(Received for publication, August 3, 1995; and in revised form, November 15, 1995)
From the
In this investigation, we examine the interaction between the
human immunodeficiency virus type I integrase and oligonucleotides that
reflect the sequences of the extreme termini of the viral long terminal
repeats (LTRs). The results of gel filtration and a detailed binding
density analysis indicate that the integrase binds to the LTR as a
high-order oligomer at a density equivalent to 10 ± 0.8
integrase monomers per 21-base pair LTR. The corresponding binding
isotherm displays a Hill coefficient of 2, suggesting that the binding
mechanism involves the cooperative interaction between two oligomers.
This interaction is quite stable, exhibiting a prolonged half-life (t 13 h) in the presence of Mn
cations. Complexes were less stable when formed with
Mg
(t
1 h). The role of
Mn
appears to be in the induction of the
protein-protein interactions that stabilize the bound complexes. In
terms of the 3`-end processing of the LTR, similar catalytic rates (k
0.06 min
) were
obtained for the stable complex in the presence of either cation.
Hence, the apparent preference observed for Mn
in
standard in vitro integration assays can be attributed
entirely to the augmentation in the DNA binding affinity of the
integrase.
The integration of retroviral DNA involves two separable DNA strand cleaving events at positions that consequently form the sites of union between the viral and host DNA. The first occurs in the cytoplasm of the infected cell and results in the removal of the two outermost bases from each 3`-end of the pre-integrated linear DNA genome(1, 2, 3) . This cleavage activity occurs invariantly adjacent to a conserved CA dinucleotide, embedded within a short inverted repeat sequence present at the termini of the viral LTRs. The second strand breakage involves the coordination of the recessed viral 3`-OH as a nucleophile in an enzyme-catalyzed attack on the phosphodiester backbone of the host target DNA. Concurrent with the exposure of a host 5`-phosphate, a single, direct transesterification with the attacking viral 3`-OH end results in the insertion of the viral DNA at this site(4) .
Whereas the integration reaction is mediated in vivo by a large nucleoprotein complex known to contain several retroviral proteins(5) , both the 3`-end processing and strand transfer activities can be carried out in vitro using short duplex oligonucleotides that mimic one of the viral LTR termini and the purified retroviral integrase protein alone(6, 7, 8, 9) . Hydrolysis of the viral substrates by the integrase is site-specific in the sense that it consistently cleaves 3` of the conserved CA dinucleotide, even when the 3`-LTR is artificially extended as single-stranded DNA(9) . However, although several studies have confirmed that a level of sequence specificity is required for efficient cleavage and integration(9, 10, 11, 12) , this is not reflected in a corresponding DNA binding preference for the cognate LTR(6, 11, 13, 14, 15, 16, 17) .
Several pertinent observations have been made with regard to the
dominant nonspecific DNA binding properties of retroviral integrases.
Deletion mapping studies have located the major determinant of the
nonspecific DNA binding domain to a region within the C terminus of the
integrase(18, 19) . Although poorly conserved and of
variable length in sequence comparisons between retroviral integrases,
this region is rich in basic residues (Arg, Lys) and may interact
nonspecifically with the DNA via general electrostatic interactions
with the phosphate backbone. A central catalytic core domain of the
integrase, which possesses a highly conserved D,D-35-E
motif(15, 20) , proposed to form the polynucleotidyl
transfer catalytic site(21, 22, 23) , also
binds DNA but with an apparent requirement for branched structures that
mimic the Y-type intermediate resulting from a single LTR integration
event into a target DNA(17) . The DNA binding properties of the
core domain thus reflect the ability of this region to recognize and
resolve such DNA structures by catalyzing the removal of the branch arm
(LTR) and resealing the broken (target) strand in a process termed
disintegration (24) . It has been proposed that a second highly
conserved region, a HHCC motif located near the N terminus of the
integrase, may form a Zn-binding finger with
similarities to those of the transcription factor IIIA-like DNA binding
proteins(25) . However, whereas this region has been shown to
bind Zn
in vitro(22) , no evidence
has been presented to define a direct interaction with the substrate
DNA in binding studies. Rather, this region has been implicated in
forming protein-protein interactions required for the divalent
cation-induced oligomerization of the integrase(26) .
Interference with this motif, either by mutagenesis (16, 21, 23, 27, 28) or
chemical modification(28) , is detrimental to the 3`-end
processing and strand transfer activities of the integrase, but such
modifications do not appear to block disintegration or the nonspecific
interaction with DNA. The observation that the modification of this
domain prevents the formation of the stable interaction between the
integrase and the LTR suggests that protein-protein interactions may be
essential in the assembly of the functional
complexes(26, 29) . The involvement of subunit
interactions in the assembly of an active oligomeric form of the HIV-1 (
)integrase has also been demonstrated by genetic (30) and in vitro complementation (31, 32, 33) studies of integrase mutants.
The refractory nature of this retroviral protein to analysis by conventional biochemical strategies, such as mobility shift and DNase footprinting techniques, has previously precluded a detailed, quantitative account of the biophysical properties of integrase nucleoprotein complexes. Recently, highly constrained solution conditions allowing a specific mobility shift to be detected between the HIV-1 integrase and the LTR were defined(34) . Notably, these required low concentrations of the DNA substrate and the omission of the divalent cation essential for the catalytic processing of the LTR. In the present investigation, we describe the application of laser-mediated single-pulse UV cross-linking and nuclease digestion techniques to obtain a quantitative description of the stable binding of the HIV-1 integrase to the LTR, under solution conditions more closely reflecting those optimized for 3`-end processing and DNA integration assays. The implications of this data on the quaternary structure of the nucleoprotein complex and the influence of divalent cations on the stability of this interaction are examined.
where denotes the fractional saturation of the DNA
lattice and
represents the quantum yield of adduct
obtained at saturation of the lattice with ligand, a constant factor
dictated by the photochemical reactivity of the oligonucleotide
sequence occluded and the number and nature of protein-DNA contacts
formed. The implicit requirement that
is independent
of the value of
, and hence remains constant throughout the
binding isotherm, is a point that will be validated under
``Results.''
where, in this simplified case, the Hill coefficient, n, corresponds to the number of integrase oligomers that
interact upon binding to the DNA substrate. To account for these
cooperative interactions, the titration data, in terms of fractional
saturation (), were fitted directly by computer-assisted least
squares procedures to the appropriate isotherm corresponding to the
above mechanism:
where [D] refers to the total DNA
concentration and [I]
represents the
concentration of free protein required to titrate the DNA to half
saturation (for convenience, titrations are expressed in terms of
integrase monomers, and consequently the equilibrium binding parameters
obtained from the corresponding isotherms are also reported in
monomeric units). For a first-order (independent site) binding
mechanism (n = 1), the isotherm reduces to a hyperbola,
and the dissociation constant (K
) is given by
[I]
. Where n = 2,
second-order binding is observed, i.e. the degree of
polymerization of integrase oligomers is increased by two upon transfer
from solution to DNA. In this case, the value
[I]
is equivalent to
&cjs3484;K
. Transformed into the familiar
Scatchard form, we obtain following the linear expression:
To obtain the concentration of free integrase,
[I], binding isotherms (constructed in terms of
total integrase monomer, [I]) were
corrected for the stoichiometric number of binding sites (N)
(again expressed in monomer units). Since the concentration of bound
integrase, [I]
, is given by the fraction
(
) of the total concentration of binding sites (N
[D]
) occupied, we then
may write the following:
For a series of titrations performed at different concentrations
of DNA, at any given value of , plotting
[I]
versus
[D]
gives a straight
line from which [I]
and N may
be determined directly from the intercept and slope of the line,
respectively. The binding isotherm is generated by repeating this
analysis throughout the range of
values, essentially as
described previously(41) .
Dissociation kinetics were analyzed by the first-order exponential decay expression:
where D is the concentration of bound DNA
at time 0, and k is the corresponding dissociation rate
constant.
Figure 1: Gel filtration analysis of the HIV-1 integrase. Samples of the HIV-1 integrase, diluted to the appropriate NaCl concentration, were subjected to gel filtration on Superdex 75 in 400 mM (thick solid line) or 150 mM (thin solid line) NaCl. The broken line shows the elution profile of calibration standards of given molecular weight and Stokes radius. i, dextran (void volume); ii, albumin (67,000 Da, 35.5 Å); iii, ovalbumin (43,000 Da, 30.5 Å); iv, chymotrypsinogen A (25,000 Da, 20.9 Å); and v, ribonuclease A (13,700 Da, 16.4 Å).
Figure 2: Laser-mediated UV cross-linking of the HIV-1 integrase to the U5 LTR. Integrase was incubated with the 21-bp duplex U5 LTR (25 nM) for 10 min at 21 °C prior to exposure to a 5-ns pulse of 266 nm UV light, as stated under ``Experimental Procedures.'' Cross-linked adducts were resolved from uncross-linked DNA by SDS-polyacrylamide gel electrophoresis. The autoradiograph shows part of a typical titration experiment performed by varying the integrase concentration between 0 and 3 µM.
Figure 3: Higher order assembly of oligomers in the cooperative binding of HIV-1 integrase to the U5 LTR. Data points represent the quantification (by phosphorimage densitometry) of several gels such as the one shown in Fig. 2. The sigmoidal curve, shown in semi-log form (panel A), represents the best fit of the data by non-linear least squares procedures in terms of the total HIV-1 integrase concentration and corresponds to a Hill coefficient of 1.90 ± 0.2 (see ``Experimental Procedures''). In panel B, the data are transformed, in terms of fractional saturation, to Scatchard form for a first-order (top) and second-order (bottom) reaction. In this case, the data points are corrected for the bound protein assuming a density of 10 monomers per LTR at saturation (as defined in Fig. 6). The Hill coefficient is 1.94 after transformation.
Figure 6:
Binding density analysis of stable
complexes. Stable complexes formed between the U5 LTR and integrase
were discriminated from uncomplexed DNA by nuclease digestion after a
5-min pre-incubation period at 37 °C. Digestions were performed for
30 min. Panel A, titrations were performed at two DNA
concentrations: 25 nM (left) and 100 nM (right). The positions of full-length (21-mer), cleaved
(19-mer), and free nucleotides (nt) are indicated. Panel
B, from left to right, titration curves obtained
at 12.5, 25, 50, and 100 nM U5 LTR. The broken line represents the theoretical binding isotherm in terms of the free
enzyme concentration. Inset, correlation observed between
nuclease and UV cross-linking titrations performed with 12.5 nM U5 LTR under identical conditions at 37 °C. Data points
represent UV cross-linking data. The solid line represents the
best fit of the corresponding nuclease titration. Panel C,
example of binding density obtained by plotting the total concentration
of integrase (IN) required to achieve
half-maximal saturation (protection) of the DNA substrate (at
= 0.5) against the concentration of DNA protected. The gradient (r
= 0.99) giving the binding density (N) is 10.5 ± 0.8, and the intercept, giving the free
enzyme concentration at half-maximal saturation, is 68 ± 24
nM.
The yield of cross-linked
product is dependent on two factors: the fraction of the total DNA
binding sites occupied by the integrase and a quantum yield factor
() defined by the photochemical reactivity of the
occluded site (as detailed under ``Experimental
Procedures''). As shown below, the titration curves obtained in
this way correlate well with those obtained by nuclease digestion
techniques when measured under identical conditions (see inset to Fig. 6B), thereby confirming that
remains constant over the entire fractional saturation curve. The
absolute value of
is however dependent on the
photochemical reactivity of the DNA sequence occluded by the bound
protein, which varies dependent on its base composition. This is seen
readily from the comparison of the maximal cross-linking obtained with
single-stranded homopolymers (Table 1). The photochemical
reactivity of the four nucleotide bases may be ordered (relative to
poly(dT) = 1) as T (1)
C (0.09) > A (0.04)
G (0). Thus, information on the binding affinity of the integrase is
obtained from the entire fractional saturation curve as described above
and not from the absolute value of DNA cross-linking.
The binding of both LTR and nonspecific sequences is notably weaker
in the absence of a divalent cation or when Mn is
replaced by Mg
. In the absence of a detectable
specificity for the cognate LTR sequences, we measured the affinity of
binding to poly(dT) as a function of the ionic strength (Fig. 4). Several features of this nonspecific interaction are
noteworthy. The binding affinity is markedly sensitive to the
concentration of monovalent cations. Consistent with the
polyelectrolyte theory(43) , log K
(the
observed association constant, in this case reported in terms of total
monomer) decreases linearly with log [NaCl] (in the range
0.05-0.2 M NaCl). For simple equilibria, this approach
may be used to extract information on the number of
DNA-phosphate:Na
ion pairs extruded by the binding of
a protein ligand to DNA. However, it should be kept in mind that other
factors, such as the influence of NaCl on the oligomeric state of the
integrase, may also affect the apparent affinity. Nonetheless, the data
suggest that binding to single-stranded DNA may involve an important
electrostatic contribution. Such ``salting-out'' effects on
binding were also observed at the higher concentrations of
Mg
, although an initial stimulation of binding was
observed with this cation. In contrast, no significant difference in K
was noted when the concentration of MnCl
was varied between 0.1 and 10 mM. This demonstrates that
the Mn
cation may stimulate binding at considerably
lower concentrations than have been reported previously for the optimum
enzymatic processing of the LTR substrates(27) .
Figure 4:
Log-log plot of the dependence of K on the concentration of mono- and divalent
cations for the binding of HIV-1 integrase to poly(dT)
.
The equilibrium association constant (K
) was
obtained from titrations performed by laser UV cross-linking (as
described under ``Experimental Procedures'' and in the text).
The dependence of this function on the concentration of NaCl (triangles), MnCl
(open circles), and
MgCl
(closed circles) is shown. NaCl titrations
were performed in the presence of 10 mM MnCl
. All
other titrations contained 50 mM NaCl.
Figure 5:
Resistance of the HIV-1 integrase-U5 LTR
complex to digestion by a 3`-specific nuclease (the nuclease protection
assay). Integrase (390 nM) was pre-incubated with the U5 LTR
(12.5 nM) under standard 3`-end processing reaction conditions
(detailed under ``Experimental Procedures'') in the presence
or absence of 250 nM competitor DNA (poly(dT))
for 10 min at 37 °C prior to the addition of 0.8 units of
phosphodiesterase I, and the incubation continued for a further 25 min.
Experiments were conducted with the U5 duplex (left panel)
bearing the radiolabel on the strand participating in cleavage. The
pre-incubation was performed in the absence (lanes 1 and 3) or presence (lane 2) of competitor DNA. In lane 3, competitor DNA was added after the pre-incubation
period and 5 min before the nuclease. In lane 4, the integrase
and nuclease were pre-mixed before addition of the U5 LTR. The
positions of the full-length (21-mer), 3`-processed (19-mer) strands
and free nucleotides (nt) are shown (the appearance of a
-3 base product with the U5 duplex is due to the 3`-end
processing of a quantity of -1 base substrate carried over from
the oligonucleotide synthesis after gel purification). Corresponding
activities with the U5 LTR in single-stranded form are shown in the right panel. Lanes 5-8 were with the U5a
(cleavage) strand, and lanes 9-12 were with the
complementary U5b strand (see Table 1). Lanes 5 and 9, incubations performed with integrase alone. Lanes
6-8, and 10-12, nuclease was added after
pre-incubation of the integrase with the DNA. Competitor DNA was added
either after (lanes 6 and 10) or during (lanes 7 and 11) the pre-incubation step or omitted entirely (lanes 8 and 12).
Figure 7:
Cooperative stimulation of the formation
of stable complexes by Mn. Integrase (350
nM) was pre-incubated with the U5 LTR (12.5 nM) for 5
min prior to incubation with phosphodiesterase for 10 min at 37 °C.
The plot shows the consequence of altering the concentration of
MnCl
(squares) and MnCl
(triangles) on the extent of DNA
protected.
A sigmoidal response, in terms of activity versus cofactor
concentration, is predicted to arise from a cofactor-induced
oligomerization of a protein where the oligomeric form interacts
preferentially with the substrate. In the case of the integrase,
Mn clearly enhances binding in a non-hyperbolic
fashion. The Hill coefficient (n
) measured for the
Mn
-induced transition observed in Fig. 7is
4.2 ± 1.0. A similar value (n
= 5.0
± 1.2) was obtained after the proteolytic removal of the His-tag
sequence from the recombinant protein, confirming that this property is
inherent to the integrase. Consequently, the kinetics of 3`-end
processing are also markedly non-linear with respect to the
Mn
concentration (data not shown). These data are
thus consistent with the cooperative binding mechanism and indicate
that the metal ion promotes the protein-protein interactions required
for the stable binding of the DNA substrate.
A further remarkable
feature of the transition is the concentration range over which it is
effected. The apparent binding constant (K) for
Mn
estimated from several such titrations, for both
His-tagged and factor Xa cleaved proteins, is 64 ± 10
µM. Whereas the optimum in vitro 3`-end
processing and disintegration activities of the integrase are obtained
at high (non-physiological) concentrations of Mn
,
comparatively poor activity is exhibited with
Mg
(27, 37, 45) . To further
investigate the role of the divalent cation in the formation of the
stable complex, the respective dissociation and 3`-end processing
kinetics of nuclease-challenged complexes were compared (Fig. 8). Complexes formed with Mn
are
considerably more stable than the corresponding complexes formed with
Mg
, exhibiting half-lives of approximately 13 and 1
h, respectively. However, the first-order rate constant obtained for
the 3`-end processing reaction is similar in the presence of either
cation. By eliminating the uncomplexed DNA, we follow only the
enzymatic activity of the stable complex. The rate constant obtained
then gives a direct measurement of k
, which,
under these conditions, was 0.046 ± 0.011 min
and 0.069 ± 0.008 min
for
Mn
and Mg
, respectively. Such low
rates are consistent with the delayed appearance of processed
3`-termini in virus-infected cells, which accumulates over a period of
several hours (1) , and is probably an intrinsic function of
the catalytic properties of the integrase. Nonetheless, we may conclude
that the overall higher efficiency of 3`-end processing in
Mn
observed in vitro may be explained simply
by the augmentation in the stability of the interaction with the
substrate and not to an inherent preference for this cation for
catalytic function.
Figure 8:
Comparison of the kinetics of dissociation
and 3`-end processing exhibited by stable complexes formed in the
presence of Mn or Mg
. Integrase
(390 nM) was pre-incubated with the U5 LTR for 3 min at 37
°C prior to addition of phosphodiesterase I. Incubations were
stopped at appropriate time intervals and analyzed by gel
electrophoresis to discriminate the percentage of the original
substrate remaining and the extent of 3`-end processing of the stable
complexes (as detailed under ``Experimental Procedures''). Left panel, kinetics of dissociation (circles) and
3`-end processing (triangles) in the presence of 5 mM MnCl
. Right panel, kinetics of dissociation (circles) and 3`-end processing (triangles) in the
presence of 5 mM MgCl
. The time reported for
dissociation is with respect to addition of the nuclease. For 3`-end
processing, this refers to total incubation time. Data points are
fitted by non-linear least squares analysis to the appropriate
first-order expression.
In this report, we have described the development of novel
procedures to probe the interaction between HIV-1 integrase and DNA in vitro as the preliminary step in a detailed study of the
structural architecture of these nucleoprotein complexes. Whereas the
first approach, laser UV cross-linking, detects binding via the
intimate protein-DNA contacts formed within an occluded photoreactive
site, for nonspecific binding it is indiscriminate as to the position
of that site on the DNA molecule. On the other hand, protection of the
DNA from hydrolysis by exonucleases detects principally the stable
contacts formed with the ends of the DNA substrate. The fact that
identical binding isotherms are generated by these two different
techniques, combined with the absence of intermediate length nuclease
digestion products, strongly implies that the entire DNA molecule,
including both ends, is occluded upon the formation of the stable
nucleoprotein complex. Since non-occluded regions and unbound ends
appear to be absent (both of which, if present, could potentially have
introduced anti-cooperative effects(40) ), the binding isotherm
is expected to be influenced only by the positive cooperativity
involved in this mechanism. In this case, the corresponding Hill
coefficient should be representative of the number of oligomers
participating in the assembly of the stable complex (see
``Experimental Procedures''). For both techniques, the
second-order binding observed with the 21-bp LTR is consistent with a
mechanism involving the cooperative dimerization of a high-order
oligomeric form of the protein. From a density of 10.5 ± 0.8
monomers per LTR and a Hill coefficient of 2, the oligomeric form of
the integrase in solution is estimated to be pentameric, though, within
the limits of reasonable experimental error, the average degree of
oligomerization (M) in solution may be extended to incorporate
tetrameric or hexameric forms (4 M
6). Clearly, the
resultant high-order nucleoprotein complex should exhibit a substantial
molecular mass (>300 kDa). This may, in part, provide an explanation
for the sedimentation properties of such complexes (13) and may
indicate the reason why such complexes are not particularly suited to
gel retardation studies.
We have evaluated the cooperativity for the
binding of short oligonucleotide duplexes only. In this limiting case,
the observed cooperativity may be restricted, by reason of the number
of bases occluded by a single oligomer, to a single cooperative
(apparent dimerization) event. Recent experiments have enabled us to
observe the complexes formed with larger DNA fragments directly by
electron microscopy. In the presence of Mn, large
clusters of bound integrase molecules, extending inwards from the DNA
ends, have been detected, indicating that the associated cooperativity
parameter may be quite large. (
)These findings appear to
comply with the extended, nucleosome-like DNase I footprints observed
for the interaction between the avian myeloblastosis virus integrase on
plasmid-borne LTRs(46) . Accordingly, the cooperativity
parameters will require further investigation with longer DNA
substrates. We may, however, infer from these data, that in the absence
of other cellular factors, the integrase oligomer binds contiguously to
naked DNA rather than to distinctly separated sites. For the reasons
explained above, the binding of the short LTR substrates thus presents
itself as a useful model system with which to examine binding under
conditions of limited cooperativity.
In agreement with several
previous
reports(6, 11, 13, 14, 15, 16, 17) ,
no sequence specificity was observed in the binding of the viral LTR
nor was a preference apparent for the double-stranded substrate in UV
cross-linking titrations. Stable complexes are, however, not formed
with single-stranded DNA, suggesting the underlying mechanism of
binding may be different for these two forms of DNA. Our recent data
suggest that the binding density of integrase for single-stranded DNA
(poly(dT)) is considerably lower than that observed for the U5 LTR
duplex. ()We expect that the differential dissociation rates
obtained with such complexes may reflect this observation. We are
currently investigating this in more detail.
We have observed that
Mn can stimulate binding at concentrations between
one and two orders of magnitude lower than that previously reported as
the optimum concentration for 3`-processing for both the HIV-1 (27) or Moloney murine leukemia virus (45) integrases.
An explanation for this disparity could be in the existence of two
distinct divalent cation binding sites on the integrase oligomer: one
involved in mediating protein-protein interactions, and hence
stimulating cooperative binding, and another incorporated at the
catalytic site. Indeed, a region distinct from the catalytic site, the
HHCC finger domain, has recently been implicated in the oligomerization
of the HIV-1 integrase in the presence of divalent cations, a property
further shown to be essential for stable complex
formation(26) . Such interactions probably underlie the
cooperativity of DNA binding, much as the coordination of
Zn
by the
HX
CX
CX
C
motif (47) of the T4 bacteriophage gene 32 protein (gp32) has
been inferred to stimulate cooperative binding to single-stranded DNA
via enhanced protein-protein interactions(48) . The presence of
Zn
is not necessary to obtain cooperative binding of
gp32 but stimulates it approximately 30-fold(48) . By analogy,
the coordination of a metal ion by the integrase HHCC motif may
stabilize a particular conformation of this subdomain and thereby
enhance a property already inherent in the integrase to self-associate.
Although the precise Mn
interaction site remains to
be resolved, the general assumption would appear to be correct. Stable
complexes form in the absence of the divalent cation but only
efficiently at much higher protein concentrations.
Thus,
the cation appears to favor the polymerization process by a mechanism
that lowers the activation energy barrier to this association. A
similar observation has been made for the self-association equilibrium
of the RecA protein, which is stimulated in the presence of
Mg
(49) . The capacity of such proteins to
associate in the absence of DNA appears to be a recurring theme for
proteins that are known to form direct and functional interactions in
the context of a highly structured DNA-protein complex.
We have
demonstrated that the ternary nucleoprotein complex formed with
Mn is considerably more stable than the corresponding
complex formed with Mg
. The absolute rate of 3`-end
processing exhibited by the stable complex is, however, similar with
either cation. This allows us to deduce that the marked preference
exhibited in vitro for Mn
is simply a
consequence of the enhanced stability of binding to the LTR.
Furthermore, it was reported (after submission of this manuscript) that
an increase in the efficiency of the 3`-end processing of the LTR
substrate in Mg
can be achieved either by altering
the solution conditions (notably to include PEG) (50) or by
increasing the length of the DNA substrate(51) . In both cases,
these effects can be explained simply by a concommitant stimulation in
the binding interaction with the DNA substrate and add further evidence
to support our conclusion that the bias for Mn
in
standard in vitro reactions operates at the level of DNA
binding and not catalysis. The requirement for Mn
is
not seen with the pre-assembled viral nucleoprotein complex purified
from infected cells, which undergoes efficient integration in vitro in the presence of Mg
(52) . Clearly,
since the preference exhibited for Mn
in vitro is to stimulate efficient binding of the recombinant protein to
the DNA substrate, the necessity for this cation has already been
circumvented when using the preformed integration machinery. Other
cellular components, potentially host and/or viral accessory proteins,
may be required to regulate the assembly of the authentic nucleoprotein
complex. In this respect, the presence of several viral proteins other
than integrase has been noted in the pre-integration
complexes(5) . In the absence of such factors, the
physiological role of the metal ion-induced interaction between
integrase oligomers remains unclear. It is possible that the integrase
binds to the viral DNA ends as a structured and highly stable
aggregate. It also appears likely that some interaction should take
place between integrase molecules bound at the extreme ends of the
viral genome to allow the coordinated integration of the viral termini
at a discrete target site in the host genome. This is accomplished in
the analogous transposition mechanism of bacteriophage Mu by
coordination of distant MuA binding sites into a tetrameric MuA
synaptic complex(53) . By comparison, a direct interaction
between the distally bound integrase complexes may bring the LTR
termini together to form the legitimate strand-transfer complex.
Indeed, our recent observations
and those of Mazumder et al.(33) suggest that the assembly of complexes is
not restricted to lateral, cooperative interactions, but may also
involve intermolecular interactions between bound complexes.
Undoubtedly, a greater understanding of the process of retroviral
integration will necessitate a more detailed exploration of the
structural architecture and cofactor requirements of the functional
integrase nucleoprotein complexes. We expect that the approaches
outlined in this report will greatly assist these studies.