©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Binding of the HIV-1 Nucleocapsid Protein to the Primer tRNA, inVitro, Is Essentially Not Specific (*)

(Received for publication, June 7, 1994; and in revised form, October 19, 1994)

Yves Mély (1)(§) Hugues de Rocquigny Monica Sorinas-Jimeno (1) Gérard Keith (2) Bernard P. Roques Roland Marquet (2) Dominique Gérard (1)

From the  (1)Laboratoire de Biophysique, URA 491 du CNRS, Université Louis Pasteur de Strasbourg I, Faculté de Pharmacie, B.P. 24, F-67401 Illkirch Cedex, France, the (2)UPR du CNRS 9002, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, F-67084 Strasbourg Cedex, France, and the Département de Chimie Organique, INSERM U266, CNRS UA 498, U.F.R. des Sciences Pharmaceutiques et Biologiques, 4 avenue de l'Observatoire, 75270 Paris Cedex 06, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The nucleocapsid protein NCp7 of human immunodeficiency virus, type 1, is a key component in the viral life cycle. Since, the first common step of all reported NCp7 activities corresponds to a nucleic acid-binding step, the NCp7 binding parameters to the natural primer tRNA(3) were investigated. Using NCp7 intrinsic fluorescence, we found that (i) in 0.1 M NaCl, NCp7 bound noncooperatively to tRNA(3) with a K = 3.2 times 10^6M association constant and a n = 6 binding site size, (ii) four ionic interactions were formed in the NCp7bullettRNA(3) complex, and (iii) nonelectrostatic factors provided about 60% of the binding energy. These binding parameters were not significantly altered when the natural tRNA(3) was replaced by either an in vitro synthetic tRNA(3) transcript, the heterologous yeast tRNA or the structurally unrelated 5 S RNA from Escherichia coli. Moreover, the environment of the intrinsic fluorescent reporters (Trp and Trp) was similar in the various complexes. Finally, experiments performed at low protein concentration provide no evidence of high affinity binding sites. Taken together, our data strongly suggested an essentially nonspecific binding of NCp7 to tRNA(3) and thus did not seem to support a direct role of NCp7, per se, in the selection of tRNA(3) from the pool of cellular tRNAs.


INTRODUCTION

All retroviruses encode a gag gene product that is processed by the retroviral protease to give several structural proteins including the nucleocapsid protein. The nucleocapsid protein NCp15 of the human immunodeficiency virus type I (HIV-1) (^1)is derived from the Pr55 Gag polyprotein and is further processed into NCp7 and p6 proteins in mature HIV virus(1) . The mature NCp7 proteins, of known three dimensional structure(2, 3, 4) , contain two Cys-X(2)-Cys-X(4)-His-X(4)-Cys retroviral-type zinc fingers, also referred as CCHC motifs(5) , which have been shown to be saturated by zinc in mature virus preparations (4, 6) . The NCp7 protein has been proposed to be a key component of the retrovirus life cycle. In mature virions, numerous copies of NCp7 are associated to the genomic RNA dimer to form the ribonucleoprotein complex(7) . In vitro, NCp7 has been shown to activate the retroviral RNA dimerization (7) and the annealing of the primer tRNA(3) to the initiation site of the reverse transcription(8, 9) , suggesting a critical role for NCp7 in both genomic RNA packaging and reverse transcription. Furthermore, NCp7 was also shown to bind to strong stop cDNA and promote the annealing of this cDNA to the 3` end of the genomic RNA (10) . Finally, NCp7 was reported to possess nucleic acid unwinding (11) and strand renaturation (12) activities.

Clearly all NCp7 activities rely on interactions with nucleic acids. Hence, to further understand the biochemical basis of NCp7 biological functions, it was critical to investigate the first step of these interactions, the binding step. Most reports suggest that nucleocapsid proteins (NC) bind preferentially to single-stranded RNAs(13, 14, 15) . However, this point has been controverted in the case of the avian myeloblastosis virus NC(16) . Sequence-dependent binding has been reported for a -containing RNA transcript (15, 17) and for oligodeoxynucleotide sequences analogous to the packaging signal (4, 18) , but this dependence was rather weak (less than 10-fold). In contrast, NCp7 has been specifically cross-linked to an U-track in the genomic RNA 5` end (7, 19) and in the anticodon domain of tRNA(3)(20) , suggesting that strong specific binding sites may exist. Moreover, NCp7 was shown to interact with both the heterologous tRNA(4) and a mixture of tRNA (11) in a strong Mg-dependent way, but the binding parameters have not been reported. Finally, peptides corresponding to the amino-acid sequences of the two isolated zinc fingers have also been reported to bind to tRNA but with marginal affinity (21) .

In this context, to further delineate NCp7 functions, the binding parameters of the zinc-saturated 72-amino acid synthetic NCp7 to tRNA(3) were investigated in various NaCl and MgCl(2) conditions. Moreover to assess the importance of the sequence and the three-dimensional structure of tRNA(3) in the binding process, we investigated the binding parameters of NCp7 to two modified forms of tRNA, the heterologous tRNA and the structurally unrelated RNA 5 S from Escherichia coli. Finally, performing experiments at low protein concentrations, we tested the existence of a putative high affinity binding site in the anticodon domain.


MATERIALS AND METHODS

Proteins

Solid phase synthesis of the 72-amino acid NCp7 protein (Fig. 1) was carried out as described previously (22) . To preserve the highly oxidizable cysteine residues (purity >99%), the lyophilized peptides were dissolved in freshly degassed buffer and poured immediately into anaerobic quartz cells, which maintain an inert argon atmosphere. In all experiments, zinc was added to the protein in a 2.5 molar ratio. An extinction coefficient of 12,700 M cm at 280 nm was used to determine the protein concentration.


Figure 1: Amino acid sequence of NCp7.



Nucleic Acids

Natural tRNA(3) with sequence identical to that of human tRNA(3) was prepared and purified from beef liver(23) . Dethiolated tRNA(3) (dStRNA(3)) and a tRNA(3) lacking modified nucleotides (utRNA(3)) were obtained as described previously(24) . tRNA from brewers' yeast was purchased from Sigma.

All RNAs were checked for purity and integrity on denaturing polyacrylamide gels. Extinction coefficients at 260 nm of 6.25 times 10^5M cm and 9.6 times 10^5M cm were used to determine the concentration of the various tRNA and RNA 5 S, respectively.

Fluorescence Measurements

Titrations of NCp7 with the various nucleic acids were performed while monitoring the quenching of the intrinsic tryptophan fluorescence of NCp7 in an SLM 48000 spectrofluorometer. Excitation wavelength was set at either 280 or 295 nm, and emission wavelength was set at 350 nm. The temperature was controlled at 20.0 ± 0.5 °C, and the fluorescence intensities were corrected for dilution, buffer fluorescence, and screening effects by added nucleic acids(25) . The absence of light scattering due to high molecular weight aggregate formation during titration was systematically checked by comparing the measured absorbances at 260 and 280 nm with the calculated ones using the extinction coefficients of NCp7 and the nucleic acid at these wavelengths.

Relationship between the Fraction of Bound Protein and the Extent of Fluorescence Quenching

The general method of analysis of Bujalowski and Lohman (26) was used to determine rather than assume the relationship between the fluorescence quenching and the fraction of bound protein from a titration of NCp7 with RNA. Let L(t) be the total protein concentration and D(t) be the total nucleotide concentration for which we measure the fluorescence quenching Q = (I(0) - I)/I(0), where I(0) and I correspond to NCp7 fluorescence intensities in the absence and in the presence of D(t), respectively. This method uses the fact that, at equilibrium, the value of the quantity Q (L(t)/D(t)) is dependent only upon the binding density = L(b)/D(t) (mol of bound protein/mol of nucleotide). As a result, when several titrations are performed at different protein concentrations and the data are plotted as Q (L(t)/D(t)) versusD(t), then for all titrations, and the free protein concentration, L(f) are constant at a given value of Q (L(t)/D(t)). From the set of L(t) and D(t) values, determined from each titration at a given Q (L(t)/D(t)), one can deduce L(f) and from a plot of L(t)versusD(t) by using the relationship L(t) = L(f) + bulletD(t). Repeating this at a number of values of Q (L(t)/D(t)) yields as a function of L(f) from which the concentration of bound protein L(b) = bulletD(t) can be calculated. Finally, Q is plotted versusL(b)/L(t) (Fig. 2) to prove the proportionality between these two quantities. The plot of QversusL(b)/L(t) also allows one to straightforwardly determine the maximum extent of fluorescence quenching Q(max) when all NCp7 proteins are bound to RNA, from a linear extrapolation to L(b)/L(t) = 1.


Figure 2: Determination of Q(max) and demonstration that Q/Q(max) = L(b)/L(t) for NCp7 binding to tRNA(3). The buffer was 50 mM Hepes, 100 mM NaCl, pH 7.5. The fraction of protein bound, L/L, was obtained as described in the text. The protein concentrations were 0.4 (circles), 0.6 (stars), 1.0 (triangles), and 1.2 (squares) µM.



Determination of NCp7-RNA Binding Parameters

To determine the NCp7-RNA binding parameters, the intrinsic fluorescence quenching, Q, of a fixed concentration, L(t), of NCp7 was monitored in the presence of increasing concentrations, D(t), of nucleotides. An example of such a titration is given in Fig. 3A. Once it has been rigorously proven that L(b)/L(t) is directly proportional to Q, then one can simply calculate the concentration of bound and free protein, L(b) and L(f), respectively, using and .



Figure 3: Determination of NCp7-tRNA(3)-binding parameters. PanelA, binding isotherm. The fluorescence intensity (squares) of 1 µM NCp7 was recorded as a function of added tRNA(3) concentration expressed in nucleotides. The fluorescence intensity, I, was then converted in the fluorescence quenching parameter, Q (circles), according to Q = (I(0) - I)/I(0), with I(0) corresponding to the NCp7 fluorescence in the absence of tRNA(3). The solidline represents the best fit of for values of K, n, and Q(max) given in Table 1. The dashedline is drawn assuming, besides the nonspecific binding sites, the existence of a single high affinity binding site with a K(2) affinity 10^6-fold higher than K. PanelB, determination of the binding site size. The experimental data of Fig. 3A, expressed as fractional fluorescence quenching are reported versus the molar ratio of total [NCp7] to total [tRNA] expressed in nucleotides. The apparent binding site size, n, is given by the intersection of the two dashedlines and is about 8.





The binding site size, n, the observed affinity, K, and the cooperativity parameter, , were then recovered using the equations of McGhee and von Hippel (27) for noncooperative

or cooperative binding

with R = {[1 - (n + 1)]^2 + 4(1 - n)}.

Nonlinear least-squares fit of these equations to experimental data was performed using the algorithm of Kowalczykowski et al. (28). In the noncooperative model, all the parameters (n, K, and Q(max)) were allowed to vary. In the cooperative model, Q(max) was fixed, whereas n,K, and were allowed to vary.

Salt and pH Dependence of NCp7bulletRNA Complexes

The ion and pH dependence of K in the presence of an excess of monovalent (M, X) or divalent (M, 2X) salt, at constant temperature and pressure, may be written as

where K(T)(M) is the thermodynamic association constant in the presence of cation M, r is the number of independent and identical sites, with a binding constant K(H), on the protein that must be protonated as a prerequisite for binding, a is the number of independent and identical binding sites for anions with a binding constant K(X), m` is the number of ion pairs between protein and nucleic acid, and (M) is the fraction of cation M thermodynamically bound per phosphate group(29) .

To analyze the ion dependence of K at constant pH, is differentiated with respect to log [M].

Salt-reversal of the binding of the protein to an excess of nucleic acid was monitored by following the protein fluorescence increase brought about by the addition of a concentrated solution of either NaCl, CH(3)COONa or MgCl(2). Assuming that n and were constant, the K values were calculated from or for each salt concentration. Then, reporting Kversus [M] in a log-log plot (30, 31) allows one to determine the parameters m`, a and K(X) from .

Similarly, to analyze the pH dependence of K at a given salt concentration, is differentiated with respect to pH.

Hence, increasing amounts of either diluted HCl or NaOH were added to NCp7 in the presence of an excess of nucleic acid. The K values for each pH value were calculated as in the salt-reversal experiments and then reported versus pH in a semi-log plot to recover r and K(H).

Characterization of the Putative High Affinity Binding Site

To characterize the putative high affinity binding site, the titration curves were performed in the classical ``forward'' sense by adding increasing protein concentrations to a fixed nucleic acid amount. As the signal originates from the protein, similar amounts of protein were added in parallel to a blank without nucleic acid. As protein concentrations used in these experiments were very low, adhesion of the protein to the quartz cells was suspected to be no more negligible. Experiments were thus performed either in the absence or in the presence of 0.04% polyethylene glycol 20000 (Merck) as described in the case of Moloney murine leukemia virus NC(14) .


RESULTS

Relationship between the Fraction of NCp7 Bound to tRNA and the Extent of Fluorescence Quenching

The addition of tRNA(3) to NCp7 induced a very large fluorescence decrease. Irrespective of the initial protein concentration, the residual fluorescence of NCp7 at the highest concentrations of tRNA(3) added was less than 15% (Fig. 3A), suggesting that both Trp residues (at positions 37 and 61) were largely quenched in the protein-tRNA(3) complex. To straightforwardly analyze this fluorescence quenching in terms of fraction of bound protein, the linearity of the relationship between these two quantities was rigorously assessed according to the general method of Bujalowski and Lohman (26) (see ``Materials and Methods''). As reported for NCp7bulletpoly(A) complexes(12) , a critical problem was the appearance of aggregates during titration due to the formation of high molecular weight complexes. This aggregation was found to be basically dependent on the protein concentration and could be avoided if protein concentrations lower than 1.5 times 10M were used. In these conditions, the relationship between Q and L(b)/L(t) (Fig. 2) was clearly linear and thus the binding parameters could be confidently deduced from a titration at a single protein concentration. Moreover, a linear extrapolation to L(b)/L(t) = 1 allowed one to straightforwardly determine a 0.87 (±0.02) value of the maximum extent of fluorescence quenching, Q(max), when all NCp7 proteins are bound to tRNA.

Determination of NCp7-tRNA(3) Binding Parameters

The binding experiments were performed in 50 mM Hepes pH 7.5 and 0.1 M NaCl. Mg was omitted in a first step since the simultaneous presence of both Mg and Na largely complicated the thermodynamic analysis(32) . A typical titration of NCp7 with tRNA(3) is seen in Fig. 3A. Analysis of these data according to and yielded the binding parameters given in Table 1. The binding was noncooperative, and Q(max) was in very good keeping with the value obtained from Fig. 2.

The binding site size could be independently confirmed using the intersection of the tangential to the low [tRNA(3)]/[NCp7] ratios (where the binding is approximately stoichiometric) with the straight line Q/Q(max) = 1 in Fig. 3B. The intersection is about 8 (±1) in good keeping with the calculated value.

Salt and pH Dependence of NCp7bullettRNA(3) Interaction

To provide further molecular information about the binding process, the pH and ion concentration dependence of K was investigated. The addition of concentrated NaCl or CH(3)COONa to NCp7bullettRNA(3) complexes brought about a partial reversal of the protein fluorescence quenching, suggesting that the complex formation partly depends on ionic interaction (Fig. 4A). Assuming that the binding site size does not change with the salt concentration, the K value for each salt concentration was recovered from and was reported versus salt concentration in a log-log plot (Fig. 4B). A linear decrease of logK in a rather large range of Na concentrations was observed for both NaCl or CH(3)COONa suggesting from that for both anions, K(X)[X]1, and thus reduced to




Figure 4: Salt-reversal of tRNA(3)bulletNCp7 interaction. Protein and tRNA(3) concentrations were 1 and 0.3 µM, respectively. PanelA, reversal of tRNA(3)bulletNCp7 interaction by NaCl (circles), NaCH(3)CO(2) (squares) and MgCl(2) (triangles). The fractional fluorescence quenching of NCp7 was measured as a function of added salt. PanelB, log-log plot of the dependence of K upon cation concentration for the data of Fig. 4A. K was calculated using (see ``Materials and Methods'') for a binding site size, n = 6.2. Lines drawn are least-squares fits of through the points. The slopes logK/log[cation] were -2.8, -2.7, and -2.0 for NaCl, NaCH(3)CO(2), and MgCl(2), respectively. The intercepts at 1 M cation were 3.7, 4.6, and 2.7, respectively. PanelC, dependence of logK at 0.1 M NaCl upon f([MgCl(2)]). The abscissa, f([MgCl(2)]) was calculated as described in the text for a 3-50 mM MgCl(2) concentration range. The slope and the y axis intercept were 4.7 and 3.4, respectively.



Despite a significant shift toward higher cation concentration in the case of acetate, the slope was identical for both anions.

To further delineate the contribution of anion binding, a salt-back titration was performed in the presence of MgCl(2) only. As expected, Mg competed more efficiently than Na (Fig. 4, A and B) with NCp7 binding to tRNA(3) and thus shifted the range of Cl concentrations to substantially lower concentrations. Despite this shift, the slope ratio, (logK/log[Mg])/(logK/log[Na]), was in keeping with the

The pH dependence of K at 0.1 M NaCl was then investigated to provide molecular information about the number of sites, r, with a binding constant, K(H), on the protein that must be protonated as a prerequisite for binding. Clearly no pH dependence could be observed for K in the pH 5.5-9 range (Fig. 5) suggesting from that either r = 0 or K(H) > 10M. Thus at pH 7.5, reduced to


Figure 5: pH dependence of the tRNA(3)bulletNCp7 interaction. Protein concentration was 0.6 µM in 50 mM Hepes, 0.1 M NaCl. K was calculated from assuming that n was constant. The least-squares line drawn through the points had a zero slope.



and thus logK(T)(M) corresponded to the intercept of Fig. 4B at 1 M cation M. The K(T)(Na) and K(T)(Mg) values were consistent according to the relationship(29) : logK(T)(Na) = logK(T)(Mg) + (m`/2)logK using a thermodynamic binding constant of Mg for tRNA, logK = 0.5, close to that observed for DNA (29) . As a final check of the consistency of our preceding conclusions, a salt-back titration was performed with MgCl(2) in the presence of 0.1 M NaCl. According to Record et al.(32) and our previous conclusions, the following equation should apply

with logK = logK - 2 log [Na]. Thus a plot of logKversusf([MgCl(2)]) = -( log [Na] + log1/2[1 + (1 + 4K[Mg])]) should give a straight line with a slope, m`, and a y axis intercept, logK(T)(Na). This was clearly the case (Fig. 4C), and both m` and logK(T)(Na) were in good agreement with the values of Table 1.

Dependence of the Binding Parameters on the Type and the Structure of the Nucleic Acids

Since the modified and hypermodified nucleosides of tRNAs are well known to be critical in numerous biological properties of tRNAs, we checked their influence in the binding process by investigating the binding parameters of NCp7 for dethiolated natural tRNA(3) (dStRNA(3)) and unmodified synthetic tRNA(3) (utRNA(3)). For both dStRNA(3) and utRNA(3), the maximum extent of fluorescence quenching and the binding parameters were largely similar to those of tRNA(3) (Table 1). The only noticeable difference was a 2-fold affinity decrease for utRNA versus tRNA(3). Unfortunately, salt-reversal experiments were not accurate enough to pinpoint the origin of this slight difference. Thus our data suggested that the modified bases were not critical in the NCp7bullettRNA(3) binding process. To check if there was any influence of the primary structure at all, the binding of NCp7 to the heterologous yeast tRNA was investigated (Table 1). The maximum extent of fluorescence quenching of NCp7 was clearly independent of the tRNA nature. In contrast, both the binding site size and the affinity for tRNA were slightly reduced. These moderate differences with tRNA(3) were significant as the binding experiments were repeated more than 10 times with protein concentrations ranging from 4.10 to 1.2. 10M. Salt-reversal titrations suggested that the differences in K were primarily related to differences in K (1 M) and thus on nonelectrostatic interactions. Finally, to check the influence of the RNA tertiary structure on the binding process, we investigated the binding of NCp7 to RNA 5 S from E. coli, which differed from tRNA(3) in both primary and tertiary structures (33) (Table 1). The binding site size for RNA 5 S was similar to the tRNA(3) one, but the affinity was about 4 times lower due essentially to differences in the number of ion pairs formed and thus to electrostatic interactions.

Comparison of tRNA(3)- and tRNA-binding Parameters in Various Salt Concentrations

To further validate the comparison between tRNA(3) and tRNA binding to NCp7, we investigated the binding parameters of NCp7 for both tRNAs at various NaCl concentrations in the absence or in the presence of Mg. To increase the validity of this investigation, the binding parameters were directly obtained from titration curves (like those in Fig. 3A) rather than simply from salt-reversal experiments. In doing so, we were able to determine not only K but also the binding site size and the cooperativity parameter. Moreover, as a control, we checked that the protein fluorescence was unaffected by NaCl or MgCl(2) in the concentration range used. The results, collected in Table 2for tRNA(3) and Table 3for tRNA were largely similar. For both tRNAs, the binding site size was constant over the NaCl and MgCl(2) concentrations investigated, whereas, in contrast, the cooperativity parameter was not, since a moderate cooperativity appeared at high concentrations of MgCl(2) (geq10 mM) in the presence of 0.1 M NaCl or at high concentrations of NaCl (geq0.3 M). As K and were highly correlated, the product K times was preferred to K to compare the binding affinities of both tRNAs. The (K times )/(K times ) ratio was always between 0.25 and 6 (Table 3), suggesting that whatever the NaCl or MgCl(2) concentrations used, no large preferential binding of NCp7 to tRNAversus tRNA could be achieved.





Binding Parameters of the Putative High Affinity Binding Site

As cross-linking experiments suggested the existence of a single high affinity binding site located in the anticodon domain of tRNA(3)(20) , experiments were performed to test the existence of such a site. To this end, we assumed that this putative site, of size n, binds NCp7 according to a classical Scatchard equation; (2) = [L]/[N] = K(2)L(f)/(1 + K(2)L(f)), where [L] is the concentration of protein bound to this site, [N] is the concentration of the high affinity site and thus of tRNA(3), L(f) is the concentration of free protein, and K(2) is the association binding constant. The other nucleotides of tRNA(3) were assumed to bind to NCp7 independently and nonspecifically with an association binding constant, K(1), according to . As the anticodon domain is approximately located in the middle of the tRNA(3) sequence, the nonspecific binding sequences on both sides were of similar length. If effects of finite lattice length are neglected, it can be easily demonstrated that this is equivalent to a unique lattice of length N - n, where N is the total number of nucleotides of tRNA(3).

At the protein concentrations used in the former sections of this paper, theoretical binding curves indicated that the modifications in the binding curves induced by the presence of a single strong binding site (even with a K(2)/K(1) ratio of 10^6) were below experimental uncertainty (Fig. 3A). Thus the affinities measured in these conditions were clearly those of the nonspecific binding sites. To magnify the putative strong-binding site, lower protein concentrations were needed. Unfortunately, fluorescence was then low and furthermore the low-binding density points, which were the most informative, were the last ones in the titration and thus the less accurate ones (essentially due to screening effect correction). To circumvent this latter drawback, titrations were performed in the classical ``forward'' sense using a fixed concentration of tRNA(3) as described under ``Materials and Methods.'' Predictive analysis indicated that the best compromise between fluorescence intensity and magnification of the strong binding site was for [tRNA(3)] = 10M. Moreover as the signal originates from the protein, a similar titration on a blank without nucleic acid was performed in parallel.

The quenched fluorescence, I, of the sample and the unquenched fluorescence, I(0), of the blank were then used to calculate the observed fluorescence quenching Q = (I(0) - I)/I(0). Assuming that the maximum extent of fluorescence quenching Q(max) was identical to that obtained in ``reverse'' titrations, the Q/Q(max) ratio was plotted versus log[NCp7] and compared with theoretical curves generated assuming the existence of a single high affinity binding site (Fig. 6). In the presence of 0.1 M NaCl and 3 mM MgCl(2), this comparison clearly suggests that the K(2)/K(1) ratio was lower than 100. As errorbars were rather high, further precision on K(2) value could not be achieved. Addition of 0.04% polyethylene glycol 20,000 did not significantly change the data, suggesting that adhesion of the protein to the quartz cells was marginal. Similar conclusions on K(2)/K(1) ratios were obtained either in the absence of Mg, in 0.2 M NaCl, with slightly higher tRNA concentrations, or if tRNA(3) was replaced by tRNA (data not shown).


Figure 6: Detection and parameterization of the putative tRNA(3) high affinity binding site. The tRNA(3) concentration was 1.10M in 50 mM Hepes pH 7.5, 0.1 M NaCl, 3 mM MgCl(2). The solidline is the theoretical curve drawn using the binding parameters of Table 2, at these salt concentrations. Dashedlines correspond to theoretical curves assuming the existence of a single high affinity binding site with, from top to bottom, either 1000-, 100-, or 10-fold higher affinity than the nonspecific binding sites. The standard deviations for quadriplate measurements are indicated by the errorbars, and the average value is represented by circles.




DISCUSSION

The nucleic acid-induced intrinsic fluorescence quenching of the nucleocapsid protein, NCp7 of HIV-1 was used in this study to quantify the interaction of NCp7 with the homologous tRNA(3) on one hand and various heterologous RNAs on the other hand. The binding site size of NCp7 for tRNA(3) was close to that observed for the NCp7bulletpoly(A) interaction(12, 34) , the Moloney murine leukemia virus NCbulletpoly(rU) interaction (14) or the avian myeloblastosis virus NC interaction with various single- or double-stranded nucleic acids (16) .

At 0.1 M NaCl, NCp7 bound to tRNA(3) noncooperatively with an affinity K close to the affinity of the Moloney murine leukemia virus NC for poly(A) (14) or to the K times product of the avian myeloblastosis virus NC for its target genomic RNA (16) at the same NaCl concentration. The observed association constant was very sensitive to the ionic environment and especially to the type and concentration of ions. Analyzing this dependence according to the theory of Record et al.(29, 30, 31, 32) , we found that the binding of NCp7 to tRNA(3) released about three monovalent or two divalent cations from tRNA(3). This cation release corresponded to the formation of about 4 ionic interactions between NCp7 and the phosphate groups on tRNA(3). Moreover, the comparison of Mg and Na effects on K indicated that the primary effect of Mg on the NCp7bullettRNA(3) interaction was a competitive effect with NCp7 for binding to tRNA(3). Thus Mg did not seem to play an additional specific role in complex formation. In contrast to cations, no anions were released upon the binding of NCp7 to tRNA(3). Thus, the origin of the 10-fold increase in K when chloride was replaced by acetate might be related, as in the case of lac repressor-operator (35) and nonspecific DNA (29) interaction, to some indirect effect on the protein at sites not directly involved in nucleic acid binding. This effect induced an 8-fold increase in the thermodynamic binding constant without modifying the number of ionic interactions between NCp7 and tRNA(3). The lack of pH dependence of K in the pH range 5.5-9 suggested that neither the His residues nor the NH(2)-terminal amino group were involved in the electrostatic interactions of the NCp7bullettRNA(3) complex. Similarly, Lys amino groups were probably not dominant as well, unless their pK were significantly greater than 10. Consequently, we speculate that, as for the interaction of NCp7 with the -RNA transcript(17) , the numerous Arg residues may play a central role in the NCp7bullettRNA(3) interaction. Finally, from the thermodynamic binding constant obtained from extrapolation of the log-log plot to 1 M NaCl, we deduced that nonelectrostatic interactions corresponded to a free energy of about 5 kcal/mol at 20 °C. Thus, under approximate in vivo conditions (0.1 M NaCl, 3 mM MgCl(2), pH 7.5) about 60% of the binding energy was provided by nonelectrostatic factors.

The observed binding constant of NCp7 to tRNA(3) was not modified when mcm^5s^2U was dethiolated and only slightly decreased when all modified bases were replaced by their unmodified counterparts. Thus the modified bases of tRNA(3) were only of marginal importance for interaction with NCp7 in sharp contrast to interaction with either reverse transcriptase (9, 36) or HIV-1 primer binding site(24) . Moreover, as the K of NCp7 for the heterologous tRNA and the unrelated RNA 5 S from E. coli, at 0.1 M NaCl, were only 3- and 4-fold lower than for tRNA(3), respectively, we suggest that the binding of NCp7 to tRNA(3) is essentially not specific. This conclusion is further assessed by (i) the comparison of NCp7 binding parameters to tRNA(3)versus tRNA at various NaCl and MgCl(2) concentrations, which suggested that the affinity ratio between these two tRNAs was independent of the ionic concentrations used, (ii) the similar fluorescence quenching of NCp7 in the various complexes, which suggested that no specific environment of both intrinsic fluorescence reporters (Trp and Trp) was achieved in the NCp7-tRNA(3) complex and, (iii) the similarity of the number of ionic interactions and the thermodynamic binding constant with those governing the interaction of NCp7 with poly(A) (11) or Moloney murine leukemia virus NC with a fluorescent poly(A) derivative(14) . Interestingly, a low cooperativity factor appeared at high concentrations of NaCl or MgCl(2) but was not specific as well since it was observed with either tRNA, poly(A) (11) , or RNA 5 S (data not shown). Finally, highly similar conclusions were inferred if the 72-amino acid-long NCp7 protein (Fig. 1) was replaced in the binding experiments by its NH(2)-terminal 55-amino acid-long cleavage product (data not shown), thought to be the ultimate mature NC form(37) .

Concerning the existence of a putative high affinity binding site located in the anti-codon domain of tRNA(3)(20) , our data suggest that if such a site exists, its affinity is at most 100 times higher than the nonspecific one, irrespective of the concentrations of NaCl or MgCl(2). This affinity increase is very low in comparison with many other sequence-specific proteins as, for instance, the lac repressor system where the affinity ratio between specific and nonspecific sites was about 10^8 in similar ionic conditions(29) . Even if we assumed that the binding constant of the putative high affinity binding site corresponded to our upper-bound estimation, simulations indicated that in the presence of equimolar concentrations of tRNA and tRNA, the ratio of NCp7 bound to tRNA(3)versus tRNA never exceeded six, irrespective of the concentrations of protein or tRNA (data not shown). A similar conclusion probably applied for other tRNA species since NCp7 was shown to induce an uniform CD signal variation from a mixed population of tRNA molecules(11) . Despite the slight binding differences between tRNA(3) and tRNA, only the former is one of the four major-abundance tRNA species identified in HIV-1 virions(38) . Hence, as more than 100 different tRNA species may be present with different abundances in the HIV-1 host cell, no direct role of NCp7 per se in the selection of tRNA(3) from the pool of cellular tRNAs is expected. Thus we suggest that if NCp7 intervenes in the tRNA(3) selection, it should be either by cooperating with the reverse transcriptase (20) or more likely in a precursor form (like Gag-pol polyproteins) where the other parts of the precursor cooperate with the NCp7 sequence to select the primer(39) .

From in vitro studies, NCp7 has been inferred to activate primer tRNA(3) annealing to the genomic RNA primer binding site in the virion(40) . To further assess the reliability of this NCp7 activity, which has been shown to be concentration-dependent(40) , we estimated from our data the number of NC bound to each tRNA(3) in the virion. For this purpose, using the reported dimensions of the HIV-1 club-shaped inner core (41) and the number of NC inside(42) , we calculated a 6.4 times 10 m^3 inner core volume and thus an about 5 mM NC concentration. Assuming that the two 9213-nucleotide-long genomic RNA molecules represented about 50% of the total RNA in weight, (^2)we estimated the total nucleotide concentration to be about 100 mM. In these conditions, from the tRNA(3)-binding parameters at 0.1 M NaCl and 3 mM MgCl(2), and their moderate temperature-dependence in the 20-37 °C range (data not shown), we infer that almost all NCp7 proteins are bound to the nucleic acids in the virion. Furthermore, from the avian myeloblastosis virus NC data (16) and our data on tRNA(3) and RNA 5 S, we might reasonably assume that the nonspecific binding sites were of similar affinity irrespective of the ribonucleic acid type. Hence we calculated that at least four NC were bound to each tRNA(3); a ratio that has been shown to provide an efficient hybridization of tRNA(3) to the genomic RNA at least in vitro(40) .


FOOTNOTES

*
This work was supported by grants from the Agence Nationale de la Recherche sur le SIDA (ANRS), CNRS, and the Université Louis Pasteur. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-88-67-69-28; Fax: 33-88-67-40-11.

(^1)
The abbreviations used are: HIV-1, human immunodeficiency virus, strain 1; NC, nucleocapsid protein; dStRNA(3), dethiolated tRNA(3); utRNA(3), tRNA(3) lacking modified nucleotides.

(^2)
J. L. Darlix, personal communication.


ACKNOWLEDGEMENTS

We thank Pascale Romby and Catherine Isel for the gift of E. coli 5 S RNA and the plasmid carrying the tRNA(3) gene, respectively. We also thank Jean-Luc Darlix for the gift of a synthetic 3`-CA-deficient tRNA(3) species used for preliminary binding assays, Chantal and Bernard Ehresmann for fruitful discussions and support, and M. Wernert for expert editorial assistance.


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