From the Department of Medicine, University Hospitals
of Cleveland and Center for AIDS Research at Case Western Reserve
University, Cleveland, Ohio 44106-4984 and ¶ Institut
de Biologie Moléculaire et Cellulaire du CNRS, 15, rue René
Descartes, 67084 Strasbourg Cedex, France
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ABSTRACT |
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Recently, tRNALys-3 was
cross-linked via its anticodon loop to human immunodeficiency virus
type 1 (HIV-1) reverse transcriptase (RT) between residues 230 and 357 (Mishima, Y., and Steitz, J. A. (1995) EMBO J. 14, 2679-2687). Scanning the surface of this region identified three basic
amino acids Lys249, Arg307, and
Lys311 flanking a small crevice on the p66 thumb subdomain
outside the primer-template binding cleft. To assess an interaction of
this region with the tRNA anticodon loop, these p66 residues were
altered to Glu or Gln. p66 subunits containing K249Q, K311Q, K311E, and a dual R307E/K311E mutation formed a stable dimer with wild type p51.
All mutants showed reduced affinity for tRNALys-3 and
supported significantly less ()-strand DNA synthesis from this primer
than the parental heterodimer. In contrast, these variants efficiently
synthesized HIV-1 (
)-strand strong-stop DNA from oligonucleotide
primers and had minimal effect on RNase H activity, retaining
endonucleolytic and directed cleavage of an RNA/DNA hybrid. Structural
features of binary RT·tRNALys-3 complexes were examined
by in situ footprinting, via susceptibility to
1,10-phenanthroline-copper-mediated cleavage. Unlike wild type RT,
mutants p66K311Q/p51 and p66K311E/p51 failed to
protect the tRNA anticodon domain from chemical cleavage, indicating a
significant structural alteration in the binary RT·tRNA complex.
These results suggest a crevice in the p66 thumb subdomain of HIV-1 RT
supports an interaction with the tRNALys-3 anticodon loop
critical for efficient (
)-strand DNA synthesis.
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INTRODUCTION |
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Initiation of reverse transcription is a complex step in the
retroviral life cycle. With human immunodeficiency virus type 1 (HIV-1),1 several specific
events, occurring prior to virus entry, precede processive synthesis of
()-strand DNA. The first of these is preferential packaging of
tRNALys isoacceptors and a dimer of the RNA genome into the
assembling virion by gag-pol and gag precursors
(p160gag-pol and p55gag, respectively) (2-4).
tRNALys-3 is selectively placed on the viral RNA, serving
as primer for (
)-strand DNA synthesis, by hybridization of 18 nt at
its 3' terminus to the primer binding site (PBS), a complementary
sequence immediately adjacent to the unique 5' region (U5) (5-9).
Beyond this interaction, PBS-bound tRNALys-3 has been shown
in vitro to interact with additional sequences of the viral
RNA genome, most notably an -AAAA- sequence in the U5-inverted repeat
(IR) loop that contacts the U-rich anticodon loop (10, 11). These
events are most likely established in an immature virus particle, prior
to protease-mediated maturation of p55gag and
p160gag-pol. However, mature HIV-1 proteins are
required for initiation of (
)-strand DNA synthesis from
tRNALys-3 (12, 13). In vitro, initiation of
(
)-strand synthesis involves addition of up to 6 deoxynucleotides to
the tRNALys-3 3' terminus (14-16), after which such
products are elongated to full-length (
)-strand strong-stop DNA
containing the U5 and repeat (R) sequences of the genome. Recently, it
was shown that although the initiation event, i.e. addition
of two deoxynucleotides to tRNALys-3, can be accomplished
in the mature virus (13, 17), the level of (
)-strand strong-stop DNA
in HIV-1 was ~100-fold less than the amount of tRNALys-3
alone or tRNALys-3 extended by 2 nt, suggesting further
elongation of (
)-strand DNA in the virion is a rare event (12,
13).
HIV-1 proviral DNA synthesis is accomplished by reverse transcriptase
(RT), a multifunctional, heterodimeric enzyme (p66/p51) containing a
catalytically competent 66-kDa subunit and an inactive 51-kDa subunit
(Fig. 1A). Although all retroviral RTs perform comparable
functions, many complex steps, such as initiation of ()-strand
synthesis, require virus-specific substrates and catalysts (17, 18).
For example, we recently observed that of several retroviral enzymes,
only HIV-1 and avian myeloblastosis virus RT efficiently initiated
(
)-strand DNA synthesis from unmodified or natural
tRNALys-3 primers annealed to the HIV-1 genome (18). This
finding was unexpected, since the RTs of HIV-2, simian immunodeficiency
virus, feline immunodeficiency virus, and equine infectious anemia
virus used in our study also exploit tRNALys-3 as primer
for initiation of reverse transcription (19). However, disrupting the
U5-IR loop-tRNALys-3 anticodon loop complex in the HIV-1
template-primer substrate restored the capacity for (
)-strand DNA
synthesis to the heterologous enzymes (18). Interaction of this A-rich
sequence 5' to the PBS with the tRNA primer appears unique to HIV-1,
since no barrier to tRNA-primed (
)-strand DNA synthesis by
heterologous RTs from PBS-containing RNA fragments of the equine
infectious anemia virus or feline immunodeficiency virus genome was
observed (18).
We and others (14-16, 18) have demonstrated that wild type HIV-1 RT is
required to disrupt the U5-IR stem and U5-IR loop-anticodon loop
complex by extending tRNALys-3 by 1-6 deoxynucleotides,
after which processive synthesis from the extended tRNA yields
full-length ()-strand strong-stop DNA. The first HIV-1 RT mutant
displaying a defect in this initiation program contained a 13-amino
acid deletion at the C terminus of p51 while retaining a full-length
p66 subunit. This mutant, p66/p51
13 RT, exhibits reduced affinity
for the free replication primer in a binary complex and an inability to
catalyze (
)-strand DNA synthesis from PBS-bound tRNA while
accomplishing the same event from a PBS-bound RNA primer (20, 21). In
addition, the 13-residue p51 truncation indirectly influences the
architecture of the p66 RNase H domain, evidenced by a dysfunctional
RNase H activity, which surprisingly could be restored by addition of
HIV-1 nucleocapsid protein (21, 22). For the present study, our goal
was to identify amino acids in p66 HIV-1 RT that might be specifically
implicated in the interaction with tRNALys-3 and subsequent
initiation of (
)-strand DNA synthesis without compromising any other
enzymatic function.
Although HIV-1 RT has been shown to interact with tRNALys-3
at all three loops (23-26), its affinity for most tRNA isoacceptors is
similar (27, 28), suggesting structure is the primary determinant of
this bimolecular interaction rather than sequence. Recently, a
127-amino acid cyanogen bromide (CNBr) cleavage fragment of p66 HIV-1
RT (residues 230-357 and depicted in white in Fig.
1A) was cross-linked to synthetic tRNALys-3
thiolated at U36 of the anticodon loop (1). By analyzing
this peptide fragment in the context of the three-dimensional structure
of HIV-1 RT (29, 30), we tentatively identified three basic residues
(Lys249, Arg307, and Lys311)
conserved in several HIV-1 isolates (31) and adjacent to a small
hydrophobic crevice in the thumb subdomain of p66 (Fig. 1A)
as targets for mutagenesis. In contrast to wild type RT, these mutants
showed greatly reduced affinity for tRNALys-3 and failed to
protect regions in the anticodon stem-loop from chemical cleavage, the
latter of which was evaluated via a novel in situ
footprinting approach. Although mutant enzymes retained the capacity to
initiate ()-strand DNA synthesis from PBS-bound tRNALys-3, subsequent elongation events were severely
reduced. General defects to polymerase function were ruled out by the
observation that the same mutants retained their ability to (i)
synthesize (
)-strand DNA from PBS-bound RNA and DNA primers and (ii)
catalyze synthesis-dependent and -independent modes of
RNase H activity. Collectively, our findings suggest that mutating
Lys249, Arg307, or Lys311 of the
thumb subdomain of the catalytically competent p66 subunit induces
structural alterations in p66/p51 HIV-1 RT which disrupt interactions
with its cognate tRNA replication primer.
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EXPERIMENTAL PROCEDURES |
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Mutagenesis and Purification of Recombinant HIV-1 RT--
p66
HIV-1 RT encoded by the vector pRT (20) was selectively substituted at
amino acids Lys249 (Lys249 Glu and
Lys249
Gln), Arg307 (Arg307
Glu and Arg307
Gln), Lys311
(Lys311
Glu and Lys311
Gln), and at
both 307 and 311 Arg307
Glu plus Arg311
Glu using the USE mutagenesis kit (Amersham Pharmacia Biotech). Mutant
p66 polypeptides were expressed in the Escherichia coli strain M15::pDM1.1 following induction with
isopropyl-1-thio-
-D-galactopyranoside (20) and then
mixed with recombinant bacteria expressing a polyhistidine-extended p51
subunit and homogenized (32, 33). Clarified supernatants following
ultracentrifugation were incubated with Superflow
Ni2+-nitrilotriacetic acid-Sepharose (Qiagen, Chatsworth,
CA) for 12 h at 4 °C. Spin columns were packed with
protein-bound nitrilotriacetic acid-Sepharose, washed with buffer, and
then eluted with a 0-0.5 M imidazole step gradient (32,
33). Fractions containing stoichiometric ratios of p66 and p51 were
pooled, dialyzed to remove imidazole, and further purified on
S-Sepharose by elution with a 0-0.5 M NaCl gradient. All
mutant RTs were devoid of nuclease contaminants and judged to be at
least 90% pure by SDS/polyacrylamide gel electrophoresis.
Primers and RNA Templates--
Four primers were utilized for
this study and placement of the radiolabel differed for the various
experiments. 18-nt DNA and RNA primers were 5'-end-labeled with
[-32P]ATP and T4 polynucleotide kinase according to
standard procedures. Synthetic tRNALys-3 for
electrophoretic mobility shift analyses was internally labeled with
-[32P]UTP during its in vitro synthesis
with T7 RNA polymerase (Ambion). Fully modified, natural
tRNALys-3 was labeled by extension with a single
deoxynucleotide (
-[32P]dCTP) utilizing the Klenow
fragment of E. coli DNA polymerase on a 60-nt DNA template
homologous to the region surrounding the PBS in HIV-1 genomic RNA (21).
3'-[32P]dCMP-labeled tRNALys-3 was purified
as described (21). In situ footprinting employed natural and
synthetic tRNALys-3 3'-end-labeled with
[32P]pCp by RNA ligase. Labeled nucleotides were obtained
from NEN Life Science Products and molecular biology reagents from
Boehringer Mannheim. Finally, a 237-nt HIV-1 PBS-containing RNA
template was prepared by in vitro transcription of
HgaI-linearized pHIV-1 PBS vector as described (21). This
template lacks the dimer initiation sequence located between the PBS
and gag initiation codon.
Gel Mobility Shift Analyses and Kd Determination-- Wild type and mutant enzymes were incubated for 10 min at room temperature with 3'-end-labeled natural, 32P internally labeled synthetic tRNALys-3 or an end-labeled tRNA/PBS RNA duplex, in binding buffer (25 mM KCl, 50 mM Tris, pH 7.4, 10% glycerol, and 1 mM dithiothreitol), then fractionated through a 5% non-denaturing polyacrylamide gel. Following electrophoresis, gels were dried and visualized by autoradiography. The stability of RT bound to free tRNALys-3 or tRNALys-3-template complexes was evaluated using a 4:1 ratio of RT (16 nM) to tRNA (4 nM), i.e. an RT concentration below the Kd for wild type RT binding to free tRNALys-3. Higher ratios of RT:tRNA resulted in a complete shift of free tRNALys-3 by wild type RT, whereas lower ratios prevented an analysis of tRNALys-3 binding by mutant RTs (data not shown).
In order to determine dissociation constants, the concentration of synthetic tRNALys-3 in reactions remained constant at 0.1 nM, whereas the RT concentration was increased from 0.1 nM to 1.3 µM. The binary RT·tRNA complex was separated from free tRNA on a 5% non-denaturing polyacrylamide gel, and the amount of shifted complex calculated from phosphorimaging analyses of dried gels and plotted against RT concentration. A hyperbolic curve was fitted to the plots, and dissociation constants were estimated from the RT concentration resulting in 50% shifting of synthetic tRNALys-3. Dissociation constants were determined as the average of three independent experiments.1,10-Phenanthroline-Copper Footprinting of RT·tRNALys-3 Complexes in Situ-- Higher concentrations of RT (200 nM) and 32P 3'-end-labeled tRNALys-3 (33 nM) were necessary to increase the amount of the binary RT·tRNA complex resolved by non-denaturing polyacrylamide gel electrophoresis as described above. Wet gels were exposed to Kodak Biomax MS film for 20 min to visualize the shifted complexes and free tRNA. Gel slices containing these bands were excised, immersed in 100 µl of 50 mM Tris, pH 7.4, and then probed with chemical reagents according to Kuwabara and Sigman (34).
Solutions of 40 mM 1,10-ortho-phenanthroline (OP), 28 mM 2,9-dimethyl-1,10-phenanthroline, both in 100% ethanol, and aqueous solutions of 9 mM CuSO4 and 58 mM 3-mercaptopropionic acid were immediately prepared prior to chemical footprinting. 40 mM OP and 9 mM CuSO4 were mixed at equal volumes and diluted 1:10 with water. 10-µl aliquots of this solution and 3-mercaptopropionic acid were added to the solution containing the gel slice, which was incubated for 5 min at room temperature. Chemical modification was quenched with 10 µl of 28 mM 2,9-dimethyl-1,10-phenanthroline. Modified tRNA was eluted from gel slices overnight at 37 °C after adding 270 µl of gel elution buffer (0.5 M ammonium acetate, 1 mM EDTA, 0.1% SDS). Cleaved tRNA was precipitated with ethanol, resuspended in loading buffer (90% formamide, 1 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol), and then fractionated on a 12% denaturing polyacrylamide gel. Gels were dried and visualized by autoradiography. Ladders of tRNALys-3 cleavage products generated by NaOH treatment were used as size markers. In order to control for nuclease contamination, RT·tRNA complexes and free tRNA were eluted from unprobed gel slices and fractionated in parallel with the experimental samples.RNA-dependent DNA Polymerase
Activity--
RNA-dependent DNA polymerase activity of
wild type and mutant enzymes was measured using an HIV-1 PBS-containing
RNA template to which a 32P 5'-end-labeled 18-nt DNA or RNA
primer or 77-nt chimera of natural tRNALys-3 containing
radiolabeled dCTP at its 3' terminus (21) was hybridized. Primers were
annealed to the 237-nt RNA template prior to addition of HIV-1 RT (57 nM), the appropriate Mg2+-containing buffer,
and dNTPs (18, 21). Reaction mixtures were incubated at 37 °C for 1, 10, and 80 min and then quenched with loading buffer. ()-Strand DNA
products were resolved on a 6 or 12% denaturing polyacrylamide gel and
visualized by autoradiography.
RNase H Activity-- RNase H activity was determined as described previously (20). Briefly, HIV-1 RT (57 nM) was incubated with a 36-nt DNA primer (40 nM) annealed to a 32P 5'-end-labeled 90-nt RNA template (40 nM) prior to the addition of Mg2+. Aliquots were removed at 10 s and 1, 10, and 30 min and supplemented with gel loading buffer. Hydrolysis products were fractionated on a 10% denaturing polyacrylamide gel which was subsequently dried and subjected to autoradiography.
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RESULTS |
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Potential tRNALys-3-binding Sites in HIV-1 RT-- Mishima and Steitz (1) have successfully cross-linked synthetic tRNALys-3, containing thiouridine at position 36 of the anticodon loop, to p66/p51 HIV-1 RT. CNBr cleavage of this complex indicated covalent linkage to a 127-amino acid fragment encompassing residues 230-357 of the p66 subunit (represented in white in Fig. 1), corresponding to the entire thumb and a portion of the connection subdomain. A prominent feature of anticodon loop of mammalian tRNALys-3 are two heavily modified nucleotides at positions 34 (5'-methoxycarbonylmethyl-2'-thiouridine, mcm5 s2U34) and 37 (2'-methylthio-N6-threonylcarbamoyl adenosine, ms2t6A37) (35). By using a space-filling model of HIV-1 RT (29), we tentatively identified 5 of 13 positively charged amino acids in this cross-linked fragment (Lys249, Lys259, Lys287, Arg307, and Lys311) which might lie on the surface of the p66 thumb subdomain. Of these Lys249, Arg307, and Lys311 appear clustered around a small crevice in the thumb opposite to the primer-template binding cleft (Fig. 1). Two proposed flaps, designated flap a and flap b in Fig. 1, have hydrophilic residues on the surface (marked by dotted line), whereas the crevice floor contains mainly hydrophobic residues.
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Affinity of HIV-1 RT Mutants for tRNALys-3--
To
determine the effects of the p66 point mutations on tRNA binding, we
performed gel mobility shift analyses with the wild type enzyme and the
above-mentioned variants. For comparison, the selectively deleted
mutant p66/p5113 was included. The results of our analysis are
presented in Fig. 2. A complex
characteristic of wild type HIV-1 RT bound to natural
tRNALys-3 (23, 37) is shown in Fig. 2A. At the
RT:tRNA ratio which was necessary to visualize complexes containing
mutant RTs, two retarded species (labeled a and b
in Fig. 2A) were identified for the wild type enzyme, the
slower-migrating of which (labeled a in Fig. 2A)
most likely represents a dimer of the binary
complex.2 Reduced stability
of the binary complex was clearly evident with all mutants. With
p66R307E/K311E/p51 RT and, to a lesser extent,
p66K311Q/p51 and p66K311E/p51 RTs, a third
species (labeled c in Fig. 2A) migrating faster than the tRNA·RT complex was observed. The migration position of this
complex correlates with a binary complex of p51 RT and tRNALys-3 previously described by Richter-Cook et
al. (37), suggesting these mutations might induce dissociation.
Although retaining a stable heterodimeric organization, data of later
sections indicate p66R307E/K311E/p51 RT has substantially
reduced DNA polymerase and RNase H activity, consistent with a more
pronounced structural perturbation.
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In Situ Footprinting Analysis of RT·tRNALys-3
Complexes via 1,10-Phenanthroline Copper Cleavage--
To determine
how mutations in the p66 thumb subdomain might affect an interaction
with the replication primer, free tRNALys-3 and binary
RT·tRNALys-3 complexes were excised from a non-denaturing
polyacrylamide gel and probed in situ with
1,10-phenanthroline copper (OP-Cu) according to the procedure of
Kuwabara and Sigman (34). Cleavage products were eluted from the gel
slice and fractionated by high resolution denaturing polyacrylamide gel
electrophoresis, the results of which are given in Fig.
3. OP-Cu preferentially intercalates and cleaves single-stranded RNA. Both the A conformation of double-stranded RNA and bulky ribonucleoside modifications (such as those in the anticodon domain of natural tRNALys-3) can prevent
intercalation of OP-Cu (38). Thus, enhanced OP-Cu cleavage in both the
anticodon and TC loops was observed in some single-stranded regions
of synthetic tRNALys-3 (Fig. 3A) although less
pronounced with the natural tRNALys-3 counterpart (Fig.
3B).
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()-Strand DNA Synthesis Supported by tRNALys-3-Viral
RNA Duplexes--
Previous studies indicated that mutant or non-HIV-1
RTs with reduced affinity for tRNALys-3 do not support
efficient synthesis of (
)-strand strong-stop DNA from
tRNALys-3 annealed to an HIV-1 RNA template (18, 21). Since
p66K249Q/p51, p66K311Q/p51,
p66K311E/p51, and p66R307E/K311E/p51 RT showed
reduced affinity for tRNALys-3, we compared their ability
to synthesize (
)-strand DNA from oligonucleotide primers and natural
tRNALys-3 annealed to an HIV-1 PBS-containing RNA genome.
The substrates employed in this assay and the predicted (
)-strand
strong-stop products are schematically represented in Fig.
4.
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()-Strand Strong-stop Synthesis from PBS-bound RNA and DNA
Primers--
Although the RT mutant p66/p51
13 is incapable of
supporting tRNALys-3-primed (
)-strand strong-stop DNA
synthesis, it efficiently copies the same viral RNA template when this
is substituted by RNA or DNA primers (18). Since important tRNA-viral
RNA loop-loop interactions outside the PBS (10, 11, 15) cannot be
established under the latter conditions, substitution of
oligonucleotide primers removes the intermolecular structural barrier
to initiation. We therefore elected to evaluate our mutants in the
context of RNA- and DNA-primed (
)-strand strong stop DNA synthesis,
the results of which are presented in Fig.
6.
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Evaluation of RNase H Activity--
The multifunctional nature
of HIV-1 RT allows mutations influencing DNA polymerase function
to be evaluated in the context of RNase H activity. The importance of
monitoring both activities was recently evidenced by an RT mutant
containing a point mutation at position 232 of the DNA polymerase
catalytic center (Y232A) which directed RNase H cleavage almost
exclusively to template nucleotide 8 rather than
17 (36). In light
of this, the RNase H activities of all mutants were evaluated on a
defined heteropolymeric RNA-DNA hybrid that monitors the hydrolysis
products of endonuclease (DNA synthesis-dependent) and
directional processing (DNA synthesis-independent) events. Substrate
for this analysis is outlined in Fig.
7A and the cleavage products
derived from each enzyme in Fig. 7B.
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DISCUSSION |
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Initiation of HIV-1 reverse transcription is a multi-step process,
involving intricate interactions between the retroviral polymerase and
its cognate tRNA primer, intertwined with the HIV-1 RNA genome at sites
including and surrounding the PBS. The anticodon loop of
tRNALys-3 appears essential to these interactions and
concomitant initiation of ()-strand DNA synthesis from the PBS (10,
11, 15, 16, 18, 21, 23). Independent experiments have demonstrated that this domain can be chemically cross-linked to RT in a binary
nucleoprotein complex (1, 23). Moreover, the anticodon loop has the
capacity for intermolecular base pairing with the A-rich U5-IR loop of HIV-1 RNA to provide an additional level of control/selectivity (10,
11, 15, 16, 18, 21). Although this A-rich sequence is not
phylogenetically conserved in non-primate lentiviruses or other
retroviruses (18, 42), it does appear that this element is important in
HIV-1 reverse transcription and replication (43). For example, a mutant
virus with a deletion of these four adenosines supported
substantially less (
)-strand DNA synthesis (43). Long term cultures
of this mutant HIV-1 resulted in the replacement of two or three
adenosines and the restoration of near wild type levels of (
)-strand
DNA synthesis and virus replication (43). The significance of the tRNA
anticodon loop during (
)-strand DNA synthesis is quite evident in
studies showing that the use of a tRNAHis isoacceptor as a
replication primer on a mutated HIV-1 RNA genome (i.e. a PBS
complementary to the 3' end of tRNAHis) was greatly
facilitated by substitutions of U5-IR loop bases for those
complementary to the tRNAHis anticodon loop (44).
In this study, we have combined cross-linking (1) and crystallographic
data of the p66/p51 heterodimer (29, 30, 45) to identify p66 residues
potentially involved in tRNALys-3 binding events, beyond
the duplex formed with the PBS, which promote efficient initiation of
()-strand synthesis. Our studies have focused on three residues
conserved in a large number of HIV-1 isolates, Lys249,
Arg307, and Lys311, which appear to line a
crevice of the p66 thumb subdomain (residues 244-322). We show here
that substitutions at these positions affect the following:
(a) the affinity of RT for tRNA; (b) the manner in which the tRNA primer is accommodated in a binary complex with RT;
and (c) the transition from initiation to elongation of
tRNA-primed (
)-strand synthesis. The same mutations have minimal
impact on either oligonucleotide-primed (
)-strand synthesis or RNase
H activity, suggesting their effects are localized to the manner in
which tRNALys-3 is utilized by HIV-1 RT.
From cross-linking and biochemical studies on HIV-1
RT-tRNALys-3 interactions (23-26, 35), we had predicted
that these mutations in the p66 component of p66/p51 RT might alter the
conformation of binary RT·tRNA complexes. Indeed, enhanced reactivity
over the entire anticodon domain was highlighted with two mutants via in situ footprinting with the chemical nuclease OP-Cu. Based
on the reactivity of OP-Cu toward single-stranded RNA, including bulges
and loops (46), the data of Fig. 3 suggest that much of the anticodon
domain has been unwound following substitution of p66 residue
Lys311 with either Glu or Gln. This effect is clearly
evident with synthetic tRNALys-3 and to a lesser extent
with the fully modified natural species (which may be attributable to
the presence of hyper-modified bases in the latter) (38). In addition
to altered protection of the anticodon domain, the TC loop of
synthetic tRNA is rendered hypersensitive when bound to
p66K311Q/p51 or p66K311E/p51. The tRNA
structure depicted in Fig. 3C indicates intramolecular pairing between bases of the T
C and D-loops, which also appears to
be disrupted following substitution of Lys311. Similarities
in the OP-Cu hydrolysis profiles of mutant p66K249Q/p51 and
wild type RT complexed with tRNALys-3 suggests this residue
may not play the same role in tRNA binding, although a contribution is
suggested by its 6-fold reduction in affinity for free tRNA (Fig.
2B). The inability of mutant RTs to extend PBS-bound
tRNALys-3 into full-length (
)-strand strong-stop DNA
(Fig. 5A) appears unrelated to the initiation event, since T + 3
T + 5 products accumulate in all cases (Fig.
5C). Moreover, the observation that (i) the affinity of
mutant enzymes for a tRNALys-3-viral RNA duplex (Fig.
5B) and (ii) DNA- and RNA-directed (
)-strand synthesis
(Fig. 6B) are largely unaltered, an unfavorable interaction of the tRNA anticodon domain with our RT mutants during or immediately following initiation might account for the reduced efficiency with
which this primer is utilized.
A consequence of pre-annealing tRNALys-3 to the viral
genome will be the establishment of intermolecular loop-loop
interactions (10, 11, 15, 16, 18, 21) prior to RT binding. Under such
conditions, viral template sequences ahead of the DNA polymerase catalytic center, together with the 18-base pair PBS/tRNA duplex, will
become major determinants of the strength of the preinitiation nucleoprotein complex (evidenced by the gel mobility shift data of Fig.
5B). Thus, reduced affinity of RT for free tRNA appears unlikely to account for the inability of our mutants to overcome the
tRNA-directed transition from initiation to elongation. An alternative
explanation for loss of tRNA-primed synthesis would be that mutant
enzymes, having initiated ()-strand synthesis, cannot disrupt the
U5-IR loop-tRNA anticodon loop interaction, with the consequence of a
stalled RT. Such a general defect also seems unlikely, considering the
same mutants efficiently extend a PBS-bound RNA primer through several
highly structured regions of the viral genome (Ref. 47 and Fig.
6A). In light of this, an explanation for our findings might
lie in the position the polymerase domain occupies on the template as
the tRNA primer is extended at the DNA polymerase catalytic center.
Independent lines of evidence indicate that the p66 N-terminal fingers
subdomain (residues 1-84 and 120-150) encompasses the single-stranded
template overhang approximately 5-7 bases ahead of the polymerase
catalytic center (48, 49). In the context of the tRNA-viral RNA
structure proposed by Isel and co-workers (10, 11, 15), extending
tRNALys-3 by three deoxynucleotides would translocate the
fingers subdomain such that it is positioned directly over the
intermolecular U5-IR loop-tRNA anticodon loop duplex. Should the
fingers participate in destabilizing ("unwinding") inter- and
intramolecular template duplexes for presentation at the polymerase
catalytic center, adding 3-5 dNTPs to tRNALys-3 3'
terminus would be accompanied by disruption of this intermolecular duplex. Once the tRNA anticodon loop is released from viral RNA within
the p66 fingers, it may be involved in an unfavorable interaction with
an alternate subdomain of the mutated RT (as depicted in Fig. 3),
thereby inducing stalling. Such an inhibition would suggest the need
for a specific interaction between wild type RT and the tRNALys-3 anticodon loop during initiation. Alternatively,
once released from the A-rich loop of HIV-1 RNA, these mutants fail to
fulfill a second interaction with the anticodon domain necessary for
allosteric activation of the replication complex. While speculative,
preliminary data suggest that tRNA-primed synthesis by our mutants is
inhibited on a viral template lacking the U5-IR loop -AAAA- sequence,
which prevents intermolecular loop-loop interactions from being
established and again leaves the tRNA anticodon domain available to
freely interact with RT.3
Moreover, an additional site for interaction of the tRNA anticodon loop
is suggested by recent studies on the strand transfer complex (50).
These hypotheses could be evaluated experimentally by determining the
location of the tRNA anticodon loop in "pseudo-initiation complexes" where a chimera of tRNA containing the first 3-5
deoxynucleotides of ()-strand DNA is hybridized to the viral template
(18, 21). Experiments of this nature are presently underway.
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ACKNOWLEDGEMENT |
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The use of core support facilities of the Center for AIDS Research at Case Western Reserve University is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant AI31147 (to S. F. J. Le G.). The use of core support facilities of the Center for AIDS Research at Case Western Reserve University was supported by National Institutes of Health Grant AI36219.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 a postdoctoral fellowship from Health and Welfare, Canada.
To whom correspondence should be addressed: Dept. of Medicine,
University Hospitals of Cleveland and Center for AIDS Research at Case
Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4984 Tel.: 216-368-6989; Fax: 216-368-2034; E-mail:
sfl{at}po.cwru.edu.
1 The abbreviations used are: HIV, human immunodeficiency virus; nt, nucleotides; PBS, primer binding site; RT, reverse transcriptase; OP-Cu, 1,10-phenanthroline-copper; IR, inverted repeat.
2 P. Dumas and B. Ehresmann, unpublished observations.
3 E. J. Arts, unpublished observations.
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