Structure of the HIV-1 Rev response element alone and in
complex with regulator of virion (Rev) studied by atomic
force microscopy
Jesper Pallesen
1,2,
*, Mingdong Dong
1,3,
, Flemming Besenbacher
1,3
and Jørgen Kjems
1,2
1 Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Denmark
2 Department of Molecular Biology, University of Aarhus, Denmark
3 Department of Physics and Astronomy, University of Aarhus, Denmark
Introduction
HIV-1 belongs to the group of Lentiviridae, which
share a common genetic structure, including a set of
essential regulatory proteins, trans-activator of tran-
scription (Tat) and regulator of virion (Rev). They
control the level of transcription and the appearance
of unspliced and singly spliced viral RNA in the cyto-
plasm, respectively [1]. Regulation by Rev requires the
presence of a cis-acting RNA target sequence termed
the Rev response element (RRE), which is located in
the envelope gene of HIV-1 [1]. The Rev protein medi-
ates the appearance of unspliced and singly spliced
RNA in the cytoplasm by facilitating nuclear export of
these RNAs.
Initially, at low Rev concentrations, only fully
spliced HIV-1 mRNAs are exported to the cytoplasm,
where they encode regulatory proteins, most impor-
tantly Tat and Rev. Subsequently, when the Rev
concentration reaches a certain threshold value, Rev
binds and oligomerizes onto RRE and recruits the
nuclear export factor exportin-1 in complex with the
Ras-related nuclear protein ⁄guanosine triphosphate
complex (RanGTP) [2–5]. Export of singly spliced and
Keywords
atomic force microscopy; oligomerization;
Rev; RNA; RRE
Correspondence
J. Kjems, Department of Molecular Biology,
University of Aarhus, C. F. Møllers alle
Build. 1130, DK-8000 Aarhus C, Denmark
Fax: +45 8619 6500
Tel: +45 8942 2686
E-mail: jk@mb.au.dk
Present addresses
*Columbia University, New York City, NY,
USA
Harvard University, Cambridge, MA, USA
(Received 28 January 2009, revisied 10 May
2009, accepted 3 June 2009)
doi:10.1111/j.1742-4658.2009.07130.x
The interaction of multiple HIV-1 regulator of virion (Rev) proteins with
the viral RNA target, the Rev response element (RRE), is critical for
nuclear export of incompletely spliced and unspliced viral RNA, and for
the onset of the late phase in the viral replication cycle. The heterogeneity
of the Rev–RRE complex has made it difficult to study using conventional
structural methods. In the present study, atomic force microscopy is
applied to directly visualize the tertiary structure of the RRE RNA alone
and in complex with Rev proteins. The appearance of the RRE is compati-
ble with the earlier proposed RRE secondary structure in dimensions and
overall shape, including a stalk and a head interpreted as stem I, and stem-
loops II–V in the secondary structure model, respectively. Atomic force
microscopy imaging of the Rev–RRE complex revealed an increased height
of the structure both in the stalk and head regions, which is in accordance
with a binding model in which Rev binding to a high affinity site in stem
IIB triggers oligomerization of Rev proteins through cooperative binding
along stem I in RRE. The present study demonstrates that atomic force
microscopy comprises a useful technique to study complex biological struc-
tures of nucleic acids at high resolution.
Abbreviations
AFM, atomic force microscopy; RanGTP, Ras-related nuclear protein ⁄guanosine triphosphate complex; Rev, regulator of virion; RRE, Rev
response element; Tat, trans-activator of transcription.
FEBS Journal 276 (2009) 4223–4232 Journal compilation ª2009 FEBS. No claim to original US government works 4223
Fig. 1. Secondary structures of the 370 nucleotides version of the Rev response element (RRE). (A) Lowest Gibbs energy secondary struc-
ture of RRE (672.9 kJÆmol
)1
). (B) Alternative secondary RRE structure in which stem-loops III and IV have collapsed into a common stem-
loop (664.1 kJÆmol
)1
). The difference in Gibbs energy for the alternative secondary structures is relatively small (8.8 kJÆmol
)1
) and equilibrium
between the two has been suggested [45]. In both (A) and (B), estimated lengths of stem I (L
I
) and of stem II–V (L
II–V
) are shown and the
primary Rev binding site is boxed.
AFM of the HIV-1 RRE, Rev and Rev–RRE J. Pallesen et al.
4224 FEBS Journal 276 (2009) 4223–4232 Journal compilation ª2009 FEBS. No claim to original US government works
unspliced RNAs to the cytoplasm is triggered, which
leads to translation of structural proteins, Gag ⁄Pol
and Env [1]. The Rev–RRE interaction thereby
specifies an essential regulatory switch in the HIV-1
replication cycle.
The structure of the RRE (or fragments hereof)
and its interaction with Rev protein have been stud-
ied in vitro by a number of different techniques,
including gel retardation assays [6–15], RNA foot-
printing and chemical modification interference analy-
sis [6,16–18], CD experiments [19], systematic
evolution of ligands by exponential enrichment
[20,21], crystallography and NMR [22–28]. These
studies have shown that RRE consists of approxi-
mately 350 nucleotides forming a complex structure
[6] and two related secondary structures have been
proposed (Fig. 1) [6,29–31]. Common to both models
are the presence of stem I as well as stem-loops II
and V, whereas discrepancies exist for the region
encompassing stem-loops III and IV. NMR and crys-
tallographic studies have resolved the 3D structure of
a single high affinity Rev binding site located within
stem IIB of RRE [23,27].
The Rev protein is a small 13 kDa protein contain-
ing 116 amino acids [32,33]. The full tertiary structure
of Rev is still unknown; however, the N-terminal
domain is known to be of predominantly a-helical nat-
ure [22,32]. The N-terminal domain is suggested to
fold into a helix–loop–helix conformation [32,34]; one
of the a-helices has been shown to interact directly
with the major groove of the primary Rev binding site
in stem-loop IIB in RRE [22]. Furthermore, the N-ter-
minal domain of Rev contains a nuclear localization
signal [30], which overlaps the Rev RNA binding site
[9,35,36]. Consequently, the nuclear localization signal
is presumably exposed only when Rev is not inter-
acting with its target RNA.
The Rev C-terminal domain is a more flexible struc-
ture [32]; it is known to contain a nuclear export signal
[37,38], which directly interacts with the exportin-
1⁄RanGTP complex [2–5]. Although the primary bind-
ing site for Rev in an internal loop of helix IIB in
RRE has been characterized, the mechanism for the
subsequent Rev oligomerization remains largely
unknown. On the basis of RNA footprinting data, it
has been suggested that binding of Rev to stem IIB
triggers a cooperative oligomerization of several copies
of Rev along RRE stems IIA and I, and that the olig-
omerization is facilitated by RNA–protein interactions
as well as protein–protein interactions [6,10,14,17,39–
42]. In vitro, at least eight copies of Rev are detected
in complex with RRE and, for steric reasons, it is spec-
ulated that up to 11–12 copies of Rev may bind to
RRE [6]. Despite many years of intense research, no
insight into the 3D structure of the entire Rev–RRE
complex has been obtained. In the present study, the
molecular structure of single 370 nucleotides version of
HIV-1 RRE was studied using high-resolution atomic
force microscopy (AFM), enabling, for the first time, a
direct visualization of the entire RRE RNA alone and
in complex with Rev protein.
Results
RRE 3D morphology characterized by AFM
To study the physical appearance of single RRE mole-
cules, a renatured 370 nucleotides version of RRE was
deposited on a piece of freshly cleaved flat mica crystal
pre-covered with a spermine layer, and was subsequently
imaged by AFM. The AFM images depicted in Fig. 2A
revealed a uniform spatial- and size distribution of RRE
molecules on the surface, with higher-resolution AFM
images of representative RRE structures being shown in
Fig. 2B–D. The structure appeared as a globular struc-
ture (the head) with a stalk extending from it. RRE size
parameters are shown in Fig. 2E. The AFM images may
be influenced by the finite size of the AFM tip; an effect
often referred to as AFM tip convolution. Nevertheless,
under the present conditions, the molecular dimensions
as determined by the AFM images were similar to those
predicted for the 3D structure extrapolated from the
RRE secondary structure model; the average cross-sec-
tional length of 43.6 ± 4.6 nm is in good agreement
with the dimensions expected from the secondary struc-
tures presented in Fig. 1. On the basis of the height con-
trast between the head and the stalk, the length of the
stalk and diameter of the head were measured to be
22.2 ± 2.9 nm and 17.0 ± 3.6 nm, respectively.
AFM and biochemical characterization of the Rev
protein and the Rev–RRE complex
To optimize the conditions for Rev–RRE complex for-
mation, the effect of titrating the Rev concentration
was studied by native PAGE (Fig. 3). As the Rev
concentration increases, multiple higher-order com-
plexes are formed, implying that Rev oligomerizes
onto RRE as described previously [17]. At a Rev :
RRE molar ratio of 20 : 1, essentially all RRE
molecules are shifted (Fig. 3, lane 5) and this reaction
condition was chosen for further AFM analysis of the
Rev–RRE complex.
To enable identification of unbound Rev, an AFM
line scan was performed on Rev protein alone. Rev
appeared mainly as small globular dots (Fig. 4A).
J. Pallesen et al. AFM of the HIV-1 RRE, Rev and Rev–RRE
FEBS Journal 276 (2009) 4223–4232 Journal compilation ª2009 FEBS. No claim to original US government works 4225
The height was estimated to 2.3 ± 0.4 nm (Fig. 4B).
Next, Rev–RRE complexes formed at a 20 : 1 molar
ratio were subjected to AFM imaging in a physiologi-
cally relevant buffer. An overview of the Rev–RRE
complexes is depicted in Fig. 5A, and magnifications
of the Rev–RRE complexes are shown in Fig. 5B–D.
It is observed that the overall elongated morphology
described for the RRE RNA alone is preserved for the
Rev–RRE complex. However, the Rev–RRE complex
is wider and higher than the RRE alone and the clear
distinction between head and stalk structures has
disappeared. The total length of the Rev–RRE
complex was measured to be 42.8 ± 5.6 nm (Fig. 5E),
which is very close to the estimated length of RRE
alone (43.6 ± 4.6 nm). Apart from Rev–RRE com-
plexes, the AFM images also revealed an excess of
unbound Rev, which was readily identified by its
resemblance to the structures observed when imaging
the Rev protein alone.
Rev binding to RRE led to a dramatic height
increase for the observed molecules and, to quantify
this observation, the heights of the stalk and head
were measured for RRE and for Rev–RRE, respec-
tively. By convention, the highest point in the AFM
cross-sectional profile was tentatively used to esti-
mate the height of the head, whereas the next local
height maximum was used to estimate the stalk
height. The results are shown in Fig. 5F, and it is
Fig. 3. Rev–RRE complexes. Lane 1: renatured RRE RNA alone.
Two alternative RRE structures and a faint band corresponding to
an RRE dimer can be seen. Lanes 2–7: Rev–RRE complex forma-
tion as a function of Rev:RRE molar stoichiometry (5 : 1, 10 : 1,
15 : 1, 20 : 1, 25 : 1 and 30 : 1). Lane 5 exhibits a complete shift
of RRE into a distribution of Rev–RRE complexes and was chosen
as the condition for AFM imaging.
Ltot, Total length LI, Stalk length LII-V, Head length
43.6
22.2
17.0
0.0
10.0
20.0
30.0
40.0
50.0
60.0
nm
RRE length
0.0 -
1.0 -
2.0 -
3.0 -
nm
20 nm
LI
Ltot
LII-V 20 nm 20 nm
200 nm
A
B
E
CD
Fig. 2. AFM scan of the RRE. (A) An overview image showing a
homogenous distribution of similarly structured RRE molecules
(inset: vertical height scale is 0–3 nm). (B–D) Representative RRE 3D
structures described by high-resolution AFM. The visible RRE struc-
tural characteristics include a wider head region and a protruding
stalk. (E) Average total length, stem I (stalk) length and stem-loops
II–V (head) length was estimated along the axes shown in (B).
AFM of the HIV-1 RRE, Rev and Rev–RRE J. Pallesen et al.
4226 FEBS Journal 276 (2009) 4223–4232 Journal compilation ª2009 FEBS. No claim to original US government works
seen that the stalk height for RRE was measured to
be 0.5 ± 0.1 nm, increasing to 3.1 ± 1.1 nm in the
Rev–RRE complex, whereas the heights of the head
of the RRE and Rev–RRE were determined to
0.7 ± 0.1 nm and 4.5 ± 1.2 nm, respectively. We
can thus conclude that the binding of the Rev pro-
tein to RRE results in a dramatic increase in the
height, although with little effect on the overall
length of the complex.
The height difference between RRE and Rev–RRE
may be interpreted as the volume of Rev protein
bound to RRE. We therefore estimated the length of
the Rev protein coating on RRE by measuring the
length of the part of the Rev–RRE complex that was
greater than 0.5 nm for the stalk and 0.7 nm for the
head. The length of this large substructure of the Rev–
RRE complex was 40.0 ± 5.8 nm (Fig. 5G). The mea-
sured and calculated dimensions of RRE, Rev and
Rev–RRE are summarized in Table 1.
Discussion
Previous studies of the full-length RRE and Rev–RRE
complexes have so far employed biochemical and
biophysical techniques only [22–28]. These studies
have led to the proposal of the two related RRE
secondary structure models, as depicted in Fig. 1
[6,7,10,11,13,16,17,31,32,43,44]. This is consistent with
the findings of the present study, which shows that the
renatured RRE sample migrates as two bands (Fig. 3,
lane 1). The two conformers may be in equilibrium
A
B
E
G
F
CD
Fig. 5. AFM scan of Rev–RRE in solution. (A) An overview of the
distribution of Rev–RRE complexes in the sample (inset: vertical
height scale is 0–3 nm). (B–D) High-resolution AFM scanning of
Rev–RRE complexes. (E) Average total length of Rev–RRE com-
plexes. (F) Height measurements of stalk (L
I
) and head (L
II–V
). (G)
Length of Rev protein coating of RRE (L
Rev oligomer
). For additional
details, see Experimental procedures.
A
B
Fig. 4. AFM analysis of Rev protein. (A) Rev alone appeared as
small globular dots in the AFM scans. (B) Rev height was esti-
mated to be 2.3 ± 0.4 nm.
J. Pallesen et al. AFM of the HIV-1 RRE, Rev and Rev–RRE
FEBS Journal 276 (2009) 4223–4232 Journal compilation ª2009 FEBS. No claim to original US government works 4227