of large absorption b a n d s with m a x i m a (~, expressed in Mlcm-1) a r o u n d 695 (a = 473), 642(c = 550), 615 n m (e = 348) c o r r e s p o n d i n g to the d-d electronic t r a n s i t i o n s of t h e m e t a l (fig.4) a n d in t h e 350 (a = 1374), 310 n m (a = 2560} r a n g e c o r r e s p o n d i n g to S-Co 2+ c h a r g e t r a n s f e r b a n d s . E x p e r i m e n t s p e r f o r m e d w i t h c o n c e n t r a t i o n s of Co 2+ g r e a t e r t h a n a 1:1 m o l a r ratio s h o w e d no f u r t h e r i n c r e a s e in absorption. Addition of ZnC12 to the Co 2+ complex abolishes the Co 2+ d-d t r a n s i t i o n b a n d at 600-700 nm. A t t e m p t s to m e a s u r e t h e E G T A - i n d u c e d d i s s o c i a t i o n c o n s t a n t s of t h e Co 2+ ions from the zinc finger(s) in p s e u d o first o r d e r c o n d i t i o n s (excess of EGTA>50 eq.) w a s h i n d e r e d by t h e complete d i s a p p e a r a n c e of t h e a b s o r p t i o n b a n d at 642 n m w h i c h o c c u r r e d too r a p i d l y to be m o n i t o r e d w i t h a c l a s s i c a l UV s p e c t r o m e t e r . T h e r e f o r e a q u a l i t a t i v e e v a l u a t i o n of t h e s t a b i l i t y of t h e Co 2+ c o m p l e x e s w a s a c h i e v e d b y competition s t u d i e s with 10 eq. of EGTA, l e a d i n g to a m e a s u r a b l e biphasic t i m e - d e p e n d e n t r e d u c t i o n of the a b s o r p t i o n at 642 n m with the following a p p a r e n t d i s s o c i a t i o n r a t e s (fig.5) : 1.04x10 -1 a n d 1.17x10 -3 sec -1 for NCp7, 3 . 0 9 x 1 0 -2 a n d 5.25x10 -4 sec -1 for (13-301NCp7, 4 . 4 0 x 1 0 -2 a n d 2 . 4 1 x 1 0 -3 sec -1 for (34-51)NCp7.
RESULTS AND DISCUSSION
Until now, the s t u d i e s devoted to N C p l 5 or its f r a g m e n t s have b e e n done with p r o t e i n s purified from virions or infected cells o b t a i n e d from o v e r p r o d u c t i o n of a r e c o m b i n a n t NCp15 e x p r e s s e d in E.coli. This s t u d y r e p o r t s t h e first s y n t h e s i s of NCp7 u n d e r c o n d i t i o n s w h i c h allow t h i s m i n i p r o t e i n to be o b t a i n e d in large q u a n t i t e s a n d w i t h a h i g h degree of p u r i t y . The c r u c i a l p a r a m e t e r s for s u c c e s s f u l s y n t h e s i s of NCp7 were d e t e r m i n e d by modifying the s t a n d a r d p r o g r a m of the synthetizer. T h u s , all p e p t i d e s were p r e p a r e d u s i n g F m o c c h e m i s t r y on HMP r e s i n (110 mg, 0,9 m m o l / g ) b u t c o u p l i n g t i m e s w e r e i n c r e a s e d progressively from 20 to 60 m i n as a f u n c t i o n of t h e growing size of t h e peptide. In the case of NCp7, double coupling (1 m m o l p e r coupling) was s y s t e m a t i c a l l y achieved from the first to the 22 n d a m i n o acid. Then, after the 22 n d , 44 t h a n d 60 t h c o u p l i n g steps, h a l f of t h e r e s i n w a s r e m o v e d b o t h to r e d u c e t h e p e p t i d e resin v o l u m e a n d to i n c r e a s e t h e a m o u n t of Fmoc a m i n o acid a d d e d to the resin from 10 to 80 eq. After TFA t r e a t m e n t , t h e c r u d e p r o d u c t w a s p u r i f i e d b y r e v e r s e p h a s e HPLC (fig.2) yielding to 124.6, 117.7 a n d 173 m g of p u r e NCp7, (13-30)NCp7 a n d (34-51)NCp7, respectively. The p e p t i d e s were j u d g e d to be >99% p u r e (yield a r o u n d 60%). Amino acid a n a l y s i s a n d s e q u e n c i n g s h o w e d a good a g r e e m e n t b e t w e e n p r e d i c t e d a n d e x p e r i m e n t a l c o m p o s i t i o n of t h e p e p t i d e s (table 1). 1013
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NCp7 8.94 min.
13-30 NCD 7 '89 min.
34-51 ~c~7 8.27 rain. 10
lb g I~tu'e 2.
~b
HPLC of purified NCp7 and related fragments. C o l u m n C4 Vydac 5 ~m (220 x 10 mm}. Gradient 10%-90% B. Solvent A 0. I% TFA in H 2 0 . Solvent B 0.09% TFA in C H 3 C N / H 2 0 {70/30,v/v), Flow rate 3 m l / m i n .
To p r e s e r v e t h e h i g h l y o x i d i z a b l e c y s t e i n e s u n d e r r e d u c e d f o r m s , all manipulations
were carried
out under
argon,
In these
conditions,
the
t i t r a t i o n of f r e e t h i o l s b y 5,5' d i t h i o b i s ( 2 - n i t r o b e n z o i e aeid) (24) g a v e 5 . 8 5
TABLE 1 . Amino acid c o n t e n t (uncorrected values) of purified peptides
AMINO ACID
NCp7
Asx Thr Ser Glx Pro Gly Ala Val Leu Tyr Lys His Arg Cys Phe Met Trp Ile
8.24 2.00 0.91 6.77 2.90 8.80 2.87 1.00 2.20 0.96 9.30 1.96 7.43 5.85 3.90 1.50 2.00 1.00
(13-30)NCp7
(34-5 I ) N C p 7
1.98 {2) 1.20 (1}
1.00 {1) 1.30 (1}
0,97 (i}
2,78 {3)
1.78 (2) 1.71 {2) 1.00 (I}
2.73 (3}
1.84 0.99 1.99 2.90 0.95
3.42 (4) 0.95 (i)
{8) {2) (I) (7) (3) (9) (3) (I} (2} (I) (I0} (2} (8) (6} (4) {2) (2} (1)
(2} (i) (2) (3) (I)
2.90 (3) 0.50 (I} 1.00 (I}
The theoretical amino acid c o m p o s i t i o n of each peptide is given in parentheses.
1014
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NCp7 j c
NCp7
i 12
3 I
2
s
x
T
E 0
7
B
R
~.
4-
i 600
9
M
®
Q
700 rim
Agarose gel e l e c t r o p h o r e s i s showing HIV-1 RNA dimerization (gel A) and primer tRNA annealing onto genomic HIV-1 RNA (gel B). Left lanes c o r r e s p o n d to the control with 32p tRNALys.3 (3x106 cpm/~tg) alone or in the presence of HIV-1 RNA. Lanes 1, 4 : 25 ng NCp7 ; lanes 2, 5 : 50 ng NCp7 ; lanes 3, 6 : 75 ng NCp7 showing a c o n c e n t r a t i o n d e p e n d e n t i n d u c t i o n of HIV-1 RNA dimerization and tRNA a n n e a l i n g onto m o n o m e r i c and dimeric HIV-1 RNA. Flgt~e 4 .
Visible a b s o r p t i o n s p e c t r u m of C o 2 + - N C p 7 (-), C o 2 + - ( 1 3 30)NCp7 (. . . . } and Co2+-(34-511NCp7 (.... ) between 700 and 600 n m corresponding to the d-d electronic transition.
e q u i v a l e n t s for N C p 7 a n d 2 . 9 for ( 1 3 - 3 0 ) N C p 7 a n d ( 3 4 - 5 1 ) N C p T , in good agreement with the expected number of SH groups. The
involvement
of the
nucleocapsid
protein
in retroviral
RNA
d i m e r i z a t i o n , e n c a p s i d a t i o n a n d a n n e a l i n g of t h e r e p l i c a t i o n p r i m e r tRNA o n t o t h e g e n o m i c RNA is n o w well d o c u m e n t e d
(4,8). In t h i s a r t i c l e w e
s h o w for t h e f i r s t t i m e t h a t N C p 7 c o n t a i n s all t h e s t r u c t u r a l e l e m e n t s r e q u i r e d to p r o m o t e HIV-1 RNA d i m e r i z a t i o n a n d a n n e a l i n g o f p r i m e r tRNA to t h e P B S (fig.3) w h i c h is c r i t i c a l for t h e i n i t i a t i o n
of reverse
t r a n s c r i p t i o n (8}. In c o n t r a s t , n e i t h e r of t h e two z i n c f i n g e r s w a s f o u n d to b e a c t i v e in v i t r o u s i n g t h e biological a s s a y s . T h e s e d a t a s u g g e s t t h a t b o t h N and C terminal elements surrounding the zinc fingers play an important role in RNA r e c o g n i t i o n a n d a n n e a l i n g . T h u s t h e z i n c f i n g e r s w o u l d n o t b e t h e o n l y e s s e n t i a l e l e m e n t s for t h e specific p a c k a g i n g o f t h e g e n o m i c RNA dimer. NCp7 and the finger domains (13-30)NCp7 and (34-51)NCp7 bind t h e Co 2 + i o n s in 2 / 1 a n d 1 / 1 r a t i o r e s p e c t i v e l y . T h e p o s i t i o n a n d t h e intensity of the bands
a t 6 9 5 , 6 4 2 a n d 6 1 5 n m (fig.4} a r e t y p i c a l o f a n
almost symmetrical tetrahedral arrangement intense
bands
at 349 and
314 nm
of t h e m e t a l w h i l e t h e t w o
a r e i n d i c a t i v e o f Co(II)-S- c h a r g e
t r a n s f e r (16). I n t e r e s t i n g l y t h e a b s o r p t i o n s p e c t r a of N C p 7 a n d t h e f i n g e r 1015
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
," 6[ o
5
×
4
T
2
T
time, s e c
leigttre 5 .
Time-dependent dissociation curves, followed at 642 nm, of the complexes formed between CO 2+ and (13-30)NCp7, (B} (3451)NCp7 ($} or NCp7 {-) in the presence of 10 eq. of EGTA.
d o m a i n s are very similar s u g g e s t i n g t h e l a c k of large differences in t h e g e o m e t r y of t h e complexed m e t a l for t h e t h r e e peptides. Similar r e s u l t s h a v e b e e n r e p o r t e d for t h e N C p l 0 of MoMuLV (24, 25) a n d for a r e c o m b i n a n t N C p l 5 from H1V (16). A d d i t i o n of a small excess of EGTA to NCp7 or its finger d o m a i n s allowed for t h e first time the stability of t h e complexes f o r m e d b e t w e e n Co 2+, a n d by e x t e n s i o n Zn 2+, w i t h NCp7 (fig. 5} to be e x p e r i m e n t a l l y evaluated. The affinity of Co 2+ ions is clearly higher for the proximal (1330)NCp7 t h a n for the distal finger d o m a i n (34-51)NCp7. T h u s , after 150 sec. a b o u t 80% of the complexed form of t h e distal finger h a s d i s a p p e a r e d as c o m p a r e d to only 50% of the proximal d o m a i n . In t h e case of NCp7, the dissociation rate of the Co 2+ complex is f a s t e r t h a n t h o s e f o u n d with b o t h fingers alone a n d after 150 sec the p e r c e n t a g e of b o u n d form is a r o u n d 40%. Differences in the s t a b i l i t y of t h e c o m p l e x e d zinc fingers a n d t h e negative c o o p e r a t i v i t y b e t w e e n t h e m e v i d e n c e d here, could i n d i c a t e t h e o c c u r r e n c e of spatial i n t e r a c t i o n s between b o t h finger d o m a i n s in NCp7. A similar h y p o t h e s i s h a s b e e n proposed from 1H NMR s t u d y of a s h o r t e r 55 a m i n o acid NCp7 e x t r a c t e d from a HIV-1 m u t a n t (17) a n d from l l 5 c d NMR s p e c t r o s c o p y of a r e c o m b i n a n t H1V-1 NCp15 (16). A l t h o u g h in m a n y retroviruses, t h e NC proteins have only one zinc finger, it is interesting to observe t h a t in t h e case of RSV, w h i c h c o n t a i n s two fingers, deletion of t h e p r o x i m a l d o m a i n s u p p r e s s e s a n y detectable infectivity, while deletion of t h e distal finger one is less deleterious (10,11}. The s t r u c t u r a l origin of t h e s e differences r e m a i n s u n k n o w n . In conclusion, the NCp7, b u t n o t its zinc fingers alone, w a s s h o w n to e x h i b i t v a r i o u s b i o l o g i c a l a c t i v i t i e s m i m i c k i n g t h o s e of N C p l 5 . E x p e r i m e n t s are n o w in p r o g r e s s to d e t e r m i n e t h e m i n i m a l s e q u e n c e 1016
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
required for full retention of biological activities with the aim to u s e this data in designing antiviral agents.
Acknowledgments We a c k n o w l e d g e Mary LAPADAT for a critical r e a d i n g of the m a n u s c r i p t and A. B O U J U and C. DUPUIS for typing. This work w a s supported b y the French Program (ANRS) against AIDS.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Barrd-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L. (1983) Science, 220, 868-870. Ratner, L., Haseltine, W., Patarca, R., Livak, K.J., Starcich, B., J o s e p h s , S.F., Doran, E.R., Rafalski, J.A., Whitehorn, E.A., B a u m e i s t e r , K., Ivanoff, L., Petteway Jr, S.R., Pearson, M.L., Lautenberger, J.A., Papas, T.S., Ghrayeb, J., Chang, N.T., Gallo, R.C. and Wong-Staal, F. (1985) Nature, 313, 277-284. Di Marzo Veronese, F., Rahman, R., Copeland, T.D., Oroszlan, S., Gallo, R.C. and Sarngadharan, M.G. (1987) AIDS IRes. H u m a n Retroviruses, 3, 253-264. Darlix, J.L., Gabus, C., Nugeyre, M.T., Clavel, F. and Barr6-Sinoussi, F. (1990) J. Mol. Biol. 216, 689-699. Barat, C., Lullien, V. Schatz, O., Keith, G., Nugeyre, M.T., GrfiningerLeitch, F., Barrd-Sinoussi, F., Le Griee, S.F.J. and Darlix J.L. {1989) EMBO J., 8, 3279-3285. Bieth, E., Gabus, C. and Darlix, J.C. (1990) Nuc. Acids Res. 18, 119127, Prats, A.C., Roy, C., Wang, P., Erard, M., H o u s s e t , V., G a b u s , C., Paoletti, C. and Darlix, J.L. {1990) J. Virol. , 64, 774-783. Prats, A.C., Sarih, L., Gabus, C., Litvak, S., Keith, G. and Darlix, J.L. (1988) The EMBO J., 7, 1777-1783. Aldovini, A. and Young, R.A. (1990) J. Virol., 64, 1920-1926. Meric, C. and Spahr, P.F. (1986) J. Virol. 60, 450-459. Merit, C., Gouilloud, E. and Spahr, P.F. (1988) J. Virol., 62, 33283333. Berg, J.M. (1986) Reports, 232, 485-487. Cornille, F., Mely, Y., Ficheux, D., Savignol, I., G6rard, D., Darlix, J.L., Fournid-Zaluski, M.C. and Roques, B.P. (1990) Int. J. Pept. Prot. Res. , 36, 551-558. Green, L.M. and Berg, J.M. (1990) Proc. Natl. Acad. Sci., 87, 64036407. Mely, Y., Cornille, F., Fourni6-Zaluski, M.C., Darlix, J.L., Roques, B.P. and G6rard, D. (1991) Biopolymers (in press). Fitzgerald, D.W. and Coleman, J.E. (1991) Biochemistry, 30, 51915201. South, T.L., Blake, P.R., Hare, D.R. a n d S u m m e r s , M.F. (1991) Biochemistry, 30, 6342-6349. Summers, M.F. (1991) J. Cell. Biochem. , 45, 41-48. S u m m e r s , M.F., S o u t h , T.L., Kim, B. a n d Hare, D.R. (1990) Biochemistry, 29, 329-340. Gorelick, R.J., Henderson, L.E., Hanser, J.P. and Rein, A. (1988) Proc. Natl. Acad. Sci. USA, 85, 8420-8424. 1017
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
21. Jentoft, J.E., Smith, L.M., Fu, X., J o h n s o n , M, and Leis, J. (1988} Proc. Natl. Acad. Sci., USA, 85, 7094-7098. 22. Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and Meirnhofer, J., Eds) Vol. 2, pp. 1-224, Academic Press, New-York. 23. Ellman, G.L. (1959) Arch. Biochem. Biophys. , 82, 70-77. 24. Roberts, W.J., Pan, T., Elliot, J.I., Coleman, J.E. and Williams, K.R. (1989) Biochemistry, 28, 10043-10047. 25. Green, L.M. and Berg, J.M. (1989) Proc. Natl. Acad. Sci. USA, 86, 4047-4051.
1018
