(Received for publication, December 7, 1994; and in revised form, January 25, 1995)
From the
Genomic human immunodeficiency virus type 1 (HIV-1) RNA consists
of two identical RNA molecules joined noncovalently near their 5` ends
in a region called the dimer linkage structure (DLS). Previous work has
shown that the putative DLS is localized in a 113-nucleotide domain
encompassing the 5` end of the gag gene. This region contains
conserved purine tracks that are thought to mediate dimerization
through purine quartets. However, recently, an HIV-1 RNA
dimerization model was proposed as the HIV-1
RNA
dimerization initiation site, involving another region upstream from
the splice donor site and possibly confined within a stem-loop. In the
present study, we have investigated the dimerization of HIV-1
RNA, using in vitro dimerization assays under conditions
of low ionic strength, predictive RNA secondary structures determined
by computer folding, and antisense DNA oligonucleotides in order to
discriminate between these two models. Our results suggest that purine
quartets are not involved in the dimer structure of HIV-1
RNA and have led to the identification of a region upstream from
the splice donor site. This region, comprising an autocomplementary
sequence in a possible stem-loop structure, is responsible for the
formation of dimeric HIV-1
RNA.
All retroviruses have a common feature, namely a genome consisting of two identical unspliced RNA molecules noncovalently linked(1) . It has been shown by electron microscopy that this junction is located close to their 5` ends, in a structure termed the dimer linkage structure(2, 3, 4) .
Dimerization of retroviral RNA is thought to play a crucial role in
several steps of the retroviral life cycle. RNA dimerization seems to
be closely related to the encapsidation of retroviral RNA, since cis-elements such as the dimer linkage structure (DLS) ()and the encapsidation site (E) are localized within the
same region in the genome of MoMuLV
(5) ,
RSV(6) , REV(7) , BLV(8) , and
HIV-1(9) . It has also been suggested that dimerization of
retroviral RNA is associated with reverse transcription through
interstrand switching(10) , genomic
recombination(11, 12, 13) , and
down-regulated translation(6) .
Several attempts have been
made to investigate factors responsible for the dimerization event.
Reports describe a crucial role for a trans-acting factor, the
HIV-1 nucleocapsid protein (NCp7) in the formation of RNA
dimers(9, 14) . It has also been demonstrated that in
the absence of any cellular or viral protein, HIV-1 RNA is able to
spontaneously dimerize in vitro under conditions of high ionic
strength(15, 16, 17, 18) .
HIV-1 sequences able to support this spontaneous
dimerization have been localized between nucleotides 311 and 415
encompassing the 5` end of the gag gene(9) . Published
results indicate that (i) the HIV-1 RNA dimer is very stable, (ii)
antisense RNA cannot form a dimer, and (iii) a consensus sequence,
PuGGAPuA, is found in the putative dimerization-encapsidation region of
HIV and other retroviral genomes(15) . Marquet et al.(15) and others (16, 17, 18) have
suggested that the structural motif mediating the association of two
identical viral RNA molecules should involve purine quartets. This
model is based on the dimerization of telomeric DNA which occurs via
the formation of unusual intermolecular quadruple helical structures
that are stabilized by guanine base tetrads(19) .
However,
dimerization of viral RNA by purine quartets has recently been
discussed(20, 21, 22, 23) . First,
Berkout et al.(21) reported that the HIV-2 mutant
RNA, bereft of all PuGGAPuA sites, could dimerize in vitro.
Second, Skripkin et al.(22) recently postulated that
the linking of the two HIV-1 RNA molecules is initiated
at the level of a short RNA region, located upstream from the splice
donor site and consisting of a palindromic sequence in a stem-loop
structure. However, this process requires high ionic strength and the
presence of MgCl
in the electrophoresis gel in order to be
observed(22, 23) . Similarly, based on an RNA-RNA
recognition model, Girard et al.(
)identified a
possible stem-loop structure containing a palindromic sequence, which
is probably responsible for the formation of dimeric MoMuLV RNA.
In
this report, we analyze the spontaneous in vitro dimerization
process of HIV-1 RNA, under conditions of low ionic
strength, in an attempt to enhance our understanding of the nature of
the interactions leading to dimerization and to distinguish between the
above described dimerization models. We have characterized the dimer
linkage structure by using HIV-1
RNA fragments of
different lengths, complementary DNA oligonucleotides, and heterodimer
formation.
Plasmid pBRU2 (a gift of Dr. F. Subra) contains the HIV-1 cDNA in the pKP59 vector with XbaI-AatII as the
cloning site. This plasmid has a deletion of 2701 base pairs from
+5367 to +8061 to prevent it from being infectious.
All these oligonucleotides were loaded on an 18%
polyacrylamide gel in a buffer containing 4 M urea, 50 mM Tris-borate, pH 8.3, and 1 mM EDTA, electrophoresed, and
purified by excision from the gel by UV shadowing. DNA oligonucleotides
were 5`-P-labeled with [
P]ATP
(Amersham, United Kingdom) and T4 polynucleotide kinase. The specific
radioactivity was about 10
cpm/µg DNA oligonucleotide.
RNA 224-402 was heat denatured in the presence of the
[
P]DNA oligonucleotide before starting in
vitro dimerization process. [
P]DNA
oligonucleotide
RNA complexes were analyzed by agarose gel
electrophoresis. The level of hybridization of the
[
P]DNA oligonucleotide to monomer and dimer RNA
224-402 was detected by autoradiography of the corresponding gel.
Figure 1:
Mapping of the sequences required for
HIV-1 RNA dimerization by deletion mutagenesis. a, representation of the 5` end of HIV-1
DNA.
Numbering is relative to the genomic RNA cap site (+1).The
restriction sites of interest are indicated: HindIII
(+77), SacI (+224), and RsaI (+296). PBS and ATG indicate, respectively, the primer
tRNA
-binding site and initiation codon for Pr55gag synthesis. b, HIV-1
RNAs generated in
vitro and used in this study. RNAs were generated in vitro by transcription of pDM2, pDM3, pDM6, and pDM7 linearized with the
appropriate enzyme. RNA 1 and 2 begin at position
+77, RNAs 3-5 at position +224, and RNA 6 at position +296. The deletion in RNA 5, between nt
257-266 is represented by the broken line. The column of symbols, headed RNA Dimer, indicates the
level of dimeric RNA. -, 0-5%; ±, 10-15%;
+++, 70-90% (means value from at least three
experiments). c, agarose gel electrophoresis of HIV-1
RNAs. For each sample, the RNAs are numbered as in b.
Heat-denatured RNAs (M) are shown in lanes 1, 3, 5, 7, 9, and 11, and
RNAs (D) in dimerization conditions are shown in lanes
2, 4, 6, 8, 10, and 12. Lane MK, 0.16-1.77-kb RNA ladder (Life
Technologies, Inc.).
Figure 2: Thermal stability of the dimer RNA 224-402 as a function of temperature. Samples were analyzed by 1.5% agarose gel electrophoresis and the percent of each species determined from the gel. The temperatures on the plot correspond to the temperatures shown above the gel.
The same behavior observed for RNA 77-402 and 224-402 justified our choice of RNA 224-402 as the reference fragment for the following studies.
Figure 3:
RNA secondary structure predicted for
HIV-1 RNA 224-402 by computer-assisted energy
minimization analysis. The full lines represent the antisense
DNA oligonucleotides, used in this work, which are complementary to the
RNA sequences covered.
It is
noteworthy that our computed model is in accordance with the one
proposed by G. P. Harrison for HIV-1(32) which
comprises this stem-loop with the same primary sequence.
The first question to be
addressed was: is the autocomplementary stem-loop I, which encompasses
nt 251-266, involved in the dimerization of HIV-1 RNA?
We studied the dimerization process of RNA 224-402 when incubated with increasing concentrations of oligonucleotide 257B. As shown in Fig. 4a, antisense oligonucleotide 257B, which targets the autocomplementary sequence 257-262, completely blocks dimerization. Total inhibition of dimerization is observed for RNA 224-402 when the oligonucleotide 257B ratio was equal to 1:1. The affinity constant can be estimated to be 0.1 to 1 µM of the antisense DNA oligonucleotide 257B for the RNA 224-402 target (for a strand concentration of 1 µM). Autoradiogram (Fig. 4b) shows that oligonucleotide 257B only hybridizes to RNA 224-402 in its monomeric form.
Figure 4:
Inhibition of HIV-1 RNA
224-402 dimer formation in the presence of increasing
oligonucleotide 257B concentrations. Fixed concentrations of RNA
224-402 (0.5 µg of heat denatured RNA/assay) were incubated
in a buffer with 50 mM Tris-HCl, pH 7.5, and 100 mM NaCl, for 45-60 min at 37 °C, in the presence of (lanes 2-11) 0, 0.05, 0.075, 0.1, 0.5, 0.75, 1, 2, 5,
and 7 molar equivalent(s) of the [
P]DNA oligomer
257B, complementary to nucleotides 256-336. Lane 1 shows
the monomeric form of HIV-1
RNA 224-402 (heat
denatured for 5 min at 95 °C), lane MK, as in Fig. 1. m and d indicate monomeric and dimeric
RNAs, respectively. a, 1.5% agarose gel electrophoresis. The
samples are visualized by ethidium bromide staining. b,
autoradiogram of a.
Two antisense DNA oligonucleotides were used as controls. Oligonucleotide 257 M, differing from oligonucleotide 257B by four nucleotides in its sequence, is unable to prevent the dimer formation of RNA 224-402 (Fig. 5A). It converts 50% of the dimer into the monomer when its concentration is 5-fold higher. The other control oligonucleotide T (see ``Experimental Procedures'') does not inhibit the RNA 224-402 dimerization process, even at high concentrations (Fig. 5B). It should be noted that oligonucleotide 257M is not bound to the RNA fragment since it migrates as a free oligonucleotide in the gel. Consequently, it did not anneal RNA 224-402 because of the presence of its four mutated nucleotides. Such was not the case with oligonucleotide T which hybridizes to the RNA target 224-402 (in Fig. 5B a difference can be observed in dimer shift mobility in the gel when oligonucleotide T concentrations increase).
Figure 5:
Analysis of HIV-1 RNA
224-402 dimer formation in the presence of oligonucleotides 257 M (A) and T (B) as controls. As described in Fig. 4, fixed concentrations of RNA 224-402 were incubated
in the presence of (lanes 2-8) 0, 0.05, 0.1, 0.5, 1, 2,
and 5 molar equivalent(s) of the DNA oligonucleotide 257M or T. Lane 1, monomeric form of HIV-1
RNA
224-402. Lane MK, as in Fig. 1.
The second question was: Were antisense DNA oligonucleotides 320B, 327B, 365B, and 382B, which are complementary to polypurine tracks, able to interfere with dimer formation?
Oligonucleotides 327B, 365B, and 382B are
complementary to PuGGAPuA sequences, and oligonucleotide 320B targets a
GGAGG sequence which was proposed by Sundquist and Heaphy (16) as an attractive candidate for a purine-rich region
involved in dimerization. As shown in Fig. 6A, neither
completely inhibited RNA 224-402 dimer formation, and
oligonucleotide 327B never reduced the amount of dimer by more than
50%. In every case, each [P]DNA oligonucleotide
annealed to both monomer and dimer RNA 224-402 (see
autoradiographies, Fig. 6A).
Figure 6:
Analysis of HIV-1 RNA
224-402 dimer formation in the presence of each oligonucleotide
320B, 327B, 365B, and 382B (A) or the four together (B). As described in Fig. 4, fixed concentrations of
RNA 224-402 were incubated in the presence of (lanes
1-6) 0, 0.1, 0.5, 1, 2, and 5 molar equivalent of each
[
P]DNA oligonucleotide (A) or in the
presence of the four together, each of them at a molar equivalent of
RNA (B). Oligonucleotides 320B, 327B, 365B, and 382B which
correspond, respectively, to nucleotides 312-326, 322-336,
360-374, and 377-391 of the HIV-1
sequence,
are indicated in A. Lane M shows monomeric form of
HIV-1
RNA 224-402. Monomer (m), dimer (d) of RNA 224-402, and free DNA oligonucleotides are
indicated. The results are visualized on 1.5% agarose gel
electrophoresis by ethidium bromide staining (gels above) and
autoradiograms (views below).
Each RNA 224-402
molecule contains four polypurine sequences. We postulate that any of
these sequences are able to interact indifferently with any of those in
a second RNA 224-402 molecule. We therefore incubated the four
oligonucleotides 320B, 327B, 365B, and 382B together with the RNA
224-402 (Fig. 6B) and found that dimerization
still occurred. [P]DNA oligonucleotides marked
monomeric and dimeric forms of the RNA 224-402 equally (see
autoradiogram, Fig. 6B).
To confirm
the role of the autocomplementary GCGCGC
sequence in HIV-1
dimerization, nucleotides
257-266 were deleted from RNA 224-402. This deleted RNA was
analyzed by agarose gel electrophoresis to estimate the degree of
dimerization (Fig. 1b, RNA 5). RNA
224-402DEL lost the capacity to form dimeric RNA (Fig. 1c, lanes 9 and 10) while RNA
224-402 formed up to 80% of the dimer under the same experimental
conditions (Fig. 1c, lane 6). The
10-nucleotide deletion did not alter the overall predicted secondary
structure of the molecule in spite of a modification in the primary
sequence of the stem-loop which was no longer autocomplementary (Fig. 8c). Furthermore, it is noteworthy that RNA
77-257 corresponds to an RNA molecule spliced in loop I: it had
lost the capacity to dimerize efficiently (Fig. 1, b and c, lanes 3 and 4).
Figure 8:
Model of HIV-1 dimerization
process. a, predicted structure of stem-loop I, encompassing
nucleotides 257-262, implicated in the recognition of the two
HIV-1
RNA molecules. This structure was determined on
RNAs 77-402, 224-402, and 224-296 using PCFOLD
software(28) . b, formation of a double-stranded helix
by the opening of the predicted stem-loop structure. Nucleotides
spanning 240 through 280 are indicated. c, predicted structure
of the stem-loop lacking nucleotides 257-266 which
contain the GCGCGC sequence.
We therefore
conclude that the 257-266 sequence is involved in the formation
of dimeric HIV-1 RNA.
Figure 7:
Analysis on agarose gel electrophoresis of
HIV-1 RNA heterodimers between RNA 77-402 and RNAs
224-402 (a), 224-296 (b),
224-402DEL lacking nt 257-266 (c) and
296-402 (d). Lanes M show heat-denatured RNA
77-402 and lanes D show RNA 77-402 in dimerization
conditions. Coincubations of the same amount of each of the different
HIV-1
RNAs and RNA 77-402 in dimerization
conditions are shown in lanes HD. Monomer (m) and
dimer (d) of the different RNA fragments are indicated.
Numbering corresponds to that given for RNA fragments in Fig. 1b.
Thus, the
mechanism of HIV-1 RNA dimerization is most probably
common to RNAs of different sizes, only if they contain the
257-266 sequence. The heterodimers have a dimerization region
similar to that of the homodimers.
We present here data on the in vitro dimerization of
HIV-1 RNA transcripts (strain Lai) under conditions of low ionic
strength. As shown in Fig. 1, HIV-1 RNA
77-402, a 5` leader RNA fragment, can efficiently dimerize in
vitro. This RNA does not contain the TAR domain but, according to
Berkout et al.(21) , who has implicated TAR inverted
sequences in the dimerization process, this domain should not play any
role. Likewise, shorter RNAs 224-402 and 224-296 can
dimerize up to 80-90% in low ionic strength buffer whereas RNAs
77-257 and 296-402 cannot. RNA 224-402 was chosen as
the reference fragment for the study. Computer folding analysis of RNA
224-402 showed that an autocomplementary sequence could be a part
of a stem-loop structure (loop I in Fig. 3). When this
sequence is deleted from this region, the RNA (224-402DEL) is
unable to dimerize (Fig. 1) although the predicted stem-loop
structure may be conserved (Fig. 8c). Furthermore, and
consistent with this finding, a DNA oligonucleotide, complementary to
this region, totally inhibits the HIV-1
dimerization
process (Fig. 4), whereas control oligonucleotides 257M and T do
not (Fig. 5).
A hypothetical mechanism for RNA dimerization,
based on an extensive comparison of sequences in the leader regions of
30 retroviral genomes, has been proposed(15) . This comparative
analysis suggested that the consensus sequence PuGGAPuA participates in
the dimerization process through the formation of purine quartets
involving both adenine(s) and guanine(s). As HIV-1 RNA
224-402 contains three such sites as well as the GGAGG sequence
described by Sundquist and Heaphy (16) , we tested the role of
these elements in the dimerization process. Surprisingly, RNA
224-296, derived from RNA 224-402 but totally bereft of
purine consensus sites, was able to dimerize spontaneously in vitro with the same efficiency (Fig. 1). Furthermore, this RNA
224-296 was able to form a heterodimer with the leader RNA
77-402. RNA 224-296 therefore acts as an antisense RNA
since we observed more heterodimer than the homodimer 77-402 (Fig. 7b). The remaining RNA region 296-402,
which corresponds to the DLS 311-415 of HIV-1
,
previously shown to support the necessary sequence for HIV-1
dimerization(9) , was neither able to dimerize in vitro under conditions of low ionic strength (Fig. 1), nor to
form a heterodimer with RNA 77-402 (Fig. 7d). We
therefore failed to inhibit the formation of RNA dimer 224-402 in
the presence of the antisense DNA oligonucleotides 320B, 327B, 365B,
and 382B (Fig. 6) which target the purine tracks. We observed
that the oligonucleotide 327B reduced the amount of the dimer by only
50% (Fig. 6A). The partial inhibition of dimerization
by the oligonucleotide 327B probably implicates another region that may
participate, to a certain extent, in the stabilization of the final
structure of the dimer. However, the total inhibition observed with
oligonucleotide 257B indicates that this stabilization, in itself, is
not sufficient enough to lead to the formation of the dimer.
Our
results suggest that a recognition mechanism between the two identical
RNA molecules is operating via a loop-loop interaction through
nucleotides GCGCGC
in loop I (Fig. 3). We postulate that a transient complex is formed
between complementary nucleotides in loops. The opening of both
stem-loops during the interaction could lead to a double-stranded
region via Watson-Crick base pairing, as shown in Fig. 8, a and b. This mechanism is proposed in the light of two
similar mechanisms described for (i) the formation of a duplex between
RNA I and RNA II from plasmid ColE1 (33) and (ii) the antisense
RNA CopA and its target RNA CopT in the replication of plasmid
R1(34) .
Such a mechanism for the HIV-1 DLS
signal is in good agreement with recent analytical studies on
HIV-1
RNA dimerization(22) , except for a
difference observed by us in dimer stability. This difference could be
attributed to a sequence modification when it passes from HIV-1
to HIV-1
RNA (5` . . .
G
AAGCGCGCACGG
. . . 3` to 5` . . .
G
AGGUGCACACAG
. . . 3`). The HIV-1
RNA 224-402 dimer is strongly stabilized through the GC
residues present in the dimerization sequence (Fig. 8). It
dissociates into the monomer at 53 °C (Fig. 2). This Tm
value is in good agreement with that found by Darlix et al.(9) with the full-length HIV-1
viral RNA
isolated from wild-type virus particles.
The nucleotides GCGCGC are remarkably conserved in 18 of the 21 HIV-1 strains observed(29) . Interestingly, mutations observed in the stem-loop of various HIV-1 strains were offset so that the structure and the autocomplementary sequence were maintained (except for HIVHXB2 strain).
A potential link between dimerization and encapsidation of
HIV-1 genomic RNA has been
proposed(7, 9, 31) . Kim et al.(35) have recently constructed several recombinant
HIV-1 proviral DNA clones. One of them, lacking 13 bases
upstream from the splice donor site, produced a virus with less
efficient packaging of its genomic RNA than a virus bearing mutations
between the 5` splice donor site and the gag gene. This
deletion encompasses nucleotides 241-253 which are a part of
stem-loop I shown in Fig. 3and Fig. 8a.
Understanding the structures present in dimeric retroviral RNAs is
therefore an essential prerequisite for antiviral strategies. We
therefore propose stem-loop I as a potential target in attempts to
interfere with HIV-1 replication. Since one of the current antisense
strategies is based on targeting oligonucleotides to complementary
sequences of an RNA molecule, oligonucleotide 257B is proposed for this
function. HIV-1
RNA is totally inhibited in
vitro, under conditions akin to physiological ones, by this
antisense DNA oligonucleotide complementary to the putative stem-loop I
which contains the autocomplementary GCGCGC sequence.
* This work was supported by the Agence Nationale de la Recherche sur le SIDA (ANRS). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Addendum-While this manuscript was reviewed, Laughrea et al.(24) have speculated that the 248-270 or
233-285 region forms a hairpin that is the core dimerization
domain of HIV-1 RNA. Our results and theirs are
complementary and mutually supportive: we reached the same postulated
dimerization model of HIV-1 RNA.