(Received for publication, June 7, 1994; and in revised form, October 19, 1994)
From the
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 were investigated. Using NCp7 intrinsic
fluorescence, we found that (i) in 0.1 M NaCl, NCp7 bound
noncooperatively to tRNA
with a K
= 3.2
10
M
association constant and a n = 6 binding site size, (ii) four ionic interactions were
formed in the NCp7
tRNA
complex, and
(iii) nonelectrostatic factors provided about 60% of the binding
energy. These binding parameters were not significantly altered when
the natural tRNA
was replaced by either an in vitro synthetic tRNA
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
and thus did not seem to support a
direct role of NCp7, per se, in the selection of
tRNA
from the pool of cellular tRNAs.
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) ()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
-Cys-X
-His-X
-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
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
(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 were investigated in various NaCl and
MgCl
conditions. Moreover to assess the importance of the
sequence and the three-dimensional structure of
tRNA
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.
Figure 1: Amino acid sequence of NCp7.
All RNAs
were checked for purity and integrity on denaturing polyacrylamide
gels. Extinction coefficients at 260 nm of 6.25 10
M
cm
and 9.6
10
M
cm
were used to determine the concentration of the various tRNA and
RNA 5 S, respectively.
Figure 2:
Determination of Q
and demonstration that Q
/Q
= L
/L
for NCp7
binding to tRNA
. 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.
Figure 3:
Determination of
NCp7-tRNA-binding parameters. PanelA, binding isotherm. The fluorescence intensity (squares) of 1 µM NCp7 was recorded as a function
of added tRNA
concentration expressed in
nucleotides. The fluorescence intensity, I, was then converted
in the fluorescence quenching parameter, Q
(circles), according to Q
= (I
- I)/I
, with I
corresponding to the NCp7 fluorescence in the absence of
tRNA
. The solidline represents the best fit of for values of K
, n, and Q
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
affinity 10
-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
with R = {[1 - (n + 1)]
+ 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
) were allowed to vary. In the cooperative
model, Q
was fixed, whereas n,K
, and
were allowed to vary.
where K(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
, 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
, m` is the number of ion pairs between
protein and nucleic acid, and
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, CHCOONa or MgCl
. Assuming that n and
were constant, the K
values were calculated from or for each
salt concentration. Then, reporting K
versus [M] in a log-log plot (30, 31) allows
one to determine the parameters m`, a and K
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
.
The binding
site size could be independently confirmed using the intersection of
the tangential to the low
[tRNA]/[NCp7] ratios
(where the binding is approximately stoichiometric) with the straight
line Q
/Q
= 1 in Fig. 3B. The intersection is about 8 (±1) in
good keeping with the calculated value.
Figure 4:
Salt-reversal of
tRNANCp7 interaction. Protein and
tRNA
concentrations were 1 and 0.3
µM, respectively. PanelA, reversal of
tRNA
NCp7 interaction by NaCl (circles), NaCH
CO
(squares)
and MgCl
(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
CO
, and MgCl
, 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
]). The abscissa, f([MgCl
]) was
calculated as described in the text for a 3-50 mM MgCl
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 only. As
expected, Mg
competed more efficiently than
Na
(Fig. 4, A and B) with
NCp7 binding to tRNA
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
, 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
> 10
M
. Thus at pH 7.5, reduced to
Figure 5:
pH dependence of the
tRNANCp7 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(M) corresponded to the
intercept of Fig. 4B at 1 M cation M. The K
(Na
) and K
(Mg
) values were consistent
according to the relationship(29) :
logK
(Na
) =
logK
(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
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
logK
versusf([MgCl
]) =
-(
log
[Na
] + log1/2[1 + (1 +
4K
[Mg
])
])
should give a straight line with a slope, m`, and a y axis intercept, logK
(Na
).
This was clearly the case (Fig. 4C), and both m` and logK
(Na
) were in
good agreement with the values of Table 1.
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/K
ratio of 10
)
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
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
] =
10
M. 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, of
the blank were then used to calculate the observed fluorescence
quenching Q
= (I
- I)/I
. Assuming that the
maximum extent of fluorescence quenching Q
was
identical to that obtained in ``reverse'' titrations, the Q
/Q
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
, this comparison clearly suggests
that the K
/K
ratio was lower
than 100. As errorbars were rather high, further
precision on K
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
/K
ratios were obtained
either in the absence of Mg
, in 0.2 M NaCl,
with slightly higher tRNA concentrations, or if
tRNA
was replaced by tRNA
(data
not shown).
Figure 6:
Detection and parameterization of the
putative tRNA high affinity binding site. The
tRNA
concentration was 1.10
M in 50 mM Hepes pH 7.5, 0.1 M NaCl, 3
mM MgCl
. 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.
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 on one hand and various heterologous
RNAs on the other hand. The binding site size of NCp7 for
tRNA
was close to that observed for the
NCp7
poly(A) interaction(12, 34) , the Moloney
murine leukemia virus NC
poly(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 noncooperatively with an
affinity K
close to the affinity of the Moloney
murine leukemia virus NC for poly(A) (14) or to the K
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
released
about three monovalent or two divalent cations from
tRNA
. This cation release corresponded to the
formation of about 4 ionic interactions between NCp7 and the phosphate
groups on tRNA
. Moreover, the comparison of
Mg
and Na
effects on K
indicated that the primary effect of
Mg
on the NCp7
tRNA
interaction was a competitive effect with NCp7 for binding to
tRNA
. 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
. 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
. The lack of pH dependence of K
in the pH range 5.5-9 suggested that
neither the His residues nor the NH
-terminal amino group
were involved in the electrostatic interactions of the
NCp7
tRNA
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 NCp7
tRNA
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
,
pH 7.5) about 60% of the binding energy was provided by
nonelectrostatic factors.
The observed binding constant of NCp7 to
tRNA was not modified when
mcm
s
U
was dethiolated and only
slightly decreased when all modified bases were replaced by their
unmodified counterparts. Thus the modified bases of
tRNA
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
, respectively, we suggest that
the binding of NCp7 to tRNA
is essentially
not specific. This conclusion is further assessed by (i) the comparison
of NCp7 binding parameters to tRNA
versus tRNA
at various NaCl and MgCl
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
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
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
-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(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
. 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
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
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
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
from the pool of cellular tRNAs is
expected. Thus we suggest that if NCp7 intervenes in the
tRNA
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 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
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
10
m
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, (
)we estimated the total nucleotide concentration to be
about 100 mM. In these conditions, from the
tRNA
-binding parameters at 0.1 M NaCl and 3 mM MgCl
, 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
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
; a ratio that has been shown to provide
an efficient hybridization of tRNA
to the
genomic RNA at least in vitro(40) .