(Received for publication, August 9, 1995)
From the
Reverse transcriptases from both human immunodeficiency viruses type 1 and 2 are obligatory dimers. A tryptophan-rich repeat motif that is highly conserved between these proteins, as well as in the reverse transcriptase from simian immunodeficiency virus, has been postulated to be involved in hydrophobic subunit interactions. A synthetic 19-mer peptide covering part of this tryptophan repeat motif was recently shown to inhibit human immunodeficiency viruses type 1 reverse transcriptase subunit dimerization (Divita, G., Restle, T., Goody, R. S., Chermann, J.-C., and Baillon, J. G.(1994) J. Biol. Chem. 269, 13080-13083). In the present study, we show that the same peptide can also inhibit human immunodeficiency virus type 2 reverse transcriptase subunit dimerization, suggesting that the same inhibitors might be used as agents against both viruses as well as against variants of human immunodeficiency virus type 1 that differ from the variant against which they were developed. Under appropriate experimental conditions, e.g. at acidic pH, this peptide is also able to induce the dissociation of the enzyme from human immunodeficiency virus type 1.
A significant effort has been made over the past few years
toward improving or finding new and more potent and specific inhibitors
of reverse transcriptase (RT) ()of human immunodeficiency
virus (HIV), the etiologic agent responsible for the development of
AIDS. Unfortunately, despite the high potency of some of these
compounds, resistance to these inhibitors arises very rapidly both in
cell culture experiments and during treatment of patients (Larder and
Kemp, 1989; St. Clair et al., 1991; Nunberg et al.,
1991). This corresponds to the emergence of point mutations within the
RT sequence and arises from the hypermutability of HIV (Preston et
al., 1988; Roberts et al., 1988).
Recent crystallographic data obtained with HIV-1 RT (Kohlstaedt et al., 1992; Jacobo-Molina et al., 1993; Rodgers et al., 1995; Ren et al., 1995) suggest that there are ways other than the current use of small molecules, i.e. nucleoside analogs or nonnucleoside inhibitors to inhibit enzyme activity (for review, see Nanni et al. (1993)). As originally proposed by Restle and co-workers in 1990 (Restle et al., 1990), dimerization of RT might be a good target for therapeutic intervention in AIDS. This hypothesis was based on the observation that only dimeric forms of the enzyme are active (Restle et al., 1990; 1992). The dimer interface is largely dominated by interactions between the two connection subdomains (Nanni et al., 1993; Wang et al., 1994). These protein-protein interactions have been shown to be highly hydrophobic (Becerra et al., 1991; Müller et al., 1991; Divita et al., 1995). Sequence comparison (Baillon et al., 1991), as well as intrinsic protein fluorescence studies (Divita et al., 1993) have focused on a tryptophan repeat motif that is highly conserved between HIV-1 and HIV-2 RTs, as well as in simian immunodeficiency virus RT and that was suggested to be involved in RT subunit interactions. Indeed, a synthetic 19-mer peptide, corresponding to residues 389-407 from the BH-10 molecular clone of HIV-1 and covering part of this tryptophan repeat motif was recently shown to inhibit RT subunit dimerization (Divita et al., 1994).
In the present paper, we show that the same peptide can also inhibit HIV-2 RT subunit reassociation, although it is slightly less efficient than in the case of HIV-1 RT. This peptide appears to act on the first step of the two-step process for HIV-1 and HIV-2 RT heterodimer formation described recently (Divita et al., 1995), the rapid association of the monomers into a heterodimeric form devoid of polymerase activity, and not on the second phase of the process, which consists of a slow isomerization leading to the ``mature'' active form of RT. Under appropriate experimental conditions, P1 is also able to induce the dissociation of HIV-1 RT.
Figure 1:
Peptide inhibition of the HIV-2 RT
dimerization process. HIV-2 heterodimeric RT (5 µM) was
first dissociated by 17% of acetonitrile at pH 6.5, 25 °C, and the
monomer association was initiated by a 12-fold dilution in an
acetonitrile-free buffer in the absence () or in the presence of
10 µM P1 (
), P3 (
), or P4 (
). A, kinetics of formation of active heterodimeric RT monitored
by polymerase activity assay. The data were analyzed as a first-order
reaction as described under ``Experimental Procedures.'' B, the kinetics of monomer-monomer association were monitored
by using size exclusion HPLC on an aliquot fraction containing 10
µg of RT, and the data were analyzed according to a second-order
reaction.
In
order to identify which step of the dimerization process is affected by
the presence of the peptides, this dimerization process was also
monitored by size-exclusion HPLC (Fig. 1B). As shown in
this figure, the monomer-association step is greatly affected by the
presence of 10 µM P1 and reduced by a factor of 44,
with a second-order rate constant value (k
) of
0.9
10
M
s
in the case of HIV-2 RT. In contrast, when the heterodimer
intermediate form of RT is previously formed by a 40-min incubation in
an acetonitrile-free buffer and then incubated at pH 8.0 in the
presence of 10 µM P1, the first-order rate constant of
maturation is only reduced by 10-15%. This is the result of
partial dissociation of the intermediate form of RT, which takes place
even in the absence of peptide, as observed by an HPLC size exclusion
experiment performed after 40 h of incubation (not shown).
Two other
peptides (P3 and P4), which were also derived from the connection
domain of HIV-1 RT, inhibit the association of HIV-2 RT subunits,
although these two peptides show a less marked effect than that
observed for P1. As observed for P1, both peptides affected the
monomer-association step, as revealed by the correlation between Fig. 1, A and B, with association rate
constant values of 1 10
M
s
and 0.7
10
M
s
and
isomerization rate constant values of 0.8 h
and 0.28
h
for P3 and P4, respectively (Table 1). In
contrast, peptide P2, which corresponds to the carboxyl-terminal end of
the tryptophan cluster of the connection domain did not affect HIV-2 RT
dimerization (not shown), as previously observed in the case of HIV-1
RT (Divita et al., 1994).
As shown in Fig. 2, the
inhibition of HIV-1 and HIV-2 RT dimerization is dependent on the
peptide concentration, and analysis of these curves leads to the
determination of apparent dissociation constant values for each
peptide. Essentially complete inhibition of HIV-2 RT dimerization was
obtained for a concentration of 20 µM P1, and an apparent K value of 2.7 ± 0.6 µM was
determined, which is
2-fold higher than the value of 1.2 ±
0.7 µM obtained for HIV-1 RT. In the case of P3 and P4, it
was not possible to obtain complete inhibition of HIV-2 RT
dimerization, even at the highest concentration used (100
µM, not shown). However, apparent K
values could be estimated and led to values of 25.7 ± 4
µM for P4 and >100 µM for P3, with respect
to HIV-2 RT dimerization inhibition, and 21 ± 5 µM for P3 and >100 µM for P4, with respect to HIV-1
RT dimerization inhibition.
Figure 2:
Dependence of the HIV-1 and HIV-2 RT
monomer association rate constant on the peptide concentration. HIV-1 (panel A) and HIV-2 (panel B) RT were dissociated
using acetonitrile, and the reassociation was performed as described in Fig. 1, in the presence of increasing concentrations of P1
(), P2 (
), P3 (
), and P4 (
). The dimerization
rate constants were determined by fitting the time-dependent
association curve with an equation for describing a second-order
reaction.
Figure 3:
P1
inducing HIV-1 RT dissociation. HIV-1 RT (2 µM) was
incubated at pH 7.5 (), 6.8 (
), and 6.2 (
) in the
presence of 100 µM P1 at 25 °C. After different
incubation times, the residual polymerase activity was measured by
using a standard polymerase activity assay (panel A), and the
residual fraction of dimeric enzyme was determined by size-exclusion
HPLC (panel B). As control, it was checked that in the absence
of P1 HIV-1 RT is stable for at least 24 h at the different pH values
used.
Heterodimeric HIV-1 and HIV-2 RTs represent the biologically active relevant forms found in infectious virions (Chandra et al., 1986; Di-Marzo Veronese et al., 1986; Lightfoote et al., 1986; DeVico et al., 1989), and their dimeric nature presents an interesting target for the design of new antiviral agents (Restle et al., 1990; Divita et al., 1994). Recently, we have proposed a two-step mechanism for the dimerization process of both HIV-1 and HIV-2 RTs, which involves first an interaction between the connection subdomains of the two subunits leading to an inactive intermediate followed by a slower conformational change, which corresponds to the stacking of the thumb subdomain of p51 to the RNase-H domain of the large subunit and to the placement of the finger subdomain of p51 in the palm subdomain of p66 (Divita et al., 1995). We have also shown that synthetic peptides derived from the connection subdomain of HIV-1 RT can effectively block the formation of ``mature'' heterodimeric RT (Divita et al., 1994). In the present work, we have extended this approach of inhibition of dimerization to HIV-2 RT using the same peptides derived from HIV-1 RT and also report on effective dissociation of HIV-1 RT induced by one of these peptides (P1) at acidic pH. We also present evidence that discriminates which of the two steps of the dimerization process is affected.
Attempts to reconstitute chimeric HIV RTs from separately
expressed or co-expressed isolated subunits of HIV-1 and HIV-2 RTs have
proved to be very difficult (Howard et al., 1991;
Müller et al., 1991), especially when
HIV-2 p66 is mixed with HIV-1 p51. On the basis of these results, it
has been suggested that the regions of the two enzymes involved in
dimerization might differ. At first sight, this seems to be consistent
with the observation that the maturation step is faster for HIV-2 RT
than for HIV-1 RT (Divita et al., 1995) and that this enzyme
is 10-fold more stable ()and harder to dissociate using
organic solvents than its HIV-1 counterpart (Müller et al., 1991). However, it is still possible to reversibly
dissociate the heterodimeric form of HIV-2 RT using 20% acetonitrile,
at pH 6.5. Such a controlled dissociation offers the possibility of
rapidly testing potential inhibitors of the dimerization process of
both HIV-1 and HIV-2 RTs.
In our preceding work, we have described
peptides derived from the connection domain of HIV-1 RT, which can
inhibit the dimerization of previously isolated subunits of the same
enzyme. The most efficient was a 19-mer tryptophan-rich peptide
corresponding to residues 389-407 of the BH-10 clone derived from
the HIV-1 isolate (Divita et al., 1994). Here we
have demonstrated that this peptide (P1) also constitutes a powerful
inhibitor of dimerization of HIV-2 RT subunits, which is not very
surprising since this region is highly conserved in HIV-1, HIV-2, and
simian immunodeficiency virus RTs ( Table 2and Baillon et
al., 1991). P1 can effectively block the dimerization process of
both enzymes at a concentration of 25 µM, and exhibits
relatively high affinities for the isolated subunits of HIV-1 (1.2
µM) and HIV-2 (2.7 µM) RTs.
The
correlation between polymerase activity and HPLC size exclusion data
leads to the conclusion that for both RTs, the peptide directly targets
the first step of the dimerization process, i.e. the
monomer-monomer association. The fact that P1, which is derived from
the connection subdomain of HIV-1 RT, is able to inhibit the
association process of HIV-2 RT subunits suggests that at least part of
the dimerization domains of the two enzymes exhibit some similarities.
This observation might be significant, considering the relatively high
variance between HIV-1 and 2 isolates. The first step in the
association process between subunits might be similar in HIV-1 and
HIV-2 RTs, via part of the conserved tryptophan cluster corresponding
to -helix L and/or
-sheet 19 in the three-dimensional
structure of HIV-1 RT, since P1 exhibits almost the same effect on the
subunit association of both enzymes. This is experimentally confirmed
here by the observation that the association rate constants are quite
similar for the two enzymes, both in the absence of peptide (2-4
10
M
s
) or in the presence of P1 ( Table 1and
Divita et al.(1995)). Interestingly, the projection of
-helix L of p51 subunit reveals that all of the well conserved
aromatic amino acid residues are located on the external side of the
helix and interact directly with the p66 subunit (Wang et al.,
1994).
Although P3 and P4, which are also derived from the connection subdomain of HIV-1 RT, show inhibitory properties toward HIV-2 RT dimerization (Table 1), the effect of these peptides on the two enzymes is slightly different. In the case of HIV-1 RT, the inhibitory effect of P3 is greater than that of P4, while the opposite is true in the case of HIV-2 RT.
At pH 8.0, none of the peptides used can induce RT dissociation. In fact, this is not surprising considering the relatively low affinity of P1 for the subunits (micromolar range) in comparison with the apparent dissociation constant between the subunits (nanomolar range) (Restle et al., 1990; Divita et al., 1993). In contrast, HIV-1 RT dissociation induced by P1 at acidic pH is in good agreement with the fact that the stability of both HIV-1 and HIV-2 RTs are highly dependent on the pH, being strongly reduced at low pH (Restle et al., 1990; Becerra et al., 1991; Divita et al., 1995), although HIV-2 RT is generally more stable than HIV-1 RT (Müller et al., 1991). At pH 6.2, P1 induces significant dissociation of HIV-1 RT (only 10% of RT remains dimeric after a 40-h incubation with 100 µM P1). In comparison, this peptide has a relatively low effect on HIV-2 RT at the same concentration. One can speculate that at least part of the additional contacts between subunits, other than those present at the level of the tryptophan cluster, are highly dependent on the pH in the case of HIV-1 RT and less so in the case of HIV-2 RT. Current efforts are directed toward the identification of these additional contacts between subunits in both enzymes.
Inhibition of enzymes by the use
of peptidic inhibitors of obligatory dimeric enzymes has been proposed
as an alternative to the use of small molecules or substrate analogs
(Cohen et al., 1986; Dutia et al., 1986; Restle et al., 1990; Zhang et al., 1991; Divita et
al., 1994). However, so far, such inhibitors have been considered
mostly as academic tools due to their relatively low potency in
comparison with more classical small inhibitors and also to the
problems of delivery and targeting of these compounds. A very recent
report shows that their use may not be limited to the laboratory. In
this report, it is shown that a peptidomimetic inhibitor derived from
such an inhibitory peptide by individually optimizing each amino-acid
of the parental peptide can exhibit a tremendous improvement of potency
over the starting material (Liuzzi et al., 1994). The
optimized peptidomimetic inhibitor, whose function is to block the
dimerization of Herpes simplex ribonucleotide reductase, exhibited a
subnanomolar IC and showed antiviral activity in
vivo. This result emphasizes the need to proceed with the study of
such inhibitors to identify substances of high potency, which should be
more specific than most currently used inhibitors and, perhaps more
importantly, should lower the risk of emergence or selection of
resistant mutants.