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Journal of Virology, November 1998, p. 9353-9358, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Roles of the Human Immunodeficiency Virus Type 1 Nucleocapsid
Protein in Annealing and Initiation versus Elongation in Reverse
Transcription of Viral Negative-Strand Strong-Stop DNA
Liwei
Rong,1
Chen
Liang,1
Mayla
Hsu,1
Lawrence
Kleiman,1
Patrice
Petitjean,2
Hugues
de
Rocquigny,2
Bernard P.
Roques,2 and
Mark A.
Wainberg1,*
McGill University AIDS Centre, Lady Davis
Institute
Jewish General Hospital, Montreal, Quebec, Canada H3T
1E2,1 and
Département de
Pharmacochimie Moléculaire et Structurale, U266 INSERM-URA D1500
CNRS, UER des Sciences Pharmaceutiques et Biologiques, 75270 Paris
Cedex 06, France2
Received 17 February 1998/Accepted 16 July 1998
 |
ABSTRACT |
To study the initiation of human immunodeficiency virus type 1 reverse transcription, we have used the viral nucleocapsid protein
(NC7) to anneal tRNA3Lys primer onto viral genomic RNA
and have then eliminated NC7 from this primer-template complex by
digestion with proteinase K and phenol-chloroform extraction of
residual protein. Our data show that saturating concentrations of NC7
resulted in the formation of an active tRNA-template complex that
yielded enhanced production of full-length negative-strand strong-stop
DNA [(
)ssDNA] and that this complex remained active even after the
elimination of NC7. While both of the two Zn finger motifs found within
NC7 were essential for efficient elongation, NC protein that contained a point mutation in the first Zn finger or that was devoid of both Zn
fingers yielded primer-template complexes that could still be initiated
in 1-base-extension assays. In contrast, the use of heat annealing to
produce primer-template complexes resulted in proportions of
full-length ()ssDNA lower than those seen with NC protein, and the
addition of NC protein to such preformed primer-template complexes was
able to reverse this defect only to a marginal extent.
| |
TEXT |
The human immunodeficiency virus
type 1 (HIV-1) nucleocapsid protein (NC) is processed from Gag
precursors and is able to bind tightly to viral genomic RNA (2, 6,
16, 17, 34, 37). Accordingly, NC can function as a portion of the
Gag polyprotein precursor, essential for viral genomic packaging, and,
as well, as a fully processed protein in reverse transcription steps
that occur during de novo infection (4, 11, 14). At least
three distinct roles are associated with NC in the context of reverse transcription: (i) it facilitates the annealing of the cognate primer of reverse transcriptase (RT), i.e.,
tRNA3Lys, to the primer binding site (PBS) of the
viral RNA template (3, 10, 24, 35); (ii) it stimulates
specific viral DNA synthesis by reducing self-primed reverse
transcription (15, 25, 26) or by enhancing the processivity
of RT (20, 36, 38) during the synthesis of viral
negative-strand DNA; and (iii) it promotes the first template switch in
RT reactions to yield a full-length negative-strand DNA product
(5, 15, 30, 33, 39).
Although the ability of NC to anneal primer tRNA onto the PBS has
been demonstrated mostly in in vitro systems, the biological relevance
of this process is highlighted by results showing that such tRNA
placement is impeded in viruses with mutated NCs (18). NC
probably facilitates tRNA placement in vivo while it is part of either
the Gag or Gag-Pol precursor protein.
The structures of retroviral NCs are highly conserved, and, with the
exception of spumavirus NCs, they characteristically contain one or two
copies of a CCHC motif that can bind Zn2+ with high
affinity to form Zn fingers (7, 28). The NC of HIV-1
contains two such Zn fingers flanked by regions rich in basic residues.
Mutagenesis in the Zn fingers affects both the quantity of genomic RNA
that is packaged into virions and viral infectivity (1, 8, 13,
31).
The role of NC in the annealing of tRNA to the PBS is thought to
involve the melting of RNA secondary structure, a concept supported by
the ability of NC to unwind primer tRNA (22). However, the structure-function relationship in this process is not well understood, nor is the fact that NCs in which the Zn finger regions have been deleted retain the ability to both bind and anneal RNA in
vitro (10, 24). In contrast, Zn fingers in other, nonviral nucleic acid-binding proteins can function as nucleic acid interaction domains (29).
We have studied a synthetic form of NC (i.e., NC7) (9), a
naturally observed cleavage product of the Gag precursor protein, during the annealing of primer tRNA3Lys to viral
genomic RNA. In doing so, we have distinguished the activities of this
protein in the tRNA3Lys annealing process from that of
synthesis of negative-strand strong-stop DNA [()ssDNA] by removing
NC7 from the viral RNA template by use of proteinase K and
phenol-chloroform extraction of annealed template-primer complexes;
these digested complexes were then studied in reverse transcription
reactions.
(This work was performed by Liwei Rong in partial fulfillment of the
requirements for a Ph.D. from McGill University, Montreal, Quebec,
Canada.
The manner of placement of tRNA3Lys onto the
HIV-1 RNA template can affect the efficiency of elongation of reverse
transcription.
To study the role of NC7 in the synthesis of
()ssDNA, we used a cell-free reverse transcription reaction
consisting of viral RNA template, human tRNA3Lys, RT
(p66/51) NC7, and RT (p66/51) (36). The PBS wild-type (wt) construct was generated as previously described (2),
linearized by BssHII, and used as a template in an Ambion
Mega-Scripts kit (Austin, Tex.) to produce an RNA transcript. The RNA
template thus generated is 251 nucleotides (nt) in length and includes HIV sequences (nt 17 to 254) as well as a short 13-nt stretch derived
from the PBS wt vector. tRNA3Lys was purified in our
laboratories from human placenta as previously described
(21). In vitro reverse transcription initiated by tRNA3Lys from this template yielded a full-length
()ssDNA product of 178 nt joined to the 76-nt tRNA primer. The
placement of primer tRNA3Lys onto the viral RNA
template was performed in a 10-µl reaction mixture containing 50 mM
Tris-HCl (pH 7.2), 50 mM KCl, 5 mM MgCl2, 1 pmol of
template RNA, and 1 pmol of tRNA3Lys. Various
concentrations of NC7 were added into the reaction mixtures, which were
then incubated at 37°C for 1 h. When placement was carried out
by heat annealing, the reaction mixtures were incubated as described
previously for 5 min at 85°C and then for 10 min at 55°C
(27). Bovine serum albumin was used as a control protein in
both heat annealing and NC placement reactions and did not have either
a positive or a negative effect (data not shown).
After the placement of tRNA3Lys onto the viral RNA
template by either NC7 or heat annealing, single-nucleotide extension
by incorporation of [
-32P]dCTP, mediated by 50 ng of
RT, was performed in a total volume of 20 µl. One microliter of a 2 mM mixture of all four deoxynucleoside triphosphates was added to
achieve further extension to generate full-length ()ssDNA. Reactions
were terminated at 16 min by adding EDTA (pH 8.0) at a final
concentration of 50 mM. The amounts of both full-length ()ssDNA
and total cDNA products were quantified by molecular imaging
using a program provided by the manufacturer (Bio-Rad, Mississauga,
Ontario, Canada).
In order to understand the role of NC7 during the synthesis of
(
)ssDNA, we studied the effects of varying concentrations
of this
protein (i.e., 5, 15, 30, and 45 pmol, corresponding to
1 molecule of
NC7 per 50, 17, 8, and 6 nt residues, respectively)
in comparison to
reactions in which tRNA
3Lys had been placed onto the
RNA template by heat annealing. The
proportion of full-length (
)ssDNA
product relative to total cDNA
generated was calculated as a measure of
the efficiency of elongation
of reverse transcription. The results in
Fig.
1A (lanes 1 to 5)
show that the use
of increasing concentrations of NC7 in the placement
of
tRNA
3Lys onto viral RNA resulted in an increased
proportion of full-length
(
)ssDNA in comparison to total cDNA
product. The data show an
increase in the amount of total cDNA product
up to an NC7 concentration
of 15 pmol (Fig.
1A, lanes 1 to 3), followed
by a decline when
higher NC7 concentrations were used (Fig.
1A, lanes 4 and 5).
However, at these higher NC7 concentrations, the production of
nonspecific DNA products was also decreased, while the proportion
of
full-length (
)ssDNA that was made relative to total cDNA product
continued to increase. At an NC7 concentration of 45 pmol, more
than
two-thirds of the total cDNA product was present as full-length
(
)ssDNA. Therefore, the efficiency of elongation was generally
proportional to the amount of NC7 used to form the
tRNA
3Lys-RNA template complex (see Fig.
1A, graph).
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FIG. 1.
Dose-response curve of ()ssDNA synthesis. (A)
Complexes of tRNA and viral RNA template were formed by either wt NC7
(lanes 1 to 5), mutated H23C NC (lanes 6 to 10), or mutated
ddNC (lanes 11 to 15), and reactions were carried out for 16 min as
described in Materials and Methods. The concentrations of wt or mutated
NCs used in these reactions were 0 (lanes 1, 6, and 11), 5 (lanes 2, 7, and 12), 15 (lanes 3, 8, and 13), 30 (lanes 4, 9, and 14), and 45 (lanes 5, 10, and 15) pmol per reaction. The proportions of full length
()ssDNA compared with total cDNA product in these reactions are shown
in the graph. (B) Primer-template complexes were generated by heat
annealing. Varying amounts of wt NC7 (lanes 2 to 5), mutated
H23C NC (lanes 6 to 9), or mutated ddNC (lanes 10 to 13)
were added during the primer elongation phase of these reactions.
Lane 1, control reaction performed without NC. Lanes 2, 6, and 10, lanes 3, 7, and 11, lanes 4, 8, and 12, and lanes 5, 9, and 13, reactions performed with 5, 15, 30, and 45 pmol of NC per reaction,
representing occlusion by one NC molecule of 50, 17, 8, and 6 nt,
respectively. Band positions represent the first nucleotide added at
the 3' end of tRNA3Lys, and relative quantification of
amounts of product was carried out by molecular imaging, by using a
program that provides counts per minute equivalents, as suggested by
the manufacturer (Bio-Rad Instruments).
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|
Because NC7 is thought to exert its effect on the efficiency of viral
cDNA synthesis through disruption of the secondary structure
of the RNA
template (
12), we also studied tRNA
3Lys-RNA
template complexes that had been generated by heat annealing.
In this
circumstance, varying concentrations of NC7 were added
only later,
during the primer elongation stage of these reactions.
The results
in Fig.
1B show that the presence of NC7 in these
reactions, even at
very high concentrations, resulted in only
a modest increase in the
yield of full-length (
)ssDNA. Furthermore,
the addition of NC7, at
times after the heat annealing of tRNA
3Lys to viral
RNA, never yielded full-length (
)ssDNA products that
represented more
than 25% of total cDNA synthesis. Therefore,
the increased efficiency
of elongation in the synthesis of (
)ssDNA,
in reactions in which NC7
was used to promote the formation of
the primer-template complex,
cannot be attributed solely to the
role of NC7 during primer
elongation but must also involve aspects
of NC7 function during the
placement of tRNA
3Lys onto the viral RNA template.
The role of NC7 in formation of a
tRNA3Lys-RNA binary complex with potential to yield
high levels of full-length ()ssDNA product.
To further
verify a role for NC7 in primer placement and the synthesis of
()ssDNA, tRNA3Lys was placed onto the viral RNA
template by use of varying concentrations of NC7 or by heat annealing.
Next, the proteins in these reactions were eliminated by the addition
of 1 µl of proteinase K (5 mg/ml) at 37°C for 15 min, following
which both the proteinase K and undigested residual NC were extracted
with phenol-chloroform. Then the binary tRNA-RNA template complexes in
the liquid phase were precipitated at 20°C for 6 h by using an
equivalent volume of isopropanol. After precipitation, the
tRNA3Lys-RNA template complex was redissolved in the
initial reaction buffer, and reactions continued in the presence of RT
and [-32P]dCTP.
The results in Fig.
2 show that NC7 had
again acted in a concentration-dependent manner during
tRNA
3Lys placement to promote primer elongation,
compared with results
obtained when primer-template complexes had
been generated by
heat annealing. Concentrations of NC7 of >30 pmol,
i.e., saturating
levels, were especially active in this regard. Figure
2, lanes
3 and 4, shows that overall levels of total cDNA product
dropped
significantly when a concentration of 5 or 15 pmol of NC7 was
used and that no full-length (
)ssDNA products were generated
under
these conditions. In this experiment, NC7 had been eliminated
from the
template-primer complex by exposure to proteinase K and
chloroform-phenol; primer elongation occurred in the presence
of
the annealed tRNA
3Lys-viral RNA complex, RT, and
deoxynucleoside triphosphates only.
Therefore, differences among
reactions with regard to the generation
of full-length (
)ssDNA can be
attributed only to qualitative
differences between the types of
template-primer complex formed
in the presence of varying
concentrations of NC7, with the highest
efficiency occurring when NC7
was used at saturating conditions.
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FIG. 2.
Efficiency of elongation of tRNA-template complexes
formed with varying concentrations of NCs. tRNA3Lys was
placed onto the viral RNA template in the presence of varying
concentrations of NC7 to achieve the NC7/nucleotide ratios described in
the legend to Fig. 1. The primer-template complexes were then
treated with proteinase K and phenol-chloroform as described in
Materials and Methods, after which the reactions were reconstituted
with recovered primer-template complex. Lane 1, control reaction
representing the elongation efficiency of the primer-template
complex formed by heat annealing. Lanes 2 to 6, elongation efficiencies
of complexes formed by the addition of 0, 5, 15, 30, and 45 pmol of NC7
per reaction, respectively. Band positions represent the first
nucleotide added to the 3' end of tRNA3Lys; relative
quantification was carried out by molecular imaging. The proportion of
()ssDNA relative to the total amount of cDNA produced in each
reaction is presented in the graph.
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|
The NC7 Zn fingers are important in the formation of
primer-template complexes with the potential to participate in
highly efficient elongation.
We next examined whether
structural elements within NC7 might be involved in formation of the
tRNA3Lys primer-RNA template complex, with
potential to participate in efficient elongation reactions.
Toward this end, we studied the effects of two NCs with mutations in
the Zn finger motifs, including a variant containing a His-to-Cys
modification in the first Zn finger (i.e., H23C NC) and a
variant termed ddNC, in which both of the Zn fingers are deleted
(9). Previous studies showed that a truncated form of NC,
containing amino acids 13 to 64 and including both the Zn finger motifs
as well as the H23C substitution, could no longer
participate in annealing (8, 32). In contrast, ddNC that was
devoid of both Zn fingers did retain both nucleic acid binding and
annealing activities (10, 24).
Figure
1B shows results obtained when either wt or mutated NC was added
to RT reactions in which primer-template complexes
had been formed
by heat annealing. The data show that the addition
of either wt or
mutated NC during elongation only slightly increased
the proportion of
full-length (
)ssDNA relative to total cDNA
product and that
H
23C NC was able to promote the synthesis of (
)ssDNA at
low concentrations
(5 pmol). In contrast, when the annealing of
primer to template
was performed in the presence of NCs, far less
full-length (
)ssDNA
was observed in reactions in which the mutated
forms of NC were
used (Fig.
1A, lanes 6 through 15).
We next performed single-base-extension experiments to determine
whether the inefficient generation of full-length (
)ssDNA
was
due to decreased levels of tRNA
3Lys primer-RNA
template complexes that had been formed by the mutated
NCs. The
results in Fig.
3 show that reactions
that used wt NC7
(lanes 6 to 10) or either of the two types of mutated
NC (lanes
11 to 15 and 16 to 20) yielded similar levels of
single-base-extended
product, although, in each case, less product was
generated than
that seen when heat annealing was used for purposes of
primer-template
formation (Fig.
3, lanes 1 to 5). These findings
are consistent
with earlier reports that NCs with Zn finger deletions
retain
annealing activity (
10,
24).
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FIG. 3.
Single-base extension of tRNA-template complexes. By
monitoring the incorporation of [-32P]dCTP, reactions
were observed for varying times, i.e., 0.5 min (lanes 1, 6, 11, and
16), 1 min (lanes 2, 7, 12, and 17), 4 min (lanes 3, 8, 13, and 18), 16 min (lanes 4, 9, 14, and 19), and 32 min (lanes 5, 10, 15, and 20), and
we were able to distinguish the rates at which reactions had been
initiated from different tRNA-template complexes. Complexes had been
formed either by heat annealing or in the presence of wt NC,
H23C mutated NC, or mutated ddNC, at a concentration of 30 pmol per reaction. The densities of single-base-extension products were
determined on the basis of molecular imaging.
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To test the hypothesis that the NCs containing mutations in the Zn
fingers might be less able than wt NC to promote subsequent
extension, reaction mixtures containing primer-template and NC
were subjected to proteinase K digestion and phenol-chloroform
extraction as described above, in protocols in which both wt and
mutated NC peptides were used at saturating conditions, i.e.,
30 pmol
per reaction. The results in Fig.
4 show
that the NC that
contained a mutation in the first Zn finger, i.e.,
H
23C NC, was about 2.5 times less efficient than wt NC7
with regard
to the generation of a template-primer complex
able to participate
in the highly efficient synthesis of
(
)ssDNA. In contrast, hardly
any full-length (
)ssDNA product
was formed in reactions performed
with the NC peptide in which both Zn
fingers were deleted (Fig.
4, lane 3), and RT reactions were blocked
after the formation
of short cDNA products. Significant arrest was
noted in these
reactions at nucleotide positions +1 to +5.
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FIG. 4.
Effects of mutations within the Zn finger domains of NC
on reverse transcription elongation. Either wt or mutated NC was used
to generate primer-template complexes; these NCs were then
eliminated from the complexes by digestion with proteinase K and
extraction with phenol-chloroform as described in Materials and
Methods. The primer elongation step was performed in the absence of
NC. Quantification of products was carried out as described in the
legend to Fig. 1.
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Conclusions.
The role of NC7 in reverse transcription is
thought to result from the denaturation of RNA template secondary
structure during the elongation phase of the reaction (12).
However, we have shown that the use of NC7 to promote complex formation
between tRNA3Lys and the RNA template led to a higher
proportion of full-length ()ssDNA in the total cDNA product than that
obtained when a heat-annealing procedure was used. In contrast, the
increased elongation efficiency of RT reactions in the presence of NC7
is not due solely to the activity of NC7 during primer extension
but is also related to the manner of placement of the
tRNA3Lys primer onto the HIV RNA
template. The addition of different amounts of NC7 at times after
the heat-annealed formation of template-primer complex had little
or no effect on the subsequent efficiency of elongation.
The fact that elongation efficiency was lower in reactions in which NC7
was removed provides direct evidence that NC is involved
in the
formation of a tRNA
3Lys-RNA binary complex with
potential to yield high levels of full-length
(
)ssDNA. Thus, our data
complement the notion that NC plays a
role in the disruption of
the spatial structure of the RNA template
during both tRNA placement
and elongation; however, an additional
role is also evident with regard
to promotion of efficient elongation.
Our results show that the Zn finger motifs are especially important for
the formation of tRNA
3Lys-RNA template complexes that
are necessary for fully efficient
reverse transcription reactions
during the elongation of (
)ssDNA
synthesis. These observations are
consistent with a recent report
that mutations within the Cys-His motif
are relatively unimportant
in the genomic placement of
tRNA
3Lys in vivo but are important for extension from
the tRNA primer
(
18).
In order to attain full-length synthesis of HIV-1 (
)ssDNA from
tRNA
3Lys annealed to the viral RNA template, at least
two distinct reaction
phases are necessary, i.e., initiation, to
generate products extended
by 3 to 5 nt, and elongation, to yield
longer cDNA products (
19,
23). Our study shows that
NC7 also promotes the transition from
initiation to elongation (Fig.
2). NC devoid of both Zn fingers
resulted in a primer-template
complex that could yield only initiation
products, i.e., products
extended by 3 to 5 nt, and other short
DNA products, but not fully
synthesized (
)ssDNA (Fig.
4, lane
3). This helps to explain why the
tRNA-template complex formed
by NC7 yielded a higher proportion of
full-length (
)ssDNA products
than complexes formed by heat annealing
(Fig.
2, lane 1).
| |
ACKNOWLEDGMENTS |
This research was supported by grants to M.A.W. from the Medical
Research Council of Canada.
We thank Matthias Götte, Xuguang Li, and Yudong Quan for helpful
suggestions for the performance of this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: McGill AIDS
Centre, Lady Davis Institute/Jewish General Hospital, 3755 Cote
Ste-Catherine Rd., Montreal, Quebec, Canada H3T 1E2. Phone: (514)
340-8260. Fax: (514) 340-7537. E-mail:
mdwa{at}musica.mcgill.ca.
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Journal of Virology, November 1998, p. 9353-9358, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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