(Received for publication, April 21, 1994; and in revised form, November 29, 1994)
From the
The human spuma retrovirus or foamy virus integrase (HFV IN) is
an enzymatically active protein consisting of domains similar to other
retroviral integrases: an amino-terminal HH-CC finger, a centrally
located region with the conserved D, D-35-E protein motif required for
catalytic activity and oligomerization, and at least one DNA binding
domain implicated in the 3` DNA processing activity and integrase.
Recombinant, purified HFV IN protein carrying 10 histidine residues
displays a site-specific endonuclease, an integrase, and a disintegrase
activity with oligonucleotide substrates that mimic the viral long
terminal repeat (LTR) ends. Site-directed mutagenesis of conserved HFV
IN residues of the catalytic domain had increased endonuclease and
disintegrase activities. Deletion mutants at both ends of the HFV IN
protein were generated, purified, and characterized. Unexpectedly, it
was found that the HFV integrase and disintegrase activities require an
intact NH-terminal sequence and that COOH-terminal
deletions led to an increase in disintegrase activity. The HH-CC finger
of HFV IN was exchanged with that of the human immunodeficiency virus-1
(HIV-1) IN protein. The resulting chimeric IN had a 3` processing
activity that utilized the HFV LTR instead of the HIV LTR, indicating
that the central domain is crucial for substrate recognition.
Functional complementation of the amino-terminal deletion mutant of HFV
IN was achieved by a carboxyl-terminal deletion mutant of the chimeric
IN, resulting in high levels of integrase activity.
Retroviral integration is a key step during viral replication
that at least in vitro requires only one protein, the
integrase (IN), ()and involves the covalent insertion of a
DNA copy of the viral DNA genome into a host cell chromosome (for
reviews, see (1, 2, 3, 4, 5, 6) ).
The IN proteins are encoded by the pol genes that in most
retroviruses are synthesized as gag-pol fusion proteins from
which the integrases are generated by proteolytic processing. Early
work indicated that the Rous sarcoma virus IN protein can form
dimers(7) . Recent data from several groups show that IN forms
dimers and/or
oligomers(8, 9, 10, 11, 12, 13) .
Three enzymatic activities have been shown to reside in retroviral IN
proteins. (i) A site-specific endonuclease removes the two terminal
nucleotides at the end of each long terminal repeat (LTR) prior to
integration. (ii) A DNA strand transfer activity covalently joins the
3` recessed termini of each strand to the 5` ends of target host DNA.
(iii) Recently, a third activity, the disintegrase, was found to be
detectable in vitro and is responsible for part of the reverse
reaction of integration, also called disintegration. During the
disintegration reaction, the LTR-DNA target junction is cleaved,
thereby releasing the LTR and covalently joining the nick between the
DNA strands. The disintegrase activity can be monitored by using
appropriate branched or Y-shaped model substrates(14) .
Retroviral integrases consist of several
domains(8, 9, 15, 16, 17, 18) .
The amino-terminal HH-CC finger region has been reported to bind
Zn ions stabilizing the conformation of that
domain(19, 20) . A number of apparently conflicting
reports on the functional role of the HH-CC finger domain have been
published. Recently, it was reported that this domain is capable of
binding the LTR deoxyoligonucleotides with high affinity in the absence
of divalent cations(13) . It was found that wild type
tetrameric HIV-1 IN molecules can be cross-linked to the U5 LTR,
whereas an IN form that had been mutated in one of the Cys residues of
the finger domain showed reduced oligonucleotide binding, indicating
that the HH-CC finger may be a DNA binding domain for the 3` processing
activity under the conditions used (13) . In contrast,
efficient DNA strand transfer activity was restored to a mutant Rous
sarcoma virus integrase lacking the HH-CC domain by fusion to various
short peptides(18) .
The centrally located, catalytic domain with the characteristic D, D-35-E motif overlaps and coincides with the dimerization or tetramerization domain(8, 9, 12, 13) . The carboxyl-terminal regions of HIV-1 IN were reported to contain a low affinity or aspecific DNA binding domain(16, 17) .
In view of the distinct human foamy virus (HFV) IN sequence, we are
interested in defining the minimal sequences required for the
individual enzymatic activities of the human spuma or foamy virus
integrase. The HFV IN protein has been reported to have a size of 39
kDa in wild type infected cells(21) . Recombinant HFV IN
protein expressed from Escherichia coli that carries a tag of
10 His residues at the amino terminus was purified by Ni chelate affinity chromatography and shown to possess all three
enzymatic activities of the intact HFV integrase(21) . The
41-kDa recombinant HFV IN protein has a relatively high degree of
homology compared to other retroviral IN(22) . While most of
the amino acid residues conserved in other retroviral IN proteins are
also invariant in the HFV integrase, there are some features and motifs
that set it apart(23) . In particular, the HFV HH-CC domain
itself and adjacent sequences are longer and different from those of
other complex human retroviruses. In an effort to analyze and
characterize the HFV IN protein, we used deletion mutants,
complementation analysis of differently shortened mutant IN proteins,
site-directed mutagenesis, and domain swapping to define the functions
of the HFV IN domains. Our results show that for full enzymatic
activity of the HFV integrase, sequences flanking the HH-CC finger
domain were required.
The truncated forms of the HFV IN
protein were synthesized in an analogous way as described above for the
recombinant intact HFV IN protein. A NdeI site was created at
the 5` end and a BamHI site at the 3` end. The sequences of
the primer pair used for 1-34 were
5`-ACAAAAACATATGCTTCAAG-3` as sense primer and
5`-CAGGGTCATCATATGAA-AGGA-3` as antisense primer (HFV genomic positions
5429-6598); for
1-74, sense primer
5`-AGCGCCCGCCATATGCAACAGTGTTTAATC-3`, antisense primer
5`-CAGGGTCAT-CATATGAAAGGA-3` (genomic positions 5658-6598);
323-366, sense primer 5`-CAGTATAATTGGATCCTTCTG-3`, antisense
primer 5`-CTCTCCTGGATCCATTGGCC-3` (genomic positions 5429-6410);
297-366, sense primer 5`-CAGTATAATTGGATCCTT-CTG-3`,
antisense primer 5`-AGTCCTTGGATCCAACACCT-3` (genomic positions
5429-6334). To construct the double mutant
1-34/
323-366, DNA of pET16b
1-34 and
pET16b
323-366 vectors were digested with EcoRI. The
6020-bp DNA backbone of pET16b
1-34 containing the
NH
-terminal deletion was ligated to the 540-bp
COOH-terminally truncated fragment of pET16b
323-366.
Figure 1:
Structure of oligonucleotides
substrates of the HFV and HIV-1 integrase. Substrates of disintegrase
of HFV (A) and HIV (D), integrase of HFV (B)
and HIV (E), and endonuclease of HFV (C) and HIV-1 (F). The substrates in panels B and C were
derived from the 3` end of the HFV U5 LTR (E) and the HIV U5
LTR (F). The branched substrates A and D represent random target sequences shown as horizontal strands; the
3` end of the HFV U5 LTR region and the 3` end of the HIV U5 LTR region
(I1, 21 nucleotides and I4, 33 nucleotides in length) are shown
diagonally. The asterisks mark the P label. G, autoradiogram of the reaction products of the enzymatic
activities of the HFV IN. The substrates shown in A-C were incubated in the absence (lanes 1, 3, and 5) and in the presence of the IN protein for 90 min under
standard conditions. Lane2, endonuclease with
substrate C; lane4, disintegrase with
substrate B; lane6, integrase activity with
substrate A; the brackets mark the integration
products. For further details, see ``Experimental
Procedures.''
To determine the role of the different domains for the three enzymatic activities of the HFV integrase, we constructed deletions at either terminus of the IN protein to analyze which of the three enzymatic activities, if any, was lost or perturbed.
The mutated
versions of the HFV IN were expressed in E. coli and purified
by Ni chelate affinity chromatography and cation
exchange chromatography (21) to near homogeneity. Subsequently,
the purified HFV IN proteins were assayed for endonuclease,
disintegrase, and DNA strand transfer activity by using the appropriate
oligonucleotide substrates shown in Fig. 1. The oligonucleotides
are model substrates derived from the termini of the LTRs of either the
HFV or HIV-1 DNA genome for assaying the 3` processing and integrase
activity. The branched, Y-shaped substrates consisting of four
different oligonucleotides were used for assaying the disintegrase
activity of HFV or HIV-1 IN (Fig. 1, A and D).
Quantitation of substrates and reaction products was done by
determining the intensities of individual bands of the same lane only
separately after electrophoresis. Total concentration of substrates and
reaction products were set to 100% (see ``Experimental
Procedures''). Fig. 1G shows autoradiograms of the
reaction products of the three enzymatic activities of the HFV IN
protein with the corresponding substrates C, A, and B
under standard conditions. The result shows that the three oligomers
served as substrates for the individual enzymatic activities in the
presence (lanes 2, 4, and 6) of the HFV IN
protein but not in its absence (lanes1, 3,
and 5).
Figure 2:
Structure of point mutations in highly
conserved regions of the HFV IN protein and enzymatic analysis. Panel A schematically presents the HH-CC finger domain, the
positions of point mutations in two different centrally located DYIG
and SDQG motifs of the catalytic domain, and the resulting enzymatic
activities given in percentage of activity of the intact HFV IN protein
set to 100% (for details of quantitation, see ``Experimental
Procedures''). Panel B shows the autoradiograms of the
endonuclease, panel C the disintegrase, and panel D the strand transfer reaction: wild type HFV IN (lane 1),
I106T106 (lane 2), S159
T159 (lane 3), and
S159/N161
T159/Q161 (lane 4).
The second conserved region analyzed was the TDNG motif, which is highly conserved among retroviral integrases. Point mutations of this motif led to either a complete loss or a drastic reduction of the 3` processing and integrase activities as reported for HIV-1(23, 28) . The HFV integrase differs at two positions from this motif (Fig. 2A); therefore, mutagenesis from the HFV sequence to the retroviral consensus sequence was carried out in two subsequent steps. First, the Ser residue at position 159 was changed into Thr and the enzymatic activities were determined. Subsequently and additionally, Gln-162 was changed to an Asn residue. The three enzymatic activities were separately determined for the two mutant proteins. The first amino acid exchange had nearly no effect on the endonuclease and integrase activities, but the disintegrase increased to 161%. The second additional amino acid mutation caused no major change in the integration and endonuclease activity compared with those of the single exchange mutant. Both the nucleolytic cleavage and the disintegration activity increased to 118 and 143%, respectively, of that of the intact HFV IN (Fig. 2, panels A and D).
The results obtained demonstrate that point mutations at defined positions can lead to a strong enhancement of disintegration but to reduce DNA cleavage activity. One point mutation (I106T) only slightly changed the disintegrase and affected the endonuclease and integrase activities, reducing the integrase activity to almost background values, whereas the endonuclease was still detectable at 34% of the intact HFV IN (Fig. 2, A and B).
Figure 3:
Schematic representation of locations
of terminal deletions of the HFV IN protein and analysis of the
corresponding reaction products. Topline in part
A shows full-length HFV integrase domains. The resulting
activities of the three enzymatic activities of the deleted HFV IN
protein are compiled in the right part of panel A;
intact HIV IN activities were set to 100%. The purified mutant proteins
were assayed as described under ``Experimental Procedures.'' Panels B-D, autoradiograms of endonuclease,
disintegrase, and integrase: intact HFV IN (lane 1),
1-34 HFV IN (lane 2),
1-74 HFV IN (lane 3),
323-366 HFV IN (lane 4), and
297-366 HFV IN (lane
5).
To determine the influence of the
residues located at the opposite end of HFV IN, two COOH-terminally
truncated HFV IN proteins were constructed, expressed, and purified.
Analysis of the corresponding enzymatic activities showed that the HFV
IN mutant (323-366) that lacks 43 carboxyl-terminal residues
up had an 3` processing activity of 32% compared to that of full-length
HFV IN (Fig. 3B). The integrase was at a low level but
clearly detectable. Strikingly, the disintegration activity of
323-366 increased to 230% (Fig. 3, A and B).
In another terminally truncated HFV IN mutant
(297-366) studied, 69 amino acids were deleted from the
COOH-end that should include a presumed DNA binding domain located at
approximately the same region as in HIV IN(16, 17) .
The solubility of the mutant
297-366 protein was lower
compared with the intact and the other truncated forms of HFV IN
protein. The endonuclease activity was 11% compared with that of the
intact IN (Fig. 3B); the integrase activity was barely
detectable, while the disintegrase had approximately half of the
activity of the
323-366 deletion mutant but was still active
at about the same level as intact HFV IN (Fig. 3C). An
IN mutant that had both ends shortened (Fig. 3, bottomline of panel A) did have detectable
disintegrase activity (8%) but had less than 3% of 3` processing and
strand transfer activities of the intact HFV IN (data not shown).
The results show that a full-length HFV IN protein is required for
any DNA strand transfer activity. The endonucleolytic activity was
decreased by truncating either the NH or the COOH terminus.
In contrast to HIV-1, the disintegration activity of the HFV IN
requires an NH
-terminal region upstream of the HH-CC finger
domain for full activity. It is remarkable that small deletions at the
COOH terminus increased the disintegrase whereas, the 3` processing
activities were negatively influenced by deletions at either end of HFV
IN.
Figure 4:
Endonucleolytic cleavage activity of the
chimeric integrase HFV HH-CC/HIV HH-CC IN protein. Assays were
done at different pH values; intact HFV IN activity was set to 100%. Panel A, Mg
, pH 6.2, HIV LTR substrate E (lane 1); Mg
, pH 6.2, HFV LTR substrate B (lane 2); Mg
, pH 7.6 substrate E (lane
3); Mg
, pH 7.6, substrate B (lane 4);
Mn
, pH 6.2, substrate E (lane 5);
Mn
, pH 6.2, substrate B (lane 6);
Mn
, pH 7.6, substrate E (lane 7); and
Mn
, pH 7.6, substrate B (lane 8). For the
structures of the different substrates, see Fig. 1. In panel
B, the endonucleolytic cleavage activities are
compiled.
Figure 9:
Determination of the optimal concentration
of KCl, Mn, and of the pH maximum of the HFV IN
endonuclease activity. The assays were done as described under
``Experimental Procedures'' under standard assay
conditions.
Figure 5:
Integrase activity of the chimeric HFV
HH-CC/HIV HH-CC IN protein. Standard assay conditions were as
described under ``Experimental Procedures'' with different
substrates and divalent cations. Panel A, Mg
and HIV U5LTR substrate F (lane 1), Mn
and HFV 20-mer substrate C (lane 2), Mn
and substrate F (lane 3), Mg
and
substrate C (lane 4). The brackets mark the
integration products longer than the 18-nucleotide substrate. Panel
B, both preprocessed substrates C and F were
utilized in the DNA strand transfer reaction in the presence of either
Mn
or Mg
with the HFV DNA as the
preferred substrate. Intact HFV IN activity was set to
100%.
Figure 6:
Disintegrase activity of the chimeric
HFV/HIV-1 IN protein. Assays were performed as described under
``Experimental Procedures'' in the presence of
Mn. Panel A, chimeric HIV-1 HH-CC finger IN
activity with the branched HIV substrate D (lane 1), HFV
substrate A (lane 3), intact HFV IN activity with HIV
substrate D (lane 2), and HFV substrate A (lane 4);
for the structure of the Y-shaped substrates, see Fig. 1. Panel B, disintegrase activity.
Figure 7: Complementation experiments of enzymatic activities of HFV IN mutant proteins. The purified mutant proteins were mixed at equimolar amounts and assayed as described under ``Experimental Procedures.'' The resulting enzymatic activities of the deleted HFV IN protein mixtures are compiled at right. Integrase, endonuclease, and disintegrase activities are given as the percent of wild type activity: +++, 50-100%; ++, 10-50%; +, 3-10%; -, <3%.
Figure 8:
Enzymatic activities of intact and mutant
chimeric HIV/HIV IN proteins and functional complementation of the HFV
IN deletion mutants by the chimeric HIV/HFV IN mutant proteins. Assays
were done under standard conditions. The resulting enzymatic
activities of the deleted HFV IN protein mixtures are compiled in the right part of panel A. Panel B, chim HIV/HFV
1-334 (lane 1), chim mutant HIV/HFV 291-334 (lane 2), chim HIV/HFV
291-334 plus HFV
1-34 (lane 3), chim HIV/HFV
291-334 plus
HFV
1-74 (lane4), chim HIV/HFV
265-334 (lane5), chim HIV/HFV
265-334 plus HFV
1-34 (lane6),
and HIV/HFV
265-334 plus HFV
1-74 (lane7). Integrase and disintegrase activities are given as
the percent of wild type activity: +++, 50-100%;
++, 10-50%; +, 3-10%; -, <3%.
Functional complementation of
the HIV/HFV IN mutant 265-334 was achieved with both
NH
-terminal HFV IN deletion mutants
1-34 and
1-74 (Fig. 8). This result is remarkable, since
neither the short nor the longer HFV IN protein was active by itself.
Thus, functional complementation of the integrase activity of an
inactive chimeric HIV/HFV IN deletion mutant was efficient and
accomplished by mixing it with one of the NH
-terminally
truncated HFV IN proteins.
The HH-CC finger does influence the pH
range of the endonucleolytic cleavage activity, since the chimeric
enzyme was active at pH 7.6 in the presence of Mn.
The strand transfer activity of the chimeric IN protein was active with
both the HIV and HFV preprocessed LTR substrates and both of the two
divalent cations. This result is consistent with the known fact that
integrases generally lack DNA target specificity. This is best
illustrated by comparing the disintegrase activities of the different
mutant proteins, which seem to be unmasked by COOH-terminal deletions.
In Fig. 9(A-C), the results of determining
the optima for KCl concentration, divalent cation, and pH for the 3`
processing activity are summarized. It was found that the optimal KCl
concentration was broad ranging from 20 to 160 mM (panel
A). The optimal range for Mn was between 2 and 8
mM (panel B). The pH optimum of the HFV endonuclease
was found at 6.2; at pH 7.6 activity, was not detectable (panel
C).
The results of this report, which include the analysis of point and deletion mutations, indicate that the in vitro DNA nucleolytic cleavage and strand transfer activities of the HFV IN protein cannot be separated into independent domains. This is consistent with data on the properties of HIV integrase from several groups(8, 9, 15) . Concerning the HFV IN point mutations with increased disintegrase activity, it is interesting that particular HIV IN mutants also showed elevated disintegrase activities surpassing those of wild type HIV-1 IN(23) .
Close examination and comparison of the three enzymatic activities
of the HFV IN deletion mutants with those of HIV-1 IN revealed clear
differences. Analysis of truncated HIV-1 IN proteins showed that even a
short deletion of 5 residues at both the NH and COOH
termini (8, 9, 19) abolished both
endonuclease and integrase activities of HIV-1 IN, while disintegrase
activity was still detectable after deleting of up to 50 amino acid
residues at both ends, although at a level lower than that of the wild
type IN. In contrast, the HFV IN deletion mutants displayed a different
pattern of activities. It is noteworthy that the
1-34 HFV IN
mutant lost DNA strand transfer activity but retained detectable
activity, not only of 33% disintegrase but also of 13% of the 3`
processing activity. This HFV IN mutant is directly comparable to the
HIV-1
N5 mutant(9) , since the deletions in both mutants
are located 4 and 3 residues upstream of the first His residue of the
corresponding viral HH-CC domains. The lower activity of the HFV
1-34 mutant indicates that for full activity the HFV
disintegrase requires not only the HH-CC finger but also upstream
sequences of this domain encompassing the major part of the residues
deleted. Removal of additional 40 residues as in mutant
HFV
1-74 led to a complete loss of detectable levels of the
three enzymatic activities, including the disintegrase activity. This
is in contrast to the corresponding
N38 mutant HIV IN that still
retained residual disintegrase activity(17) . The results
suggest that the HH-CC finger and flanking sequences of the HFV IN
protein contribute substantially to full disintegrase activity.
On
top of this difference with NH-terminal deletion mutant IN
proteins, HFV IN COOH-terminal deletion mutants showed properties
completely different from the corresponding HIV mutants. It was found
that COOH-terminally deleted HFV IN mutants showed a dramatic increase
in disintegrase activity that even surpassed the levels of the intact
HFV IN protein. It has been reported that 50-91 carboxyl-terminal
residues of HIV-1 IN could be deleted with some disintegrase activity
retained(17) . A 2-3-fold increase over the activity of
the intact HFV IN protein as in the case of HFV IN mutants was not
found for HIV IN deletion mutants(19) .
We assume that several factors contribute to the difference between the HFV and HIV IN activities. These factors include sequence differences of the individual domains of the retroviral IN analyzed and, in addition, functional redundancies that are likely caused by the oligomeric nature of the IN proteins.
As to the amino terminus, there are 32
additional residues in the HFV IN that include 26 residues upstream of
the metal finger domain and 6 extra residues within the finger region.
Compared to that of HIV-1, the HFV IN sequence encodes 7 additional
residues directly downstream of the C(X)C motif of
the metal finger in the region preceding the catalytic domain. The
higher disintegrase activity of the COOH-terminally truncated HFV IN
mutant
323-366 might be explained by assuming that the
mutated IN protein has a better capacity to recognize and bind to the
branched DNA substrates because of higher accessibility of the
substrates to the DNA binding domains(16, 17) . The
HFV mutant
297-366 that probably lacks the aspecific DNA
binding domain of the COOH-terminal domain has, nevertheless, full
disintegrase activity. Based upon this result, it is concluded that
another HFV IN region may function as DNA binding domain at least for
this mutant.
The chimeric HIV/HFV IN protein that contains the HH-CC
finger domain of HIV-1 had relatively high levels of integrase activity
with the preprocessed substrates but comparatively low disintegrase
activity with the HIV and particularly with HFV LTR substrates (Fig. 8B), again indicating that for full activity the
HFV disintegrase requires not only the HH-CC finger but also sequences
flanking this domain. Since the chimeric HIV/HFV IN endonuclease was
active with the HFV LTR substrate only, it appears that the HH-CC
finger does not play an essential role in the recognition of the DNA 3`
processing substrate. The chimeric protein had about 60% of the
endonuclease activity of that of the intact HFV IN. Thus, the chimeric
IN protein is functionally equivalent to at least the integrase
activity of the wild type HFV IN protein. This result is agreement with
data on functionally active Rous sarcoma virus mutants that contain
foreign peptide sequences instead of the HH-CC finger(18) . On
the basis of these results, we conclude that the specificity of the DNA
cleavage reaction is not determined by the HH-CC finger domain.
Instead, the central region of the HFV IN is responsible and essential
for the site-specificity of the endonucleolytic cleavage reaction.
While the precise boundaries of the central catalytic domain of HFV IN
remain to be defined, it seems that in analogy to other well
characterized IN proteins it extends from the
C(X)C motif approximately to residue 297. The
extended pH range of the chimeric HIV/HFV IN also indicates that the
HH-CC finger domain is in close contact to the catalytic center of the
active oligomeric forms of the IN protein. However, the precise role of
the NH
-terminal IN sequences that include the
H(X)
H motif (n = 3-6,
including IN sequences of retrotransposons) is still not fully
understood.
The disintegrase activity of the chimeric IN was 2-fold
higher with the HIV-1 LTR substrate than with the HFV LTR. Thus, the
part of the chimeric IN that contributes to HFV disintegrase activity
appeared to reside more to the amino-terminal part when compared to the
HIV-1 IN protein consistent with the results of the
NH-terminal HFV IN deletion mutants.
To analyze why HFV
IN mutant proteins deleted at either end were not capable of
complementing each other in integrase activity as reported for the
corresponding HIV IN mutants(8) , a chimeric HIV/HFV IN
COOH-terminal deletion mutant that by itself had no integrase activity
was separately complemented by two inactive HFV IN mutants that had
different truncations at the NH terminus. The result of the
positive functional complementation indicates that DNA binding sites
can be supplied in trans. In addition, the HH-CC finger domain
seems to be active when present in trans. This did not hold
for the disintegrase activity, which was not restored, probably because
of the very low level of this activity of the intact chimeric IN
protein itself.
Close inspection of the data of the complementation
assays revealed that again the amino-terminal deletions behave
differently when compared to those of the carboxyl-terminal truncated
forms of the HFV IN protein. The low efficiency of complementation of
different HFV deletion mutants is due to the requirement of the
NH-terminal sequence of the HH-CC finger domain and those
upstream of it. This was proven by the functional complementation of
the HFV mutant
1-74 by the chimeric IN mutant
297-366 that restored integrase activity to high levels. In
addition, the endonucleolytic activity of HFV IN point mutant I106T was
reconstituted to nearly wild type activity by complementation with
either NH
- or COOH-terminal HFV IN mutants (data not
shown).
Our results are in agreement with those reported for the MLV IN protein (28, 29) . MLV IN proteins with mutations in the HH-CC region were inactive in integrase function but not in disintegration activity (30, 31, 32) . In fact, the HFV IN protein sequence has a higher degree of homology to that of MLV compared to that of HIV-1 IN as reported previously(33) .