(Received for publication, November 3, 1995; and in revised form, January 25, 1996)
From the
The mechanism for the initiation of reverse transcription in
human immunodeficiency virus type 1 (HIV-1) was studied utilizing a
unique reverse transcriptase (RT) mutant altered in its noncatalytic
p51 subunit. This mutant (p66/p5113) retains full DNA- and
RNA-dependent DNA polymerase activity but has reduced affinity for
tRNA
, the cognate HIV primer. When the
ability to support(-)-strand DNA synthesis on a viral RNA
template was evaluated, this mutant initiated from an 18-nucleotide
(nt) oligoribo- or oligodeoxyribonucleotide primer complementary to the
primer binding site (pbs). However, it failed to do so from natural and
synthetic versions of tRNA
.
tRNA-primed(-)-strand synthesis could, however, be rescued by
substituting the 76-nt tRNA
with 81- and
107-nt tRNA-DNA chimeras, i.e. tRNA
extended by 5 and 31 deoxyribonucleotides complementary to the
viral genome upstream of the pbs. These findings imply that through
interactions involving its p51 subunit, RT may be required to disrupt
additional tRNA-viral RNA duplexes outside the pbs to proceed into
productive(-)-strand DNA synthesis. Alternatively, specific
interactions between tRNA
and HIV-1 RT may be
necessary for efficient initiation of(-)-strand DNA synthesis.
Initiation of reverse transcription in retroviruses is a complex
process requiring several key elements. These include virus-coded
reverse transcriptase (RT), ()a specific host tRNA, viral
genomic RNA, and auxiliary components such as the nucleocapsid protein.
Convergent evolution of retrovirus and host has most likely increased
the specificity of retroviral reactions requiring host molecules. Thus,
placement of a specific host tRNA on the retroviral genome and
utilization of the tRNA-viral RNA complex by a specific RT reflect
highly organized and distinct events. Recent studies provide increasing
evidence of extensive interplay between elements near the 5` end of the
retroviral genome and its cognate tRNA primer in controlling initiation
of(-)-strand DNA synthesis(1) . In addition to the
18-base pair intermolecular duplex of primer binding site (pbs) and
tRNA 3` nucleotides (Fig. 1, region A), biochemical and
genetic evidence (2, 3, 4, 5) suggests formation of a
viral RNA
tRNA complex involving the tRNA T
C arm and a
heptanucleotide sequence in the 5` unique (U5) sequence of viral RNA (Fig. 1, region B). Initially demonstrated for Rous
sarcoma virus (2) , sequence analysis indicates that this
complex may be generally applicable(5) . Chemical footprinting
of the leader-stem of HIV-1 genomic RNA and tRNA
(the HIV replication primer) has revealed another site of
intermolecular contact, i.e. between the tRNA U-rich anticodon
loop and the A-rich U5-IR loop of viral RNA immediately preceding the
pbs (Fig. 1, region C1) (6, 7) . In
contrast, Kohlstaedt and Steitz (8) suggest that the tRNA
anticodon loop interacts with viral RNA sequences immediately 3` to the
pbs (Fig. 1, region C2). Despite apparent
discrepancies, these findings collectively implicate higher order RNA
structures (9) in the ``preinitiation'' complex,
which must be disrupted subsequent to the onset of(-)-strand DNA
synthesis. Involvement of tRNA primers in such structures might ensure
that following sequestration into the virion, they are directed to the
pbs rather than other regions of the genome with which they display
reduced homology.
Figure 1:
Proposed regions of
intermolecular base pairing between tRNA and
the HIV-1 RNA genome outside the pbs. Left, a pbs-containing
portion of the HIV
genome (15) between
nucleotides 127 and 220. Annealing of 18 nt at the 3` end of
tRNA
and the pbs is illustrated in region
A. The anticodon loop of tRNA
is highlighted, together with HIV-1 sequences 5` (C1)
and 3` (C2) to the pbs. Regions C1 and C2 have been proposed by Isel et al. (7; see also (6) ) and Kohlstaedt and Steitz(8) , respectively, to
participate in an interaction with the anticodon loop of the primer. In
contrast, Leis et al. (4) propose a control mechanism
mediated through intermolecular base pairing between nucleotides of the
T
C loop and an octanucleotide sequence in U5 (region B).
Nucleotides representing the 3` termini of tRNA-DNA chimeras employed
in two-step DNA synthesis experiments of Fig. 5are indicated.
For illustrative purposes, only 25 3` tRNA nucleotides are represented
in the RNA/tRNA hybrid. Right, L-shaped configuration of human
tRNA
.
Figure 5:
Synthesis of(-)-strand strong stop
DNA from tRNA-DNA chimeras. A, schematic representation of the
two-step DNA synthesis experiment. The tRNA primer is hybridized to a 60-nt DNA template containing the viral
pbs and is preceded by 31 template nucleotides (step a). This
is subsequently extended by 1, 5, or 31 deoxynucleotides with the
Klenow fragment of DNA Polymerase I (step b). tRNA-DNA
chimeras are recovered by denaturing high voltage electrophoresis and
gel elution and then hybridized to the viral pbs-containing RNA
template (step c). Subsequent extension of the chimeric primer
yields a 250-nt(-)-strand strong stop DNA (step d). B, (-)-strand strong stop DNA synthesis from a 77-nt
tRNA-DNA chimera. Lane 1, wild type p66/p51 RT; lane
2, p66/p51
5; lane 3, p66/p51
9; lane 4,
p66/p51
13; lane 5, p66/p51
19. The migration
positions of the 77-nt tRNA-DNA chimera (T+1) and its 250-nt cDNA
extension product are indicated. C and D,(-)-strand strong stop DNA synthesis from an 81-nt (T+5) and 107-nt tRNA-DNA chimeras (T+31),
respectively. Lane notations are as in B. Lane C,
76-nt tRNA
.
We have demonstrated that activities of p66/p51
HIV-1 RT can be modulated through minor truncations in the C-terminal
``connection'' subdomain (10) of its noncatalytic p51
subunit(11) . In particular, reconstituted heterodimer whose
p51 subunit lacks residues Gln-Phe
(p66/p51
13) shows wild type polymerase activity from a
heteropolymeric template-primer while showing severely reduced affinity
for free tRNA
. These findings are consistent
with crystallographic data (10) proposing that the p51
connection subdomain interacts with the T
C and D arms of
pbs-bound tRNA, whose 3` terminus is extended into(-)-strand DNA
at the p66 polymerase catalytic center. The notion that
tRNA
and a template-primer duplex can
interact with different subdomains of RT is suggested by mutagenesis
studies on the ``primer grip''(12) . Altering
Trp
in the
12-
13 hairpin of this p66 motif
results in normal tRNA binding but severely reduced affinity for
template-primer (13) . (
)
Our results with RT
mutant p66/p5113, coupled with a potential contribution of
tRNA-viral RNA loop-loop interactions to initiation of (-)-strand
DNA synthesis, prompted us to evaluate how an enzyme with reduced
affinity for free tRNA
interacted with and
utilized the pbs-bound intermolecular duplex. We demonstrate here that
although p66/p51
13 RT efficiently extends 18-nt DNA and RNA
primers hybridized to a pbs-containing viral RNA template, it fails to
do so when these are replaced by natural or synthetically prepared
tRNA
. However, if the 76-nt tRNA is replaced
with 81- or 107-nt tRNA-DNA chimeras (i.e. tRNA extended by 5
or 31 deoxynucleotides at its 3` terminus), the ability of
p66/p51
13 RT to support(-)-strand strong stop DNA synthesis
is restored. This is not the case with a tRNA primer whose 3` terminus
contains a single deoxynucleotide. These findings suggest that the tRNA
binding deficiency of p66/p51
13 RT is further manifested in an
inability to disrupt loop-loop interactions of the tRNA-viral RNA
preinitiation complex. Properties of additional deletion mutants
provide further insights into structural elements of the p51 connection
subdomain, which might participate in tRNA binding.
Figure 2: A, ability of p66/p51 HIV-1 RT and selectively deleted mutants to support programmed DNA-dependent DNA synthesis. DNA synthesis was evaluated with a heteropolymeric 71-nt template/36-nt primer in the presence (lanes a and b) and the absence of the competitor heparin (lanes c and d). Lanes a and c, +4 primer extension reaction; lanes b and d, +19 primer extension reaction. Migration positions of unextended 36-nt primer (P), together with its 4- and 19-nt extension products, are indicated. B, programmed RNA-dependent DNA synthesis. For these experiments, a 90-nt template hybridized to the 36-nt DNA primer of A served as substrate. Lane notations and primer extension products are as in A.
The 497-nt
pbs-containing HIV-1 RNA template of Fig. 3A contains
repeat, U5, pbs, and 5` noncoding sequences (17) predicted to
adopt extensive intramolecular base pairing (7, 19) ,
in addition to intermolecular structures with the anticodon and
TC loops of
tRNA
(3, 4, 5, 6, 7, 8) .
Mutants p66/p51
5 and p66/p51
9 were capable of extending both
synthetic and natural variants of tRNA
into
full-length(-)-strand strong stop DNA, although with lower
efficiency than the parental enzyme (Fig. 3B, lanes
1-3). The efficiency of (-)-strand DNA synthesis from
natural tRNA was
10-fold greater than from its synthetic
counterpart, which is in keeping with predictions of Isel et al.(6, 7) from chemical probing studies of
tRNA-viral RNA duplexes. In contrast, the ability of mutants
p66/p51
13 and p66/p51
19 RT to support DNA synthesis from
either natural or synthetic tRNA
was severely
compromised (Fig. 3B, lanes 4 and 5).
A 100-fold drop in(-)-strand strong stop DNA synthesis was
estimated between reactions catalyzed by wild type enzyme and mutant
p66/p51
13, whereas product was completely absent from those
catalyzed by p66/p51
19. The experiment of Fig. 2B utilizes internally labeled nascent(-)-strand DNA and
therefore failed to address whether mutants p66/p51
13 and
p66/p51
19 RT could initiate(-)-strand DNA synthesis. In
order to address this, the analysis was repeated with internally
labeled, synthetically prepared tRNA
(Fig. 3C). The virtual absence of
stalled(-)-strand products in the vicinity of the tRNA primer in
reactions catalyzed by mutants p66/p51
13 and p66/p51
19
suggests a deficiency prior to initiation rather than during
elongation. Reducing the salt concentration from 100 to 5 mM allowed limited tRNA-primed (-)-strand synthesis by mutant
p66/p51
13 but still substantially less than with the wild type
enzyme under the same conditions (data not shown).
Figure 3:
tRNA-primed(-)-strand
strong stop DNA synthesis. A, schematic representation of the
exogenous DNA synthesis reaction. The 18-nt viral pbs sequence is bold and is preceded by 173 nt from the repeat and U5 region
of the RNA genome. Intact(-)-strand strong stop DNA is evidenced
by a 250-nt tRNA-DNA chimera. B, tRNA-primed DNA synthesis
activity of selectively deleted HIV-1 RT mutants. In these experiments,
nascent DNA was internally labeled with
P. Lane notations S and N refer to DNA synthesis primed with synthetic (i.e. unmodified) and natural (i.e. fully modified)
tRNA
, respectively. Lane 1, wild
type p66/p51; lane 2, p66/p51
5; lane 3,
p66/p51
9; lane 4, p66/p51
13; lane 5,
p66/p51
19. C, extension of
P internally
labeled synthetic tRNA
into(-)-strand
strong stop DNA by RT mutants. Lane notations are as in B. Lane C, unextended 76-nt tRNA
primer.
Figure 4:
Oligonucleotide-primed synthesis of
(-)-strand strong stop DNA. A, schematic representation
of the in vitro system. An 18-nt oligoribo- or
oligodeoxyribonucleotide hybridized to viral pbs-containing RNA yields
a 192-nt(-)-strand strong stop product. B, ability of RT
mutants to support synthesis of(-)-strand strong stop DNA from an
oligoribonucleotide primer complementary to the pbs. Lane 1,
wild type p66/p51 RT; lane 2, p66/p515; lane 3,
p66/p51
9; lane 4, p66/p51
13; lane 5,
p66/p51
19. C, oligodeoxynucleotide-primed synthesis
of(-)-strand strong stop DNA. Lane notations are as in panel B.
Data of Fig. 4C clearly illustrate that the
inability of p66/p5113 RT to initiate(-)-strand DNA
synthesis from pbs-bound tRNA cannot be attributed to the nature of the
primer 3` terminus or features of the viral RNA template. This could
imply that recognition and destabilization of an intermolecular duplex
between the viral RNA template and the T
C loop of tRNA primer by
RT (6, 7) may be a prerequisite to efficient
initiation of(-)-strand DNA synthesis.
Fig. 5B indicates that wild
type RT supports(-)-strand DNA synthesis from a 77-nt tRNA-DNA
chimera, i.e. the 76-nt tRNA containing dC at its 3` terminus.
Although the activity of p66/p515 and p66/p51
9 RT with this
primer is reduced, the levels are in keeping with the data of Fig. 3(B and C). In contrast, p66/p51
13
RT is unable to overcome the barrier to initiation of(-)-strand
DNA synthesis. If, however, the 77-nt primer is replaced by an 81-nt
tRNA-DNA chimera, (i.e. tRNA
containing the 3` extension dC-dT-dC-dT-dA) p66/p51
5,
p66/p51
9, and p66/p51
13 RT support levels of (-)-strand
DNA synthesis comparable with that of the parental heterodimer, whereas
mutant p66/p51
19 remains inactive (Fig. 5C). Region C1 of Fig. 1may be disrupted with
tRNA
extended at its 3` terminus by 5
deoxynucleotides (81-nt tRNA-DNA chimera). However, extension of
tRNA
by a single deoxynucleotide (77-nt
tRNA-DNA chimera) would be predicted to have only a minimal effect on
the loop-loop interactions between tRNA
and
the viral RNA template.
DNA synthesis by p66/p5113 RT was also
restored when a 107-nt tRNA-DNA chimera (tRNA
extended by 31 deoxynucleotides at its 3` terminus) was
hybridized to the viral RNA genome (Fig. 5D). However,
significantly increased pausing by all RTs was observed in reactions
primed by the T+31 chimera compared with those primed by the
shorter chimeras or tRNA
. The 107-nt tRNA-DNA
chimera would be predicted to anneal to regions C1 and B of Fig. 1, which have been proposed to interact with
tRNA
via its anticodon (6, 7) and T
C
loops(2, 3, 4, 5) , respectively.
The complex involving region C2 of Fig. 1may also be
destabilized by the use of a T+31 chimeric primer due to a
complete unwinding of the U5-IR stem-loop.
Figure 6:
RNase H activity of selectively deleted
HIV-1 RT mutants. A, schematic representation of the RNase H
assay. Template and primer nucleotides occupied by RT (filled
ellipsoids) in the absence of DNA synthesis are those determined
by a combination of molecular modeling and DNase I
footprinting(26) . Initial endonucleolytic hydrolysis of the
template at position -17 generates a 71-nt RNA. Subsequent
directional processing activity to template position -8 reduces
the size of this fragment to 62 nt(14) . B, RNase H
activity of selectively deleted mutants. Hydrolysis products were
visualized after 5 s (left) and 5 min (right) of
incubation with wild type or mutant enzyme. Lanes 1, wild type
p66/p51; lanes 2, p66/p515; lanes 3,
p66/p51
9; lanes 4, p66/p51
13; lanes 5,
p66/p51
19. The sizes of hydrolysis products were determined by
co-electrophoresis of DNA sequencing
ladders.
Fig. 6A provides a schematic representation of the
RNase H assay. Enzyme located over the primer 3` OH will cleave the RNA
template nucleotide -17, which defines the spatial separation of
the DNA polymerase and RNase H
domains(8, 12, 22) . In our system,
endonucleolytic cleavage results in release of a 71-nt product.
Subsequent directional processing as far as position -8 yields a
62-nt product. These two activities and their uncoupling were recently
described by Ghosh et al.(14) . In Fig. 6B, the -17 hydrolysis product can be
visualized following short incubation with wild type RT and with
prolonged incubation is replaced with the -8 product (Fig. 6B, lanes 1). Although the rate of
hydrolysis is slightly slower for the selectively deleted mutants
p66/p515 and p66/p51
9, their cleavage specificity is
retained. In contrast, the RNase H activity of p66/p51
13 RT is
quite different. Following short incubation, additional -21 and
-25 hydrolysis products were evident, reflecting a relaxed
specificity of the RNase H domain. Furthermore, with prolonged
incubation, hydrolysis was only observed as far as template nucleotide
-17. Hydrolysis products representing -17, -21, and
-25 intermediates were also produced by mutant p66/p51
19
although greatly reduced. Thus, in an analogous manner to the findings
of Ghosh et al.(14) directional processing activity
of p66/p51
13 RT appears to have been lost. However, although Ghosh et al.(14) achieved this phenotype by altering the
p66 RNase H domain, the data of this communication illustrate that the
same can be achieved by subtle alterations to the geometry of an intact
RNase H domain.
Genetic (2, 3, 4, 5) and
biochemical data (6, 7, 8) point increasingly
toward higher order RNA structures outside the pbs controlling
initiation of retroviral replication. Although alternative tRNA-viral
RNA duplexes have been proposed, each implicates loop-loop interactions
involving bases of the anticodon (6, 7, 8) or
TC arms (2, 3, 4, 5) of the
tRNA primer. For HIV, the importance of these structures has been
investigated with a unique RT mutant lacking 13 C-terminal p51 residues
(p66/p51
13) whose DNA polymerase activity on heteropolymeric RNA
and DNA templates was unaffected. However, unlike wild type enzyme,
this mutant failed to initiate(-)-strand DNA synthesis from
tRNA
annealed to an HIV-1 pbs-containing RNA
template. This feature was specifically
tRNA
-dependent, because p66/p51
13 could
extend pbs-bound RNA or DNA primers to full-length(-)-strand
strong stop DNA. This mutant will, however, catalyze synthesis of
(-)-strand DNA from tRNA
containing
deoxynucleotide extensions of 5 and 31 nt at its 3` terminus, which
increase homology with the viral genome to 23 and 49 nt, respectively.
Restoration of DNA synthesis by the two chimeras suggests disruption of
tRNA-viral RNA structures by the mutant. Alternatively (although less
likely), this could reflect increased affinity of the mutant for an
extended nucleic acid duplex of chimeric primer and RNA template.
The connection subdomain at the p51 C terminus has been proposed to
accommodate the anticodon and D stems and loops of
tRNA(10) . However, recent data
suggest that only residues in the p66 subunit interact directly with
tRNA
(23) . In all available
structures, residues 421-426 of the p51 C terminus are found as a
short
helix (
)and form a key part of the interface
between the p66 RNase H domain and the p51 connection
subdomain(8, 12, 22) . Only in crystals of
the unliganded HIV-1 RT with a full 440-residue p51 are residues
427-433 visible and shown to extend toward the p51 connection and
palm subdomains, opposite the nucleic acid binding cleft. Although the
p51 subunit may not be in direct contact with primer tRNA(23) ,
truncating its C terminus could alter the subunit interface and disrupt
the structural framework required for a proper interaction of the p66
subunit with tRNA
. These alterations must be
subtle, because (i) p66/p51
13 accommodates and extends
oligonucleotide DNA and RNA primers as efficiently as the parental
enzyme ( Fig. 2and Fig. 4) and (ii) the tRNA priming
deficiency is rectified with p66/p51
9, which restores only four
C-terminal residues to p66/p51
13.
The same nucleotides of the
anticodon loop of tRNA involved in specific
binding to RT (U
-U
) (24) interact with the A-rich U5-IR loop of viral RNA (Fig. 1, region C1)(6, 7) . By binding
to tRNA, wild type RT positioned over the primer terminus may
destabilize this loop-loop structure, as well as other tRNA-viral RNA
interactions to initiate DNA synthesis. p66/p51
13 RT, whose
affinity for the free tRNA primer is reduced(11) , appears
unable to disrupt these tRNA-viral RNA strucutres and subsequently
initiate(-)-strand DNA synthesis from pbs-bound tRNA. Annealing
of 81- (T+5) and 107-nt (T+31) tRNA-DNA chimeras to viral RNA
would selectively disrupt two proposed structures, i.e. those
between the anticodon loop/U5-IR loop (Fig. 1, region
C2) (6, 7) and the T
C loop/U5-IR stem (Fig. 1, region B)(4) , respectively. These
tRNA-DNA chimeras were efficiently utilized as primers by
p66/p51
13 RT, suggesting that constraints preventing this mutant
from extending tRNA
or a 77-nt (T+1)
tRNA-DNA chimera are released. Thus, disruption of the anticodon
loop-U5-IR loop structure may be required for p66/p51
13 to
productively use the tRNA primer.
As predicted by computer-generated
nucleic acid folding programs, extending tRNA by 5 nt is unlikely to
disrupt a proposed interaction between its TC loop and the U5-IR
stem of the viral RNA (Fig. 1, region B), suggesting
this intermolecular interaction is less critical for HIV replication
than was recently demonstrated for Rous sarcoma
virus(2, 3, 5) . In support of this notion,
inspection of several HIV-1 genomes suggests that homology to the tRNA
primer can often extend into viral sequences directly 3` to the pbs.
This would have the consequence of making the T
C loop unavailable
for hybridization to the U5-IR stem while having little effect on the
anticodon loop-U5-IR loop interaction. However, it is possible that
alternative structures have evolved to control initiation of
replication in different retroviruses. For example, the U5-IR loop of
other retroviral genomes have considerably lower probability for a
loop-loop interaction with anticodon bases of their cognate tRNA primer (4) . Furthermore, an interaction between the D arm of
tRNA
and genomic sequences 50-100 nt
downstream of the pbs has also been proposed to play a role in
initiation of(-)-strand synthesis in the retrotransposon
Ty(25) . Thus, although different structures are invoked,
recognition of an extensively base paired tRNA-viral RNA preinitiation
complex appears a mechanism common to retroviruses and retrovirus-like
elements. The results of this communication also appear to contradict
the data of Kohlstaedt and Steitz(8) , who suggested that the
interaction of an A-rich segment of the viral genome 3` to the pbs and
the tRNA anticodon loop provides an important control mechanism. A
possible reason for this discrepancy might lie in the fact that these
authors used tRNA hybridized to a short DNA template rather
than a larger portion of viral RNA genome, which may prevent
formation of stable intermolecular base paired structures proposed by
others(2, 3, 4, 5, 6, 7) .
However, it is equally conceivable that retroviral initiation proceeds
through several conformationally distinct nucleoprotein complexes prior
to productive elongation, one of which involves the A-rich loop 3` to
the pbs. Clearly, additional studies are required to more precisely
evaluate conformational changes in the initiation complex and their
susceptibility to therapeutic attack.
An alternative mechanism for
the restoration of tRNA-primed DNA synthesis to p66/p5113 by
tRNA-DNA chimeras could be that extended complementarity to the viral
RNA template renders initiation events independent of structural
features of the tRNA primer. In recent work (26) , we
demonstrated that p66/p51 HIV-1 RT accommodates 23 or 24 base pairs of
the template-primer duplex during DNA-dependent DNA synthesis. A hybrid
between 81-nt tRNA-DNA chimera and viral genome may allow the 23 base
pairs of template-primer duplex to be better accommodated by the
replicating enzyme. Inefficient use of tRNA or a 77-nt (T+1)
tRNA-DNA chimera as primer might then suggest that the duplex region of
these template-primers were too short to stably bind p66/p51
13.
However, this hypothesis would be inconsistent with our finding that
18-nt DNA or RNA primers were more efficiently extended
into(-)-strand DNA than tRNA
or any
chimera with extended homology to the RNA template.
Finally, data
presented here provide a good example of how subunit and subdomain
interactions influence the multiple activities of this highly versatile
retroviral enzyme. Although the RNase H domain of mutant p66/p5113
is intact, loss of directional processing activity suggests that the
architecture of the C-terminal p66 domain can be subtly altered by a
minor truncation of the p51 subunit. The same architectural alteration
may also affect the interaction with tRNA
.
Although the complete 440-residue p51 subunit is used here,
heterodimeric RT has been prepared from recombinant microorganisms
where a bacterial protease cleaves p66 in the vicinity of the p51-p66
junction rather than directly between Phe
and
Tyr
(10, 12) . The possibility therefore
exists that the DNA polymerase and RNase H activities of such enzymes
may be altered and escape detection by conventional analyses, which
often measure the total amount of product rather than its nature.