Nucleic Acids Research, Vol. 20, No. 1
i mm
man an
netic ani
27-31
man
Ben Berkhout
Viral Regulation Laboratory, Department of Virology, University of Amsterdam, Meibergdreef 15,
1105 AZ Amsterdam, The Netherlands
Received November 11, 1991; Accepted December 9, 1991
ABSTRACT
A comparative analysis of TAR RNA structures in
human and simian immunodeficiency viruses reveals
the conservation of certain structural features despite
the divergence in sequence. Both the TAR elements
of HIV-1 and SIV-chimpanzee can be folded into
relatively simple one-stem hairpin structures. Chemical
and RNAase probes were used to analyze the more
complex structure of HIV-2 TAR RNA, which folds into
a branched hairpin structure. A surprisingly similar RNA
conformation can be proposed for SUV-mandrill, despite
considerable divergence in nucleotide sequence. A
third structural presentation of TAR sequences is seen
for SIV-african green monkey. These results are
generally consistent with the classification of HIV-SIV
viruses in four subgroups based on sequence analyses
(both nucleotide- and amino acid-sequences). However,
some conserved TAR structures were detected for
members of different virus subgroups. it is therefore
proposed that RNA structure analysis might provide an
additional tool for determining phylogenetic
relationships among the HIV-SIV viruses.
INTRODUCTION
In eight years following the isolation of the first human
immunodeficiency virus (HIV-1, ref. 1 and 2), many genetically
distinct isolates have been reported (reviewed in 3). In addition,
a second type of human immunodeficiency virus (HIV-2, ref.
4) was found and related simian immunodeficiency viruses were
isolated from macaques (SIV-MAC, 5), sooty mangabeys (SIVSMM, 6), African green monkeys (SIV-AGM, 7), mandrills
(SIV-MND, 8) and most recently from chimpanzees (SIV-CPZ,
9). Based on genetic sequence analysis, these primate lentiviruses
were split into four groups that are all equally distantly related
to one another (3,9,10). Interestingly, the two human viruses
(HIV-1 and HIV-2) fall into different genetic groups, but both
appear to be very similar to specific simian species. The HIV-1
genome is closely related to that of SIV-CPZ (9). HIV-2, on the
other hand, is almost identical to the SIV-SMM and SIV-MAC
isolates (5,6). Two additional genetic groups are represented by
the simian viruses SIV-MND and SIV-AGM. These observations
have elicited speculation as to the evolutionary relationship of
HIV-SIV viruses and the origins of the current AIDS epidemic.
For instance, the similarity between SIV-CPZ and HIV-1
genomes may lend support for the hypothesis of inter-species
transmission of an ancestral virus from monkeys to humans. In
order to obtain further insight into such important aspects of
retroviral evolution, I have conducted a phylogenetic analysis
of RNA secondary structure motifs in the viral repeat (R) region.
This region encodes the essential TAR RNA motif, which forms
the target structure for the Tat trans-activator protein. The results
of this limited survey are generally consistent with the proposed
classification of the HIV-SIV viruses. Interestingly, the RNA
structural data do suggest some evolutionary links between
members of the different groups.
MATERIALS AND METHODS
Structure probing of HIV-2 TAR RNA
The plasmid Blue-TAR2, which contains region - 1 1 2 to +750
of the ROD isolate of HIV-2 fused to the T7 promoter, was
constructed by cloning a Pstl-Kpnl fragment of pRODIO (gift
of Dr. K.Peden) into Bluescript KS(+). T7 transcripts were
synthesized from PvuII-digested Blue-TAR2 plasmid according
to standard protocols (11). DNAase treatment and phenol
extraction was used to remove DNA and protein, respectively
and the RNA was recovered by ethanol precipitation. RNA was
dissolved (1 jtg//tl) in renaturation buffer (lOmM Tris-HCl pH7.5,
lOOmM NaCl) and incubated at 72°C for 2 minutes, followed
by slow cooling to 20°C. Renatured RNA (1 /tg per reaction)
was digested with increasing amounts of RNAase SI
( 0 - 0 . 1 - 0 . 3 - 0 . 9 U), RNAase CV (0-0.001-0.003-0.010
U) and RNAase Tl (O-O.OO1-O.OO3-O.O1O-O.O33-O.1OO
U), or treated with varying concentrations of diethylpyrocarbonate
(DEP; 0-0.5-1.0-2.0%[vol/vol]) according to published
procedures (12). The modified RNA was deproteinized by phenol
extraction and subsequently ethanol precipitated. Primer extension
assays were used to detect the sites of modification. Primer
HIV2-U5 (5'AGGAGAGATGGGAGCAC), complementary to
HIV-2 sequences +183 to +199, was end-labeled and annealedextended as previously described (13). The same primer was used
in dideoxy-sequencing of the corresponding Blue-TAR2 plasmid.
Samples were analyzed in a 6% acrylamide-8M urea gel.
28 Nucleic Acids Research, Vol. 20, No. J
one co-variation (A-U to G-C), a 3-bp deletion at the base of
the TAR stem and a 3-bp insertion at another position in the
hairpin was seen (Figure 1, middle panel). Thus, a structurally
conserved one-stem TAR hairpin, consisting of 23 basepairs, can
be drawn for all HIV-1 isolates.
Next, the TAR structure of the SIV-CPZ virus (9) was
analyzed. The TAR sequences of SIV-CPZ can easily be lined
up with those of HIV-1, with 13 nucleotide substitutions in the
first 51 nucleotides of the transcript. 10 of these nucleotide
changes were neutral with respect to RNA secondary structure
in that they represent 5 base-pair covariations in the lower part
of the stem (Figure 1, right panel). Thus, despite major sequence
variation, the HIV-1 and SIV-CPZ viruses share the same
fundamental architecture of the TAR stem-loop structure (9). For
this reason, SIV-CPZ was placed in the HIV-1 group that is
characterized by a relatively simple one-stem TAR element. The
actual sequence of SIV-CPZ TAR is more divergent than that
of any HIV-1 isolate reported so far. Moreover, the sequence
variations in SIV-CPZ do not resemble those seen in a particular
HIV-1 isolate. Among the several HIV-1 isolates, some common
patterns of sequence variation can be detected that might suggests
a close relationship. For instance, the Zairean MAL isolate
(Figure 1, number 5) shares two rather unique nucleotide
substitutions with the Ugandan virus U455 (number 14); a G to
U change at position +10 and a U-deletion in the 3-nucleotide
bulge element. Consistent with this analysis, a relatively small
genetic divergence between these two isolates was reported based
on amino acid sequences (20).
RESULTS
Structural resemblance of TAR motifs in HTV-1 and SIV-CPZ
In this report, the available HIV-SIV nucleic acid sequences (3)
were used to analyze and compare the RNA secondary structures
of the various TAR elements. This approach is useful because
it can provide additional information on the relatedness of RNA
sequences that is not detectable at the primary structure level.
For instance, a new RNA species that folds into a cloverleaf
structure will easily be recognized as a tRNA molecule, although
this might initially not have been apparent from its sequence.
Conservation of RNA structure, but not necessarily of sequence,
will be limited to regions of the viral genome that serve an
important biological function as a structured motif. Another
prerequisite for such an analysis is the availability of a detailed,
experimentally proven RNA secondary structure for the region
under study. For these reasons, the R region of the HIV-SIV
viruses was selected for this study. The HIV-1 R region contains
the TAR element, a stable stem-loop RNA hairpin that was
experimentally shown to exist in solution (Figure 1, ref. 12,14).
This structured RNA is the target for the viral Tat trans-activator
protein and both Tat and TAR are essential for HIV-1 replication
(15 — 18). Furthermore, the Tat-TAR trans-activation function
is conserved among all HIV-SIV lentiviruses.
Despite its crucial function, considerable sequence diversity
was found in TAR among a phylogenetic variety of HFV-1 isolates
(Figure 1, left panel). 17 out of 22 HIV-1 isolates tested had at
least one nucleotide change when compared to the prototype
HXB-2 TAR hairpin. However, most of these nucleotide changes
did not affect the RNA secondary structure. In many cases, the
mutation only changed the type of base-pair (e.g. G-U to A-U).
Other mutations that individually would affect base-pairing, were
compensated for by other base changes, the so-called covariations (e.g. G-U to U-A). In one isolate, HIV-1 ANT-70 (19),
major rearrangements in the primary structure were observed
that do not affect the overall secondary structure. In addition to
A complex, multi-stem TAR structure for HIV-2
Alignment of the HIV-1 R sequence with those of the other HIVSIV viruses is poor, in part because of their variability in size
(HIV-1, 97; HIV-2, 173; SIV-AGM, 117; and SIV-MND, 175
nucleotides). The structure of HIV-2 TAR was investigated by
monitoring the accessibility of nucleotides to chemical and
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Figure 1. Phylogenetic comparison of TAR RNA structures in different HIV-1 isolates and SIV-CPZ. The hairpin structure of the HXB-2 isolate was used as prototype
and compared to 22 HIV-1 viruses and SIV-CPZ (3). Nucleotide changes occurring in other isolates are indicated in bold with the number of the particular isolate
in superscript. Deletions are shown as A , insertions are indicated by + . Changes in HIV-1 isolates 1-17 (left panel). HIV-1 isolate ANT-70 (middle panel) and
SIV-CPZ (right panel) are shown. The TAR sequence of four HIV-1 isolates (SF2. MN, N1TE and SF33) was identical to that of the HXB-2 prototype.
Nucleic Acids Research, Vol. 20, No. 1 29
RNAase probes (Figure 2A). The results of this analysis are
summarized in the secondary structure model (Figure 2B). The
difference in length of R between HIV-1 and HIV-2 can be
accounted for by the duplication of the upper part of the TAR
hairpin and the addition of a branched stem-loop element at the
base of the TAR hairpin. In the discussion that follows, I will
refer to the different domains as stem-loop 1, 2 and 3 (Figure 2,
HIV-2 coordinates 20 to 54, 56 to 87 and 95-115, respectively).
The double-strand specific RNAase CV cleaved the RNA mainly
in the basepaired stem regions. The single-strand specific probes
reacted with most of the bulge and loop elements, and in addition
with the unpaired nucleotides at the junctions of helical segments
(e.g. A55, A88 and A94). One striking observation is that the
GG(G) sequence, present in all three loop elements, is highly
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Figure 2. Secondary structure analysis of HIV-2 TAR RNA. A. The HIV-2 transcript was synthesized in vitro using T7 RNA polymerase. The RNA was treated
under native conditions with limiting amounts of several single-strand specific reagents (DEP=diethylpyrocarbonate (A-specific). RNAase Tl (G-specific) and RNAase
SI) and the double-strand specific RNAase CV (Cobra Venom). Each treatment (indicated above the lanes) was performed with increasing amounts of the chemicalRNAase (see Materials and Methods for details, left lanes represent mock incubations). Cleavage-modification sites were detected using primer-extension analysis
For reference, the same primer was used in a DNA sequence reaction (lanes 9 to 12; CTAG and lane 17, G reaction only). Numbers on the left represent TAR
coordinates (position +1 to +130) and the location of the three single-stranded loop regions is schematically indicated on the right B. Putative secondary structure
of HIV-2 TAR RNA. Sensitivities of TAR nucleotides to the various reagents are indicated by symbols (see insert). Positions at which strong, non-specific reverse
transcriptase stops were observed are indicated by arrows.
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Figure 3. TAR RNA secondary structure models for the four groups of HIV-SIV viruses. The hairpin structure shown for the HIV-1/SIV-CPZ group is that of
the HIV-1 HXB-2 isolate and the structure representing the HIV-2/SIV-SMM/SIV-MAC group is that of the HIV-2 ROD isolate. The SIV-AGM and SIV-MND
sequences were initially analyzed using computer-aided RNA-folding programs (34) and base-pairing schemes were further refined by hand to maximize the structural
similarity with the structures shown for HIV-1 and HIV-2. It should be noted that no comparative support (35) exists for the SIV-AGM and SIV-MND structures.
Multiple G-U pairs are present at different positions in all the HIV-SIV TAR elements and one A-G pair (36) is proposed for SIV-MND.
30 Nucleic Acids Research, Vol. 20, No. 1
accessible to the RNAase Tl enzyme in loops 1 and 3, while
no cleavage was observed in the context of loop 2. These results
suggest that loop 2 nucleotides are involved in a more complex,
tertiary structure and may explain the observation that the second
loop contributes only marginally to Tat-mediated trans-activation
(13,30). The RNA structure in Figure 2B is also supported by
phylogenetic data (13). Because there is almost no sequence
variation among the HIV-2, SIV-SMM and SIV-MAC isolates,
an identical 3-stem structure is proposed for these viruses. Thus,
when compared to the HIV-1/SIV-CPZ group, the HIV-2/SIVSMM/SIV-MAC viruses represent a distinct group with a
complex, 3-stem TAR structure (Figure 3).
The SIV-MND TAR element is structurally similar to HIV-2
TAR
The R region of SIV-MND is comparable in size to that of the
HIV-2 group of viruses, but the sequence homology is low (40%).
It was therefore surprising that an RNA secondary structure
model can be proposed for SIV-MND with a framework that is
strikingly similar to that of HIV-2 TAR (Figure 3). Despite the
striking conservation of the basepairing-scheme, only 12 of the
47 base-pairs are conserved in sequence between SIV-MND and
HIV-2 TAR. Phylogenetic trees constructed by protein sequence
alignments indicated that SIV-MND is distinct from HIV-1 and
HIV-2, with an equal relatedness to both of these human viruses
(21). A speculative interpretation of the RNA structural data is
that SIV-MND is more similar to HIV-2 and perhaps diverged
from a common ancestral virus.
SIV-AGM folds a TAR structure of intermediate complexity
The R region of SIV-AGM, intermediate in size, can be folded
into another secondary structure (Figure 3). This structure is an
intermediate form between the one-stem hairpin of HIV-1 and
the branched 3-stem structure of the HIV-2 group and SIV-MND.
The TAR element of SIV-AGM can be thought of being derived
from a simple HIV-1 TAR by the addition of the short stemloop segment, or from a complex HIV-2/SIV-MND TAR
structure by the removal of either one of the duplicated stems
in the upper part of the structure. Hypothetically, it is possible
that SIV-AGM TAR represents an intermediate species in the
evolution from simple (HIV-1) to complex TAR structures
(HIV-2 and SIV-MND) or vice versa. Inspection of the nucleotide
sequence of the different stem-loop regions within TAR suggests
a closer relationship of SIV-AGM with HIV-2. In particular, the
upper portion of stem-loop 1 and loop 2 in SIV-AGM are identical
in sequence to HIV-2 stem-loops 2 and 3, respectively (Figure 3).
A phylogenetic tree analysis based on gag amino acid sequences
did also suggest a closer relationship of SIV-AGM with the HFV-2
group than with the other groups (9).
DISCUSSION
The data presented here show a considerable heterogeneity in
TAR sequence and structure among the primate HIV-SIV
lentiviruses. It is nevertheless reasonable to assume that the
essential role of TAR RNA in the process of Tat-mediated transactivation must be preserved among all viruses. In fact, the
observation that the different Tat proteins can cross-trans-activate
heterologous viral genomes (13,30,31) further strengthens the
idea that HIV-SIV viruses share a very similar trans-activation
mechanism. Indeed, despite the tendency of the various viruses
to structure TAR somewhat differently, the upper part of the
hairpin (HIV-1 coordinates +19 to +42), that functions as the
target in Tat-mediated trans-activation (12,29), is well-conserved.
This domain consists of a helix with a 2/3-nucleotide bulge, a
6-nucleotide loop and 4/5 base-pairs in between. Within this
structural context, critical sequence elements were described in
the single-stranded bulge (22—25) and loop (22,26) domains.
In addition, critical sequences were identified in the base-pairs
flanking the bulge (27,28). Most of these important nucleotides
are conserved among all HIV-SIV viruses.
Some other interesting aspects on TAR function emerge from
this study. All TAR structures reported sofar contain a 2 - 3
nucleotide bulge (UU, UUU or UCU). TAR RNA of SIV-MND
is remarkable in having an elongated 4-nucleotide bulge (UUGU)
in the first stem-loop (Figure 3). It should be noted that although
an unpaired C is present on the opposite side of this stem, it is
not predicted to basepair with the bulged G (34). Therefore, bulge
size seems to be a rather flexible determinant. This idea is fully
supported by recent mutagenesis data (32), showing that bulges
up to 5 nucleotides in size function properly in Tat binding. The
most critical bulge parameter is the 5' U residue (22,23), which
is conserved in all but one TAR motif (Figure 3). The presence
of a U-less bulge in the second stem of SIV-MND may reflect
the relative minor importance of the second stem-loop in the Tat
response, as was reported for the multi-stem HIV-2 TAR
structure (13,30). The apparent flexibility in bulge size does
contrast with the absolute conservation of the loop size.
Mutational data also support this notion, in that one base insertions
in the 6-nucleotide loop strongly reduce TAR activity (28).
Inspection of the loop sequences used by the various viruses
allows one to propose a consensus; C/U U/C G G/A G A/U/C
or Py Py G Pu G N.
Sequence data do allow us to get a quantitative measure of the
relationship between viruses in the form of a percentage
nucleotide- or amino acid-homology. The HIV-SIV family of
viruses was divided into 4 subgroups based on such comparative
sequence analysis (3,9). A substantially more difficult, but
perhaps functionally more significant comparison is based on
RNA folding patterns. In this report, TAR RNA elements of all
HIV-SIV viruses, widely diverged in sequence and size, were
analyzed and grouped on basis of RNA structural motifs. The
overall results of the two approaches agree to a first
approximation and thus further support the proposed classification
of the immunodeficiency viruses. However, chere are differences
in details among the proposals. All HIV-1 isolates, both of
African and American origin, fall in one group together with
the SIV-CPZ virus. A second group is formed by the closely
related HIV-2 and SIV-SMM/MAC viruses. A third group
consists of SIV-AGM, of which the overall TAR RNA shape
is an intermediate between the simple HIV-1 hairpin and the
complex HIV-2 structure. This result suggests, but does not
proof, that SIV-AGM is an evolutionary link that connects the
HIV-1 and HIV-2 groups. Structural analysis of SIV-MND RNA
does not support the presence of a separate group for this virus.
A striking structural resemblance to HIV-2 TAR RNA was
detected. Thus, with RNA structure as parameter, one would
place SIV-MND in the HIV-2 group. However, the degree of
sequence variation between the structurally related HIV-2 and
SIV-MND does not readily tell us when in time a common
ancestral virus diverged, mainly because the mutation rate of HIV
in vivo is unknown (33). Another complicating factor is the
frequent occurrence of (RNA) recombination, duplication and
deletion in the genetic material of retroviruses. Clearly additional
Nucleic Acids Research, Vol. 20, No. 1 31
sequence data and more structural analyses, perhaps including
other regions of the retroviral genome (e.g. the highly structured
RRE domain), will help us gain insight into these important
aspects of HIV-SIV evolution.
26.
27.
28.
29.
30.
ACKNOWLEDGEMENTS
I thank Keith Peden for the generous gift of plasmid pRODIO,
Margreet Valk for oligonucleotide synthesis and Wim van Est
for excellent photography work.
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