From the Department of Biochemistry and Molecular Biology,
University of Medicine and Dentistry-New Jersey Medical School,
Newark, New Jersey 07103
The existence of retroviral reverse
transcriptases as monomers or dimers is rather intriguing. A classical
example of the former is murine leukemia virus reverse transcriptase
(MuLV RT), while human immunodeficiency virus type 1 (HIV-1) RT
represents the latter. A careful scrutiny of the amino acid sequence
alignment of the two enzymes pinpoints the region tentatively
responsible for this phenomenon. We report here the construction of a
chimeric enzyme containing the first 425 amino acid residues from the
N-terminal domain of HIV-1 RT and 200 amino acid residues from the
C-terminal domain of MuLV RT. The chimeric enzyme exists as a monomer
with intact DNA polymerase and RNase-H functions.
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INTRODUCTION |
Human immunodeficiency virus reverse transcriptase is a
heterodimeric enzyme comprised of 66- and 51-kDa subunits. The p51 subunit of the heterodimer is derived from the p66 subunit due to
proteolytic cleavage and removal of the RNase-H domain (1-3). Surprisingly, the folding of p51 is remarkably distinct from that of
p66. The polymerase domain of p66 folds into an open cleft while that
of p51 assumes a closed conformation (4). It has been proposed that the
monomeric form of p66 also exists in a closed conformation
topologically similar to that of p51 and that the polymerase domain
opens up into a large cleft only upon dimerization with p51 (5). The
three distinct contact points between p51 and p66 have been discerned
from the crystal structure resolution, namely the connection domain,
the tip of the finger subdomain of p51, and the extended thumb region
that supports the RNase-H region of p66. The asymmetric heterodimer
observed in HIV-11 RT is also
found in many other retroviruses, namely, FIV, SIV, HIV-2, and EIAV.
Interestingly, murine leukemia virus reverse transcriptase is
functionally active as a monomer. Although, both MuLV RT and HIV-1 RT
exhibit similar enzymatic properties, their architectural organization
vastly differs as the former exists as a monomer whereas the latter is
a heterodimer. It has not yet been possible to elucidate the region in
the sequences of these two reverse transcriptases that necessitates
dimer formation in HIV-1 RT while posing no such requirement in MuLV
RT. In our effort to identify the region in MuLV RT (which might be
missing in HIV-1 RT) that obviate the need for dimerization of this
enzyme, we compared the primary sequence of MuLV RT with that of HIV-1
RT. We observed three major motifs containing from 11 to 28 amino acids, which are present in MuLV RT but lacking in HIV-1 RT. We therefore speculated that the conspicuous absence of these motifs in
the sequence of HIV-1 RT may have imposed the dimeric requirement for
catalytic function. To ascertain this postulation we constructed a
chimeric HIV-1 RT containing two of these motifs and the entire RNase-H
domain from MuLV RT. The resulting chimeric HIV-1 RT was found to be a
monomeric enzyme with intact polymerase and RNase-H functions. These
studies form the subject matter of the present investigation.
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MATERIALS AND METHODS |
DNA restriction enzymes and DNA modifying enzymes,
Taq DNA polymerase for polymerase chain reaction (PCR),
along with PCR buffer and HPLC-purified dNTPs were purchased from
Boehringer Mannheim. Fast flow chelating Sepharose
(iminodiacetic-Sepharose) for immobilized metal affinity chromatography
was purchased from Amersham Pharmacia Biotech, and
32P-labeled dNTPs and ATP were the products of NEN Life
Science Products. Synthetic oligomeric primers were obtained from the Molecular Resource Facility of University of Medicine and Dentistry-New Jersey Medical School and were further purified by polyacrylamide gel
electrophoresis (6). All other reagents were of the highest available
purity grade and purchased from Fisher, Millipore Corp., Boehringer
Mannheim, and Bio-Rad.
Plasmid and Clones--
Expression vector pET-24b, pET-28a and
Escherichia coli expression strain BL21 (DE3) were obtained
from Novagen. The HIV-1 RT, MuLV RT, and Klenow fragment expression
clones (pET-28a-RT66, pET-28a-RT51, and
pET-28a-MRT) constructed in this laboratory were used for PCR
amplification and construction of the chimeric HIV-1 RT (7-10).
Expression and Isolation of Wild Type HIV-1 RT, MuLV RT, and
Klenow Fragment--
Purification of Moloney murine leukemia virus
reverse transcriptase (10) and human immunodeficiency virus type 1 reverse transcriptase (11) and Klenow fragment (12) were carried out from recombinant clones according to published protocols. The homodimeric p66/66 HIV-1 RT also contains species of the heterodimeric p66/51 generated during the process of purification by bacterial proteolytic cleavage.
Construction and Expression of Chimeric HIV-1 RT--
The
chimeric HIV-1 RT containing the polymerase domain of HIV-1 RT and the
RNase-H domain of MuLV RT was constructed as follows. The 582-base pair
sequence containing the entire RNase-H domain of the MuLV RT gene
spanning from codons 471 to 670 was amplified by PCR (13). The plasmid
pET-28a-MRT containing the coding region for the full-length MuLV RT
was used as the template for PCR amplification. The sequences of the up
stream primer and the down stream primer were 5'-GTG GTA GCC CTG
GTAC CAA AAC CCG GCT ACG-3' and 5'-TAT AGG GACC CTC
GAG TGA CAT TAA CCT ATA, respectively. In the same step, we
introduced the KpnI restriction site at the 5'-end and XhoI site at the 3'-end of the RNase-H coding sequence of
MuLV RT. Using these restriction sites the amplified fragment was
restriction digested and ligated with the KpnI- and
XhoI-digested pET-24b-RT51 expression cassette to construct
the pET-24b-CRT71 clone. This construct contains a 5' T7
promoter and codes for the chimeric HIV-1 RT with the metal binding
His-Tag sequences at the C-terminal region. The screening of clones for
the appropriate insert (1858 base pairs) was carried out in HB101. The
positive clone was introduced into E. coli BL-21(DE3)pLysS
for expression of the chimeric HIV-1 RT. Induction of the enzyme
protein was carried out as described before for the wild type HIV-1 RT
(14, 15). The chimeric enzyme was purified from bacterial lysate by
immobilized metal affinity chromatography (7).
Synthetic 32P-Labeled
tRNA3Lys--
A clone for the
tRNA3Lys gene (pTL9) was obtained from Dr. S. F. J. Le Grice. A 256-base pair region of the clone containing the
T7 promoter and the gene for full-length tRNA was amplified by
polymerase chain reaction as described (16). The PCR product was used
as the template for in vitro transcription of
tRNA3Lys using T7 RNA polymerase. For
internal labeling of tRNA3Lys, 200 µM of [
-32P]UTP (specific activity: 1 µCi/60 pmol; reaction volume: 30 µl) was included in the
transcription reaction. The labeled tRNA was purified by denaturing 8%
polyacrylamide-urea gel electrophoresis. The gel-purified
32P-labeled tRNA3Lys was
stored at
70 °C in a solution containing 10 mM
Tris-HCl, pH 7.0, and 1 unit/µl of RNasin.
Glycerol Gradient Ultracentrifugation of Purified Chimeric HIV-1
RT--
Fifty micrograms of chimeric HIV-1 RT in Tris-NaCl buffer (50 mM Tris-HCl, pH 8.0, and 400 mM NaCl) was
carefully loaded onto 5 ml of 10-30% glycerol gradients prepared in
the same buffer. Gradients were centrifuged at 48,000 rpm for 22 h
in an SW48 rotor. Gradients were fractionated from the bottom, and each
fraction was assayed for polymerase activity using
[3H]dTTP as the substrate and
poly(rA)·(dT)18 as the template primer. Fractions were
also subjected to SDS-polyacrylamide gel electrophoresis to determine
the protein peak fraction. Sedimentation analyses of the marker
enzymes, HIV-1 RT, MuLV RT, and Klenow fragment were also carried out
simultaneously and compared with the sedimentation pattern obtained
with the chimeric enzyme. The HIV-1 RT preparation contained both
homodimeric p66/66 and heterodimeric p66/51 species.
RNA- and DNA-dependent DNA Polymerase
Activities--
Heteromeric synthetic 30-mer RNA and 49-mer DNA
templates corresponding to the U5-PBS region of HIV-1 RNA were primed
with 5'-32P-labeled DNA PBS primers and used to assess the
polymerase activities. In addition, homopolymeric template primer,
Poly(rA)·5'-32P-labeled (dT)18 was also used
for detecting the polymerase activity. Polymerase reactions were
carried out by incubating 2.5 nM template primer with 50 nM wild type HIV-1 RT or its chimeric derivative in a total
reaction volume of 6 µl containing 25 mM Tris-HCl, pH
7.5, 10 mM dithiothreitol, 100 µg/ml bovine serum
albumin, 2 mM MgCl2, and 100 µM
amounts of each dNTP. Reactions were initiated by the addition of the
enzyme and terminated by the addition of equal volume of Sanger's gel
loading dye (17). The reaction products were analyzed by denaturing
polyacrylamide-urea gel electrophoresis followed by
autoradiography.
RNase-H Activity Assays--
We used a 30-mer synthetic RNA
template corresponding to the PBS region of the HIV genome (3'-CAG GGA
CAA GCC CGC GGU GAC GAU CUC UAA-5') for assessing the RNase-H activity.
The RNA template was 5'-32P-labeled and purified by 8%
denaturing polyacrylamide-urea gel electrophoresis. The labeled
template was annealed with 30-mer complementary DNA. The RNA-DNA hybrid
was separately incubated with the enzyme under standard reaction
conditions. The reaction mixture (10 µl) contained 5 nM
labeled hybrid (104 Cerenkov counts/min), 80 mM
NaCl, 5 mM MgCl2, 10 mM
dithiothreitol, 50 mM Tris-HCl, pH 8.0, 0.1 mg/ml bovine
serum albumin, and 100 ng of enzyme. Reactions were carried out at
25 °C for different time points, and the reaction products were
analyzed on 16% denaturing polyacrylamide-urea gel.
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RESULTS AND DISCUSSION |
We compared the primary amino acid sequences of HIV-1 RT and MuLV
RT in order to locate the region that confers dimeric or monomeric
structure to these enzymes, respectively. Three major motifs, A, B, and
C in the primary amino acid sequence of MuLV RT, conspicuously absent
in the sequence of HIV-1 RT emerged (Fig. 1). Motif A, the longest, is comprised of
27 amino acids spanning residues 479-505 and lies in the putative
connection subdomain of MuLV RT. Motif B, found in the RNase-H domain
spans residues 597-611, while the motif C containing residues 318-328
is located in the polymerase domain. We suspected that the absence of
these motifs in the primary amino acid sequence of HIV-1 RT may have imposed the need for its dimerization. We, therefore, constructed a
chimeric HIV-1 RT containing the first 425 codons from the N-terminal of HIV-1 RT and 200 codons from the C-terminal of MuLV RT. The C-terminal fragment of MuLV RT contained part of its connection subdomain and the entire RNase-H domain, including motifs A and B. The
chimeric HIV-1 RT was cloned in pET-24b vector, expressed in E. coli BL 21(DE3), and purified by metal affinity column
chromatography (Fig. 2A). The
purified enzyme exhibited both RNA- and DNA-dependent DNA
polymerase activity (see below).

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Fig. 1.
Alignment of the primary amino acid sequences
of HIV-1 RT and MuLV RT highlighting the missing motifs A, B, and
C. The sequence alignment of HIV-1 RT and MuLV RT was performed
using the LOOK program. Motifs A, B, and C of MuLV RT are located in
the connection subdomain, the RNase-H, and the polymerase domain,
respectively. The gaps in the corresponding amino acid position of
HIV-1 RT are shown as dots.
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Fig. 2.
A, SDS-PAGE of the chimeric HIV-1 RT
containing the RNase-H domain of MuLV RT. Purified chimeric enzyme was
expressed in E. coli BL 21(DE3) and purified by one-step
purification through immobilized metal affinity column chromatography
and analyzed by SDS-polyacrylamide gel electrophoresis. Lane
1, chimeric HIV-1 RT; lane 2, wild type HIV-1 RT;
lane 3, wild type MuLV RT. B, glycerol gradient
sedimentation profile of the polymerase activities of the wild type
HIV-1 RT, MuLV RT, and chimeric HIV-1 RT. The purified chimeric HIV-1
RT as well as the wild type HIV-1 RT and MuLV RT were individually
resolved by 10-30% linear glycerol gradient ultracentrifugation at
48,000 rpm in SW50.1 rotor for 22 h. Gradients were fractionated
from the bottom and assayed for polymerase activity. C,
SDS-PAGE analysis of the gradient fractions. An aliquot of each
gradient fraction was subjected to SDS-polyacrylamide gel
electrophoresis, and the protein peak in the fraction was visualized by
Coomassie Blue staining of the gel. D, mobility shift assay
of tRNA-bound chimeric HIV-1 RT and wild type RTs by native
polyacrylamide gel electrophoresis. The internally
32P-labeled tRNA3Lys (5 × 105 Cerenkov counts/min) was incubated on ice with 10 µg of chimeric RT, MuLV RT, or wild type HIV-1 RT. The complexes were
subjected to gel retardation analysis by polyacrylamide gel
electrophoresis on a 7% nondenaturing native gel. The electrophoresis
was carried out at in a Tris borate buffer system at 4 °C at 100 volts for 4 h followed by autoradiography. Lane 1,
tRNA3Lys alone; lane 2, HIV-1
RT + tRNA3Lys; lane 3,
chimeric HIV-1 RT + tRNA3Lys; lane
4, MuLV RT + tRNA3Lys.
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To determine whether the chimeric enzyme is a dimer or monomer, a
10-30% linear glycerol density gradient ultracentrifugation sedimentation analysis was performed with the chimera and also with
wild type HIV-1 RT and MuLV RT (18). The latter two enzymes serving as
controls. The gradients were fractionated from the bottom and analyzed
for their polymerase activity and the protein peak profile (Fig.
2B and C). It was observed that the heterodimeric HIV-1 RT sedimented between fractions 12 and 15, while the chimeric RT
and MuLV RT sedimented between fractions 22 and 24 of the gradient. This observation clearly establishes the existence of the chimeric HIV-1 RT as a monomer as it sediments at a position higher than the
sedimentation position of the dimeric HIV-1 RT but closer to that of
the monomeric MuLV RT. Binding studies with
tRNA3Lys, a natural primer for HIV-1 RT,
using gel retardation assay indicated a mobility shift distinct from
the wild type HIV-1 RT on native polyacrylamide gel electrophoresis
(Fig. 2D). The chimeric HIV-1 RT migrated ahead of the wild
type HIV-1 RT, but closer to MuLV RT. This also indicated that, unlike
HIV-1 RT, the chimeric RT is a monomeric enzyme. The bands shown in the
autoradiogram appear slightly elongated due to the overloading of the
tRNA bound enzyme protein on the gel. This was essential in order to
obtain sufficient signal as all the three enzymes exhibit poor binding
affinity with the synthetic tRNA3Lys.
HPLC gel filtration of HIV-1 RT, chimeric HIV-1 RT, and MuLV RT (19)
using two BioSep 300 columns (300 × 7.8 mm; Phenomenex Inc.) also
indicated the existence of the chimeric enzyme as a monomer (data not
shown).
Although MuLV RT exists as a monomer in solution, it has been suggested
to be in a dimeric form when bound to the template primer (20). It was
therefore interesting to examine if the chimeric HIV-1 RT also
dimerizes upon binding to the template primer. We therefore
photocross-linked wild type HIV-1 RT, MuLV RT, Klenow fragment, and
chimeric HIV-1 RT with 5'-32P-labeled 37-mer self-annealing
template primer. The radioactive E-TP covalent species were
purified on DEAE-cellulose column and subjected to glycerol gradient
ultracentrifugation analysis. The sedimentation profile of labeled
E-TP complex was determined by analyzing the gradient
fractions by SDS-polyacrylamide gel electrophoresis followed by
autoradiography. The results are as shown in Fig. 3 (also see Table
I). Contrary to an earlier report (20),
the DNA-bound MuLV RT sedimented as a monomer close to the DNA-bound Klenow fragment but farther from the DNA bound dimeric HIV-1 RT. The
DNA-bound chimeric HIV-1 RT also sedimented as a monomer at the
position between that of the DNA-bound Klenow fragment and MuLV RT.
These results provide further proof that both MuLV RT and chimeric
HIV-1 RT do not dimerize upon binding to the template primer.

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Fig. 3.
Glycerol gradient ultracentrifugation
analysis of DNA-bound HIV-1 RT, MuLV RT, chimeric HIV-1 RT, and Klenow
fragment. Two-hundred microgram of individual enzyme protein was
incubated on ice for 10 min with 5'-32P-labeled
self-annealing 37-mer TP containing photoactivatable bromodeoxyuridine
base at the penultimate nucleotide from the 3' primer terminus (Table
I). The mixture was UV-irradiated at 312 nM UV for 3 min in
a Spectrolinker (9). The enzyme-TP covalent complex was applied on a
DEAE-cellulose column (1 ml) pre-equilibrated with 100 mM
NaCl in 50 mM Tris-HCl, pH 7.8. After washing the column
with the same buffer (10 ml), the E-TP covalent complex was
eluted at 400 mM salt concentration. The uncross-linked DNA
remained bound in the column and eluted at 600 mM salt
concentration. The purified E-TP covalent complex was then subjected to
glycerol gradient analysis as described under "Materials and
Methods." The sedimentation profile of DNA cross-linked enzyme was
determined by analyzing every alternate gradient fraction by SDS-PAGE
followed by autoradiography.
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The purified chimeric HIV-1 RT was examined for its DNA polymerase
activity employing homopolymeric and heteropolymeric RNA and DNA
templates primed with 5'-32P-labeled DNA primer (7). The
chimeric enzyme was found to catalyze DNA synthesis efficiently on both
RNA and DNA templates, yielding RNA:DNA hybrid and duplex DNA,
respectively (Fig. 4, A-C).
As shown in the figure, the extent of full-length product catalyzed by
the chimeric enzyme was similar to that found with wild type HIV-1 RT
and MuLV RT. Further analysis shown in Fig. 5 demonstrates the ability of the
chimeric enzyme to catalyze both endonucleolytic and processive RNase-H
functions in a manner similar to the wild type HIV-1 RT and MuLV RT.
This is so far the first report that a chimeric HIV-1 RT containing the
entire RNase-H domain from MuLV RT has been constructed with intact
RNase-H and DNA polymerase functions. Earlier Hizi et al.
(21) constructed two chimeric HIV-1 RTs and found them both to be
inactive with respect to their polymerase activities while exhibiting
low levels of RNase-H activity. Post et al. (22) have
constructed a chimeric RT by replacing the RNase-H domain of MuLV RT
with E. coli RNase-H. The resulting enzyme was found to be
impaired in catalyzing DNA synthesis, although it exhibited 300-fold
higher RNase-H activity as compared with the wild type MuLV RT. It is
not known whether chimeric MuLV RT with E. coli RNase-H
exists as a monomer or dimer. In contrast, the chimeric HIV-1 RT
constructed by us exhibits high levels of both RNA- and
DNA-dependent DNA polymerase activities in addition to
fully functional RNase-H activity.

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Fig. 4.
RNA- and DNA-dependent DNA
polymerase activities of wild type HIV-1 RT, MuLV RT, and chimeric
HIV-1 RT. Poly(rA)·(dT)18 as well as synthetic
30-mer RNA and 49-mer DNA template primed with
5'-32P-labeled DNA primers were used to assess the
polymerase activities of the wild type HIV-1 RT, MuLV RT, and the
chimeric HIV-1 RT. Reactions catalyzed by the wild type HIV-1 RT and
MuLV RT are shown for comparison. The reaction conditions are as
described under "Materials and Methods."
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Fig. 5.
RNase-H activity of chimeric HIV-1 RT.
5'-32P-Labeled 30-mer RNA annealed with 30-mer
complementary DNA strands was incubated at 25 °C under standard
conditions with the chimeric RT as well as with the wild type HIV-1 RT
and MuLV RT for the indicated time points. The cleavage product of the
RNA strand was analyzed on a denaturing polyacrylamide-urea gel.
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The region responsible for conferring monomeric structure to the
chimeric HIV-1 RT seems to be the introduction of either motif A or
motif B contributed by nonconserved connection subdomain and RNase-H
domain of MuLV RT. Interestingly, Motif A region of MuLV RT exhibits
46% similarity and 21% identity with the region spanning residues
108-141 in the N-terminal region of HIV-1 RT. This region of HIV-1 RT
contains
6,
C,
D,
7, and
7-
8 loop, which are
constituents of the finger and palm subdomains of the enzyme. The
7
and
7-
8 loop in p66 are far away from the catalytic cleft, while
in the p51 subunit they are in the vicinity of the catalytic cleft of
the p66 subunit (23). In p51, the
7-
8 loop also contains the
interaction site for a non-nucleoside inhibitor (Glu-138), TSAO.
Interaction of Glu-138 of the p51 subunit with TSAO results in
inactivation of the polymerase function of the enzyme (24). In the
three-dimensional crystal structure of the heterodimeric HIV-1 RT, the
7
8 loop of the finger subdomain of the p51 subunit is the
constituent of the subunit interaction surface. It seems to interact
with the floor (palm) of the polymerase domain of the p66 subunit,
which consequently may induce the opening of the polymerase cleft. This
contention is supported by the fact that interaction of TSAO with the
7
8 loop of p51 destabilizes the enzyme into inactive monomers
with concomitant loss of DNA binding ability and polymerase function
(Fig. 6, A-C). Interestingly, the chimeric HIV-1 RT is completely insensitive to TSAO treatment, as
both the DNA binding ability and polymerase activity of the TSAO-treated chimeric enzyme remained unaffected (Fig. 6, B
and C). Unlike, in the wild type HIV-1 RT, the
7
8 loop
in the chimeric HIV-1 RT may be inaccessible for binding to the TSAO,
thus manifesting in a complete resistance to this inhibitor.
Alternatively, due to the absence of the subunit interacting surface in
the chimeric enzyme the binding site for TSAO may not exist. We propose
that the
7-
8 loop of the p51 subunit of HIV-1 RT may structurally compensate for the missing motif A in the connection subdomain of HIV-1
RT. Insertion of this motif in the connection subdomain of HIV-1 RT is
currently being carried out to ascertain whether the resulting enzyme
is functional in the monomeric form.

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Fig. 6.
A, HPLC gel filtration profile of
TSAO-treated and untreated heterodimeric HIV-1 RT. Two-hundred
microgram of p66/p51 heterodimeric RT was treated with a 5-fold molar
excess of non-nucleoside inhibitor, TSAO, as described by Jonckheere
et al. (24). The HPLC gel filtration of TSAO-treated and
untreated heterodimeric HIV-1 RT was carried out using two BioSep 3000 columns (300 × 7.8 mm; Phenomenex Inc.) attached to HPLC (Varian
5500 HPLC unit). The elution buffer contained 150 mM NaCl
in 50 mM Tris-HCl, pH 8.0 (19). The elution time of
dissociated p66 and p51 monomers was significantly delayed by 0.67 and
1.4 min, respectively, as compared with the p66/p51 heterodimeric HIV-1
RT. B, DNA binding ability of TSAO-treated HIV-1 RT and
chimeric HIV-1 RT. Three micrograms of enzyme protein pretreated with
5-fold molar excess of non-nucleoside inhibitor, TSAO, was incubated on
ice with 5'-32P-labeled 37-mer self-annealing template
primer for 5 min. The mixture was UV-irradiated at 312 nm for 3 min in
a Spectrolinker, and the E-TP covalent complex was resolved
by SDS-PAGE (9). C, effect of TSAO on the polymerase
activity of wild type HIV-1 RT, MuLV RT, and chimeric HIV-1 RT.
Poly(rA)·5'-32P-labeled (dT)18 was used to
assess the polymerase activity of TSAO treated (+) and untreated ( )
enzymes under standard reaction conditions. The products were analyzed
on a denaturing polyacrylamide-urea gel.
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Why does the p66 subunit of HIV-1 RT need leverage from the p51
subunit? Can it support its open conformation independent of p51? One
possibility is that the elongated p66 subunit may be unable to hold its
polymerase and RNase-H domains in an unsteady horizontal position and
therefore may require support from p51. In the chimeric RT, the
positioning of the polymerase and RNase-H domains may be such that one
domain supports the other thus circumventing the need for its
dimerization. Probably, the extended connection subdomain may help in
positioning both the domains in such a fashion. This presumption is
supported by the observation that the putative connection subdomain of
MuLV RT, as determined by aligning of its primary amino acid sequence
with that of HIV-1 RT, is longer than that of HIV-1 RT, and
this may possibly be one of the factors for its monomeric
structure.
We thank M. J. Modak and N. Kaushik for
critical reading of the manuscript. We acknowledge P. N. S. Yadav and R. T. Whipple for their help in the analysis of sequence
alignment of HIV-1 RT and MuLV RT. The TSAO was generously provided by
J. Balzarini.