From the Departments of Microbiology & Immunology and
§ Biochemistry, Albert Einstein College of Medicine,
Bronx, New York 10461 and
Beth Israel Hospital,
Boston, Massachusetts 02215
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ABSTRACT |
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Human immunodeficiency virus type 1 (HIV-1)
reverse transcriptase (RT) displays a characteristic poor processivity
during DNA polymerization. Structural elements of RT that determine
processivity are poorly understood. The three-dimensional structure of
HIV-1 RT, which assumes a hand-like structure, shows that the fingers, palm, and thumb subdomains form the template-binding cleft and may be
involved in determining the degree of processivity. To assess the
influence of fingers subdomain of HIV-1 RT in polymerase processivity,
two insertions were engineered in the 3-
4 hairpin of
HIV-1NL4-3 RT. The recombinant mutant RTs, named FE20
and FE103, displayed wild type or near wild type levels of
RNA-dependent DNA polymerase activity on all templates
tested and wild type or near wild type-like sensitivities to
dideoxy-NTPs. When polymerase activities were measured under conditions
that allow a single cycle of DNA polymerization, both of the mutants
displayed 25-30% greater processivity than wild type enzyme. Homology
modeling the three-dimensional structures of wild type
HIV-1NL4-3 RT and its finger insertion mutants revealed
that the extended loop between the
3 and
4 strands protrudes into
the cleft, reducing the distance between the fingers and thumb
subdomains to ~12 Å. Analysis of the models for the mutants suggests
an extensive interaction between the protein and template-primer, which
may reduce the degree of superstructure in the template-primer. Our
data suggest that the
3-
4 hairpin of fingers subdomain is an
important determinant of processive polymerization by HIV-1 RT.
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INTRODUCTION |
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The human immunodeficiency virus type 1 (HIV-1)1 life cycle is dependent on the functions of the virally encoded polymerase, reverse transcriptase (RT). In addition to its being a target for drug development, HIV-1 RT is unusual in its structural features, versatile use of both RNA and DNA templates, high error rates (1-3), and poor processivity (4). HIV-1 RT is a heterodimer containing 66- and 51-kDa polypeptides, termed p66 and p51 (5, 6). The p51 is generated by the proteolytic cleavage of the p66 subunit (7, 8). Because of its likeness to a right hand, the subdomains of HIV-1 RT are named fingers, palm, and thumb which are joined to the RNase H domain via a connection subdomain (9, 10). The fingers, palm, thumb, and connection subdomains of p66 constitute the polymerase domain of RT and form a cleft for template-primer binding and for the polymerase active site (9-12). The p51 subunit lacks the RNase H domain, and its three-dimensional structure, in contrast to that of p66, is more globular with no cleft (13).
The precise roles played by the various subdomains in the polymerase function of HIV-1 RT are unknown. The palm subdomain contains the catalytic triad of aspartates and therefore plays a key role in catalysis (9, 10, 14). In the three-dimensional x-ray crystal structures of the apo-RT and of RT complexed with template-primer or a non-nucleoside inhibitor, the thumb subdomain has been shown to occupy different positions with respect to the fingers (9-12). Based on these observations, and the fact that the thumb domain intimately interacts with the template-primer (10), it is proposed that the thumb subdomain mediates the translocation of the enzyme along template (12). The palm, thumb, and fingers subdomains are all thought to contact the template-primer, and together they constitute the template-primer cleft of HIV-1 RT.
To understand the role of the fingers subdomain of HIV-1 RT on the
polymerase processivity, we engineered two gross alterations into the
3-
4 hairpin creating insertions within the flexible loop
connecting the
strands. Both mutants were characterized for
polymerase activity, sensitivity to nucleotide triphosphate analogs,
kinetic constants, and finally processivity. The results indicate that
the fingers subdomain plays an essential role in determining the
processivity of polymerization by HIV-1 RT. Modeling the
three-dimensional structure of RT with the extended fingers provided
insight into the possible role of the
3-
4 hairpin in increasing
the processivity of RT.
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EXPERIMENTAL PROCEDURES |
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Bacteria and Plasmids--
The Escherichia coli
strain DH5F'IQ (Life Technologies, Inc.) was used for expression of
HIV-1 RT. The sequences of the RT employed in this study were derived
from the molecular clone NL4-3 (15). The expression vector used is
pRT6H-NB/PROT containing an RTHXB2 p66 cassette and a
separate HIV-1 PR expression cassette and is a version of pRT6H-PROT
(16) containing NotI at the 5'-end. pL6H-PROT is a version
of pRT6H-NB/PROT in which the RT sequences are replaced by a polylinker
sequence.
Construction of RT Mutants-- First, the RTHXB2 sequences of pRT6H-NB/PROT were replaced with corresponding sequences from NL4-3 via polymerase chain reaction followed by digestion of the products by NotI and BglII and ligation of the fragment into the corresponding sites in pL6H-PROT. The deletion and insertion mutants were created by cassette mutagenesis as described previously (17, 18). Briefly, a gap was created in RT fingers region lacking the codons 60-79 to facilitate both insertions and deletions into the region. This intermediate clone was digested with BspMI to release the central fragment, and double-stranded adaptors were ligated to the BspMI sites to generate the deletion and insertion mutants (Fig. 1). The mutant RTs were analyzed by sequencing before subcloning the RT NotI/BglII insert into the expression construct, pL6H-PROT, which places a hexahistidine tag at the carboxyl terminus of p66.
Bacterial Expression, Lysis, and Purification of
RTs--
Bacteria carrying the appropriate expression vector were
induced for expression by the addition of 1 mM
isopropyl-1-thio--D-galactopyranoside (Sigma) as
described previously (19). The pellets of induced bacteria were lysed
with lysozyme (1 mg/ml, for 20 min on ice), sonicated, and lysates
cleared by centrifugation at 15,000 rpm (Sorvall SS-34 rotor). The
purification of the finger extension mutants was essentially similar to
the procedure we used previously (19), except that all binding,
washing, and elutions were done by the batch method. The specific
activities of the wild type, FE20, and FE103 RTs were 3030, 1515, and
3333 units/mg, respectively (1 unit is defined as the activity
equivalent to incorporation of 1 nmol of dTMP in 10 min at 37 °C
using poly(rA)·oligo(dT)).
RNA-dependent DNA Polymerase Assays--
The
RNA-dependent DNA polymerase (RDDP) assays on
heteropolymeric templates were performed in a 16-µl reaction mix
containing 53 pM 16 S rRNA template-22-mer primer
(5'TAACCTTGCGGCCGTACTCCCC3'), 50 mM Tris·Cl (pH 8.0), 80 mM KCl, 6 mM MgCl2, 1 mM dithiothreitol, 0.05% Nonidet P-40, 10 µM
[-32P]dGTP (2.7 Ci/mmol; NEN Life Science Products),
and 50 µM each of dCTP, dATP, and dTTP and incubated at
37 °C for 15 min. Duplicate reactions were stopped by spotting onto
DE81 filter squares and washed with 2× SSC (30 mM sodium
citrate, 300 mM sodium chloride (pH 7.0)) for 10-15 min
four times to remove unincorporated dNTPs. The filters were dried and
the counts/min determined via a scintillation counter (1218 Rack Beta,
LKB-Wallac, Sweden), and the picomoles of dNMP incorporated were
calculated.
ddNTP Inhibition of RTs--
The sensitivity to each of the four
ddNTPs was separately measured in the heteropolymeric RNA template
assay described above. The concentration of three dNTPs (Pharmacia
Biotech Inc.) was each at 25 µM. The fourth dNTP, for
which a competing ddNTP is also present in the reaction, was at 5 µM and was -32P-labeled (2.3 Ci/mmol). The
ddNTP concentration in each case (Boehringer Mannheim) ranged from 0.25 to 1 mM. The reactions, run for 15 min, were stopped as
described above, and dNMP incorporation was quantitated as before.
Determination of Kinetic Constants and Processivity-- The kinetic constants, Km and Vmax, were determined for RDDP activity on poly(rA)·oligo(dT). The reactions utilized 0.5 ng/µl RDDP reaction and were carried out as described above. The kinetic constants were determined as described previously (19).
The processivity assays were carried out using the heparin trap method. The two RNA templates used, from HIV-1 or influenza genomes, were derived by in vitro transcription from plasmids pKSNL/RN and pBS-M1, respectively. pKSNL/RN was constructed by ligating an EcoRV-NsiI fragment of pNL4-3 into pBluescript-KS (Stratagene) vector digested with EcoRV and PstI. pKSNL/RN was linearized with BamHI and pBS-M1 (20) kindly provided by Matthew Bui (Yale University) with AflIII prior to in vitro run-off transcription using the T3 and T7 RNA polymerase, respectively (Ambion Corp.). The sequences of the oligonucleotide primers used in combination with HIV-1 and M1 RNA template are 5'CGCTTTCAAGTCCCTGTTCGGGCGCCA3' and 5'AGTGGATTGGTTGTTGTCACCAT3', respectively. A 10-µl reaction mix containing 50 mM Tris·Cl (pH 8.0), 80 mM KCl, 6 mM MgCl, 1 mM dithiothreitol, 1.5 units of RT enzymes, and 10 nM template-primer (HIV-1 or influenza virus M1 RNA) were preincubated at 37 °C for 5 min. Subsequently, 25 µM dNTPs and 40 µg of heparin were added together and incubated at 37 °C for another 5 min. The reactions were stopped with 10 µl of stop solution (90% formamide, 10 mM EDTA (pH 8.0), 0.1% xylene cyanol, 0.1% bromphenol blue). Six microliters of the final mixture were separated on denaturing gels. A titration of the trapping activity of heparin was performed against each of the three enzymes. No differences were revealed suggesting that the ability of heparin to quench the three enzymes was very similar (data not shown).Modeling Procedures--
A model of the FE20 and FE103
finger-extension mutants was generated from coordinates of RT bound
with the non-nucleoside RT inhibitor, -APA R95845 (1HNI) (21).
Single residue substitutions were incorporated into the model to change
the HXB2 sequence used for the x-ray structure determination to the
wild type NL4-3 sequence used here. Residues that were modeled as
alanines in the crystal structure because of weak electron density were
also changed to conform the actual wild type NL4-3 RT sequence. The
loop extensions of FE20 and FE103 RTs were introduced to the model of
wild type NL4-3 RT using the loop-building function in the Homology
package. The models of the three proteins, which included all atoms of their respective residues (including the inserted residues), were minimized using steepest descents with a CVFF forcefield in two steps
using the Discover package (Biosym). In the first minimization, all
residues present in the original crystallographic coordinates were
constrained, and in the second, all non-hydrogen atoms were fixed.
Next, a model of A-type DNA was made using the Builder package
(Biosym). This model includes the 5 base pairs of A-form DNA that were
positioned based on the phosphorous positions available from the RT-DNA
co-crystal coordinates (1HMI) (10). In addition, a 10-base overhang in
the template strand was modeled assuming that the single-stranded
extension maintained an A-type conformation. Although the phosphate
backbone of the single-stranded region is flexible, it is plausible
that this DNA is in the A-form and such an assumption has been made by
others (22) attempting to model template extensions. The sequence of
the template strand is thus 5'-AAAAAAAAAAATGGC-3'. The overhang modeled
is not in the original coordinates. The double-stranded region
positioned based on experimental coordinates and 5 residues of which
were fixed throughout the minimization and dynamics calculations served as an anchor for the single-stranded region. Each protein-DNA model was
minimized in sequential rounds, in which residues 105-112 and 178-191
(which together include the catalytic triad of aspartates) were fixed
along with residues 12-15 of the template DNA and all 5 residues of
the primer strand. The protein residues were fixed to prevent
unreasonable distortions from occurring in the polymerase active site,
particularly at or near Met-184 which is part of a turn containing
unusual
and
angles. The remaining portions of the model were
restrained during minimization. Restraints were gradually lowered as
minimization proceeded. Minimizations used a steepest descents
algorithm initially and then switched to conjugate gradients when a
maximum derivative limit of 2.5 kcal/mol Å had been reached,
continuing to a final maximum derivative of 0.001 kcal/mol Å. Next,
the structures were submitted to a dynamics calculation. Again, amino
acid residues 105-112, 178-191, template residues 12-15, and all
primer residues were fixed. Additionally, residues 388-560 and all 440 residues of p51 subunit were fixed. This simplification reduced
computational times. Residues 388-560 are too far away to interact
with the model DNA or residues binding the DNA. The dynamics
calculation was carried out for 5 ps in 1-fs intervals at 298 K using a
canonical ensemble. Following the dynamics calculation, coordinates
were minimized using cff91 forcefield with the pH set to 7.5. These
structures were again minimized, with residues 105-112, 178-191, and
388-560 of p66, all residues of p51, and template residues 12-15 and
primer residues 1-5 fixed as before. Minimization was implemented to a
derivative cut-off of 0.001 kcal/mol Å, before being resubmitted to
further 11 ps of dynamics. The resulting coordinates were then
minimized in an Amber forcefield to allow simulation of the effects of
bulk solvent. A distance-dependent dielectric was employed
with an r = 4 dependence for the dielectric constant,
and 25-ps dynamics simulations were calculated. The root mean square
deviations for the wild type HIV-1RTNL4-3, FE20, and FE103
models with respect to 1HNI starting coordinants were 1.6, 1.7, and 1.7 Å, respectively, when the palm residues were superpositioned.
Superpositioning the fingers region yields root mean square deviations
of 2.7, 3.0, and 3.6 Å, and root mean square deviations of 2.4, 2.1, and 2.8 Å result from superimposing the thumb residues of the wild type, FE20, and FE103 models on 1HNI coordinants. The only collisions seen between model residues and template is that between tyrosine 183 (wild type numbering) and template residue 12 (
2 position with
respect to the template base that base pairs with incoming dNTP). The
distance between Tyr-183 C
and N-2 of the template residue 12 is 2.5 Å. This is a result of the constraints against movement imposed on
these atoms to keep the active site aspartates or the template anchor
residues from moving. No collisions are observed between finger
residues and the extended template.
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RESULTS |
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Engineering Alterations in the 3-
4 Hairpin--
Mutations
were introduced into the fingers subdomain of recombinant
pRTNL4-36H-PROT expression construct which contains
separate expression cassettes for HIV-1NL4-3 RT p66 and
HIV-1 protease (PR). Insertions in the
3-
4 hairpin were based on
the structure reported by Jacobo-Molina et al. (10). In the
original design of the finger extension, we wished to simply increase
the bulk of the flexible loop. In the absence of suitable
foreknowledge, we did not want to introduce electrostatic interactions
which might adversely affect translocation and/or processivity as a result of altered enzyme-template-primer interactions, leading to an
inactive state. Since serine residues avidly form hydrogen bonds and
glycine residues have a high degree of main chain flexibility, we
inserted a sequence that is rich in these residues to extend the size
of the flexible loop (FE103, Fig.
1).
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Insertions into Fingers Subdomain Do Not Affect the Gross
Properties of HIV-1 RT--
To assess the impact of alterations in the
3-
4 region on the polymerization function, lysates of bacteria
induced for RT expression were initially tested for
RNA-dependent DNA polymerase (RDDP) activity on both
heteropolymeric and homopolymeric RNA templates. RDDP assays on
heteropolymeric RNA template revealed that the activities of two
insertion mutants were very similar to that of wild type RT (Table
I). When homopolymeric templates were
used, the finger insertion mutants were more active on poly(rA) compared with poly(rC), a characteristic feature of wild type HIV-1 RT
(Table I).
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Finger Extension Mutants Display Altered Processivity--
Since
the insertion mutations minimally affect the polymerase activity of RT,
they were suitable to test the effect of finger alterations on the
processivity of polymerization. To facilitate an in-depth analysis, the
insertion mutants (FE20 and FE103) and the wild type
HIV-1NL4-3 RT were purified to near-homogeneity (Fig.
2). The protein preparations were tested
for contaminating RNase and DNase activities and were found to be free
of both (data not shown). The kinetic constants
Km, dTTP and Vmax were determined for these RTs in RDDP
assays on poly(rA)·oligo(dT) template-primer (summarized in Table
III). Both the Km and
Vmax for the mutants were similar to those of
wild type RT. The Vmax/Km
ratios for the mutant RTs, although reduced from wild type levels,
suggest that the catalytic efficiencies were not significantly
compromised by the large insertions in the 3-
4 hairpin.
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Modeling of the Finger Extension Mutants--
To obtain structural
insight into the role of HIV-1 RT finger extensions, we generated a
computer model of the three-dimensional structure of NL4-3 RT and its
two finger extension derivatives, FE20 and FE103. The coordinates from
the structure of HIV-1BH10 RT bound with -APA R 95845 refined to 2.8 Å (21) were used as a starting point in the
homology-based generation of protein coordinates of the
HIV-1NL4-3 RT. Positioning of the duplex DNA was guided by
the positions of the phosphorous atoms available from the structure of
HIV-1 BH10 RT complexed with duplex DNA (10).
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DISCUSSION |
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HIV-1 RT is known to display a moderate to poor degree of polymerase processivity. On heteropolymeric RNA templates, the HIV-1 RT is known to synthesize up to 105 nucleotides (26) in a single processive cycle of DNA synthesis. Although much larger products (300 bases long) can be obtained under certain conditions, these involve the use of homopolymeric RNA templates such as poly(rA) (27). Structural elements that control processivity of HIV-1 RT are currently not defined. The work reported here reveals an important role for the HIV-1 RT fingers subdomain in processive polymerization. The two insertion mutations created in the fingers subdomain, described in this report, did not lead to any gross changes in RT function other than increasing the polymerase processivity.
Previous work from several laboratories has implicated fingers
3-
4 loop in the following two functions: positioning the template-primer (28, 29) and nucleoside analog sensitivity (14). When
an extended template overhang was modeled into the three-dimensional
structure of HIV-1 RT double-stranded DNA complex, the flexible loop of
the
3-
4 hairpin was shown to contact the template approximately
three nucleotides ahead of the primer terminus (22). Nuclease
footprinting experiments have also confirmed that such an interaction
is likely (30). Biochemical support for this comes from studies that
showed greater affinity of RT to double-stranded DNAs with 6-base or
longer overhangs as compared with those with 1-base overhang (25). The
3-
4 loop of the fingers subdomain is also a hotspot for
nucleoside analog resistance mutations (14). The fact that the
3-
4 loop is distal to the dNTP-binding pocket makes this
intriguing. However, when combined with the fact that the
3-
4
loop contacts the template-primer (22), the ability of
3-
4
mutations to confer nucleoside analog resistance is consistent with an
indirect role played by template-primer contacting residues in
determining the conformation of the dNTP-binding pocket. This effect
has been termed "template repositioning" (22).
Interestingly, despite the large perturbation, we observed only a small
change (ranging from 0.14- to +3.1-fold) in sensitivity of the finger
insertion mutations to ddNTPs (Table II). A large number of nucleoside
analog resistance mutations have been identified for HIV-1 RT, and a
significant data base on their biochemical properties is available. In
a majority of the cases, the levels of decrease in sensitivity are in
the range of 10-100-fold (31-33). Thus, a change of 3-fold appears
very close to background or wild type levels. It is unclear why large
insertions that appear to increase the interaction between fingers
subdomain and the template-primer have no effect on nucleoside analog
sensitivity.
Although the mutant FE103 displayed levels of RDDP activity that were comparable to that of wild type, there was a 40% decrease in the activity on heteropolymeric RNA for the insertion mutant FE20 (Table I). However, a decrease of only 20 or 30% was observed on other templates for the same mutant (Table I). It is interesting that FE20 displays an increase in processivity in light of the fact that its overall activity is consistently lower than wild type. The fact that the finger insertions had either a small or no effect on the Km and Vmax values for RDDP activity or the sensitivities to the ddNTPs suggest that the insertions at this locus of the fingers subdomain, unlike the nucleoside analog resistance mutations known to arise during treatment, did not affect the conformation of the dNTP-binding pocket.
Although a poor processivity for HIV-1 RT is well recognized, mutations
that alter this property are rarely observed. The ()-2',3'-dideoxy,3'-thiacytidine (3TC)-resistant M184V variant (34)
and site-directed mutations in the thumb subdomain (35) decrease the
processivity of HIV-1 RT. Significant increases in processivity were
also reported for azidothymidine-resistant variant of HIV-1 RT
(including D67N, K70R, T215Y, and K219Q alterations) (36) and a
didanosine-resistant variant with the K65R alteration (37).
Interestingly, the K65R, D67N, and K70R mutations found in these mutant
RTs also map to the
3-
4 loop.
The method of preparation of the RT heterodimers employed here will
result in the presence of the insertion mutations in both p66 and p51
subunits. In the wild type HIV-1 RT heterodimer structure published,
the 3-
4 hairpin will face the template-primer duplex only as a
part of the p66 subunit. The corresponding segment of p51 is tucked
within the fingers subdomain of that subunit, well away from the
template-interacting surfaces and appears to be clearly distal to the
dimerization interface. Similarly, when the models of the two insertion
mutations were examined, the extensions appeared to be also tucked away
in the same manner (with the exception, for FE103 alone, of some
contacts with residues 401 and 427 in the p66 connection subdomain).
Thus, it is unlikely that the insertions in the p51 play a role in the
increased processivity of these mutant RTs. These mutations also did
not have an impact on the formation or the stability of the heterodimer
as shown by the stoichiometry of the two subunits during purification
via an affinity tag present only on the p66 subunit (Fig. 2).
The absence of crystallographic data for the HIV-1NL4-3 RT
and its finger extension forms makes it difficult to interpret the data
on processivity. Modeling suggests that the inserted residues of the
mutant RTs reduce the size of the channel within which the template is
held, effectively making a tighter clamp out of the fingers and thumb
subdomains (see Figs. 4 and 5). This may confer greater processivity by
improving the template-RT interactions or by helping melt structured
regions of template better than wild type RT. Analysis of the
accessible surface area for the modeled finger domains of wild type and
mutant RTs shows that the occluded surface area, that is the
solvent-accessible surface area lost as a result of complex formation,
increases by 12% for FE20 and by 14% for FE103 RTs relative to wild
type HIV-1NL4-3 RT. This is consistent with the idea that
the mutant RTs may form a stronger complex with template than wild type
RT and may be partially responsible for their greater processivity.
Analysis of the electrostatic potential, using the program GRASP (38), indicates that the surface of the fingers subdomain in proximity to the
template overhang of the mutant FE103 possesses a more positive
potential. This may allow FE103 RT fingers to form a stronger complex
with template than FE20 RT by interacting more strongly with the
template's phosphate backbone and is consistent with the greater
processivity observed for FE103 RT than for FE20 RT. Does further
reduction in the distance between the fingers and the thumb subdomains
result in a greater increase in processivity? To examine this issue, we
are currently in the process of creating a series of insertions of
progressively increasing sizes in the 3-
4 loop.
The structural elements controlling processivity and translocation of RT along the template are poorly understood. Work reported here reveals that the fingers subdomain, which is already implicated in template-primer contact and in nucleoside analog sensitivity, may play an important role in determining processivity. Several factors might contribute to the low level of processivity of wild type HIV-1 RT as follows: weak template-polymerase interactions, secondary structures, and other sequence-specific structural variations along the template. Our results suggest one of two factors may be responsible for the increased processivity of the insertion mutants as follows: a shorter distance between the fingers and the thumb subdomain, or an increased contact between the single-stranded portion of the template and the fingers subdomain. Further studies are required to delineate the factors that influence this important aspect of RT function.
The processive polymerization is an important aspect of DNA replication. HIV RT displays a characteristic low processivity that may have been evolutionarily conserved to facilitate the strand transfer reactions, a key step in retroviral DNA synthesis (39). It has been proposed that the poor processivity may be the principal cause for the high rate of nucleotide substitutions, deletions, insertions, as well as recombination observed for HIV-1 RT (39). However, during viral replication, other viral proteins such as nucleocapsid or integrase may modulate the processivity of HIV-1 RT. A positive influence of Ncp7 on the processivity of HIV-1 RT in vitro has been reported earlier (40). Additional studies are required to investigate the influence of integrase and other viral proteins on the processivity of HIV-1 RT.
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ACKNOWLEDGEMENTS |
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We thank Dr. James Sacchettini for advice on the structure-based generation of insertion mutations, Matthew Bui (Yale University) for technical assistance in the densitometric analyses, and the Cancer Center at Albert Einstein College of Medicine for the synthesis of oligonucleotides. We especially thank Drs. Steve Almo, Steve Roderick, Marshall Horwitz (all of this institution), and Aneel Aggarwal (Mount Sinai School of Medicine) for their critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grant AI-30861 (to V. R. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by an Aaron Diamond Postdoctoral Fellowship.
** To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2517; Fax: 718-430-8976; E-mail: prasad{at}aecom.yu.edu.
Data in this paper are from a thesis that was submitted in
partial fulfillment of the requirements for the Degree of Doctor of
Philosophy in the Sue Golding Graduate Division of Medical Sciences,
Albert Einstein College of Medicine, Yeshiva University.
1 The abbreviations used are: HIV-1, human immunodeficiency virus 1; RT, reverse transcriptase; RDDP, RNA-dependent DNA polymerase; dd, dideoxy.
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REFERENCES |
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