REVIEW
published: 22 March 2018
doi: 10.3389/fmicb.2018.00527
Edited by:
David Gilmer,
Université de Strasbourg, France
Reviewed by:
Jun-Ichi Sakuragi,
Osaka University, Japan
Nathan Sherer,
University of Wisconsin–Madison,
United States
*Correspondence:
Serena Bernacchi
s.bernacchi@ibmc-cnrs.unistra.fr
Jean-Christophe Paillart
jc.paillart@ibmc-cnrs.unistra.fr
Specialty section:
This article was submitted to
Virology,
a section of the journal
Frontiers in Microbiology
Received: 22 January 2018
Accepted: 08 March 2018
Published: 22 March 2018
Citation:
Dubois N, Marquet R, Paillart J-C
and Bernacchi S (2018) Retroviral
RNA Dimerization: From Structure
to Functions. Front. Microbiol. 9:527.
doi: 10.3389/fmicb.2018.00527
Retroviral RNA Dimerization: From
Structure to Functions
Noé Dubois, Roland Marquet, Jean-Christophe Paillart* and Serena Bernacchi*
Architecture et Réactivité de l’ARN, UPR 9002, IBMC, CNRS, Université de Strasbourg, Strasbourg, France
The genome of the retroviruses is a dimer composed by two homologous copies of
genomic RNA (gRNA) molecules of positive polarity. The dimerization process allows
two gRNA molecules to be non-covalently linked together through intermolecular basepairing.
This step is critical for the viral life cycle and is highly conserved among
retroviruses with the exception of spumaretroviruses. Furthermore, packaging of two
gRNA copies into viral particles presents an important evolutionary advantage for
immune system evasion and drug resistance. Recent studies reported RNA switches
models regulating not only gRNA dimerization, but also translation and packaging, and
a spatio-temporal characterization of viral gRNA dimerization within cells are now at
hand. This review summarizes our current understanding on the structural features of the
dimerization signals for a variety of retroviruses (HIVs, MLV, RSV, BLV, MMTV, MPMV. . .),
the mechanisms of RNA dimer formation and functional implications in the retroviral
cycle.
Keywords: retrovirus, RNA, dimerization, HIV, MuLV, structure, function
INTRODUCTION
During the late phase of their replication cycle, retroviruses package two homologous copies of
their genomic RNA (gRNA) in order to produce infectious viral particles. This co-packaging
of gRNA molecules is highly facilitated by the dimerization signal within the 5 -region of the
viral genome and by the viral Gag precursor in order to form new viral particles (Lee et al.,
1999; Cimarelli et al., 2000, for reviews see D’Souza and Summers, 2005; Mailler et al., 2016).
Genome dimerization is a highly conserved process amongst retroviruses, and this feature is crucial
for several important steps in the retroviral life cycle (Figure 1). First, for several retroviruses,
e.g., human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV), gRNA
dimerization is critical for selective packaging of the genome (Berkhout and van Wamel, 1996;
Mougel et al., 1996; Paillart et al., 1996a; McBride and Panganiban, 1997; Mougel and Barklis,
1997; Aagaard et al., 2004; Houzet et al., 2007). Second, the conformational changes induced by
RNA dimerization may also regulate translation of the unspliced gRNA (Kharytonchyk et al., 2016;
Boeras et al., 2017). Third, dimerization of the viral genome also plays an important role during
the reverse transcription step, allowing genome repair by strand transfer when one of the two RNA
strands is damaged (Mikkelsen and Pedersen, 2000). Finally, genome dimerization presents the
great advantage of increasing genetic diversity by allowing genetic recombination during reverse
transcription (Mikkelsen et al., 2000; Moore et al., 2009).
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Dubois et al. RNA Dimerization in Retroviruses
FIGURE 1 | Schematic overview of the role of RNA dimerization in the retroviral life cycle. The cycle begins with the entry of the retrovirus within the target cell,
followed by reverse transcription of the RNA genome into cDNA. During this step, gRNA dimerization plays an important role since RT may switch between strands,
thus allowing genome repair and/or shuffling. The pre-integration complex (PIC) is then translocated into the nucleus where it is integrated in the genome of the
target cell. The unspliced mRNAs are transcribed by the host machinery from the integrated provirus and transported to the cytoplasm. There, the single 5 capped
mRNAs serve as genomic RNAs that dimerize and are subsequently selected and packaged into the nascent virions, while mRNAs beginning with two or three
guanosine are translated by the host machinery (Kharytonchyk et al., 2016). After budding, immature particles follow a maturation step initiated by the viral protease
to produce infectious virions.
The first evidence for the existence of a dimeric genome
came from ultracentrifugation sedimentation analysis of gRNA
extracted Rous sarcoma virus (RSV) (Duesberg, 1968; Mangel
et al., 1974), even though a tetrameric organization of the
genome had also been proposed (Montagnier et al., 1969).
Subsequent sedimentation and electron microscopy analyses
supported the dimeric organization of the genome and
extended this observation to other retroviruses families such
as alpharetroviruses, gammaretroviruses, and lentiviruses (Kung
et al., 1976; Bender et al., 1978; Maisel et al., 1978; Murti et al.,
1981; Höglund et al., 1997), thus revealing the conservation
and the importance of gRNA dimerization in the life cycle of
retroviruses. Further studies showed that regions involved in
gRNA dimerization, historically referred to as the dimer linkage
structure (DLS), are typically close to the 5 -end of gRNA, and
highly structured with multiple stem-loop motifs (Prats et al.,
1990; Roy et al., 1990; Tounekti et al., 1992; Baudin et al., 1993;
Mougel et al., 1993; Garzino-Demo et al., 1995; Jossinet et al.,
2001; Abbink and Berkhout, 2003; D’Souza et al., 2004; Aktar
et al., 2013, 2014). Even though retroviral genomes are rather
large (from ∼7 to ∼12 kb), gRNA dimerization was observed
to be mediated by relatively short sequences ranging from 50to
few hundreds of nucleotides (Kung et al., 1975; Bender et al.,
1978; Maisel et al., 1978; Murti et al., 1981). In vitro analysis
showed that DLS-containing RNA fragments could dimerize at
temperatures ranging from 37 to 60◦C depending on the virus,
in the presence of monovalent (Na+ or K+ ranging from 0.1
to 0.3 M), and/or divalent (Mg2+ ranging from 1 to 10 mM)
cations (Bieth et al., 1990; Darlix et al., 1990; Prats et al.,
1990; Roy et al., 1990; Marquet et al., 1991; Paoletti et al., 1993).
However, a better understanding of the precise mechanisms
governing the dimerization process was only obtained with the
identification of the HIV-1 dimerization initiation site (DIS).
A common feature of retroviral DIS is the presence of at
least one short palindromic sequence enabling intermolecular
base-pairing, thus forming kissing-loop structures (Laughrea and
Jetté, 1994; Paillart et al., 1994; Skripkin et al., 1994; Girard
et al., 1995; Muriaux et al., 1995). In the case of HIV-1, chemical
modification interference assays allowed the identification of
the six nucleotides constituting the DIS (Skripkin et al., 1994).
Kissing-loop complexes, often referred to as “loose dimers,’
are characterized by low thermal stability (Fu and Rein, 1993;
Fu et al., 1994; Jalalirad and Laughrea, 2010), and can only
be visualized by gel electrophoresis under native conditions,
since even mild denaturing conditions were found to dissociate
RNA dimers during migration (Marquet et al., 1994; Laughrea
and Jetté, 1996; Polge et al., 2000). However, incubation of
DLS-containing RNAs at non-physiological high temperatures
(50–60◦C) was found to induce formation of RNA dimers
resistant to mild-denaturing electrophoresis conditions and were
thus called “tight dimers.” Importantly, tight dimers are also
obtained at physiological temperature in the presence of the
cognate nucleocapsid (NC) protein (Girard et al., 1996; Muriaux
et al., 1996), well-known for its RNA chaperone properties (Feng
et al., 1996; Girard et al., 1996; Muriaux et al., 1996; Rist and
Marino, 2002; Mujeeb et al., 2007; Aduri et al., 2013; for a review
Levin et al., 2005). These results lead to the notion that NC lowers
the energy barriers and promote refolding of the 5 -end region
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Dubois et al. RNA Dimerization in Retroviruses
FIGURE 2 | Motifs involved in the RNA dimerization of alpha-retrovirus. (A) Schematic representation of the 5 -end of alpha retrovirus gRNA. The functional domains
and their positions are represented: R, repeated region; U5, unique sequence in 5 ; PBS, primer binding site; AUG, gag translation initiation codon; SD, splice donor
site; L3 and DLS, dimerization motifs for ALV and RSV, respectively. (B) Predicted secondary structure of the ALV L3 stem-loop. The consensus nucleotides are
represented, with the palindromic hexanucleotide sequence highlighted in red. (C) Proposed kissing-loop complex and extended duplex conformations of ALV L3
element.
of gRNA into a more stable conformation (Feng et al., 1996;
Gorelick et al., 1996, 1999; Cruceanu et al., 2006). Studies on short
sequences harboring the DIS suggested that these conformational
changes could involve the refolding of these structures by forming
cruciform intermediates that evolve into extended intermolecular
base-pairing (Polge et al., 2000; Rist and Marino, 2002; Bernacchi
et al., 2005).
In vivo, subsequent to viral budding, immature virions
undergo a maturation step that is mandatory for viral infectivity
and is mediated by the viral protease that sequentially
cleaves the viral Pr55Gag and Pr160GagPol precursors into
the mature structural and enzymatic proteins. Concomitantly
to this proteolytic maturation, the viral genome undergoes
a maturation process (Ohishi et al., 2011; Mailler et al.,
2016). Indeed, HIV-1 and MuLV gRNA dimers extracted
from immature viral particles are less stable than those
extracted from mature virions (Fu and Rein, 1993; Fu
et al., 1994; Jalalirad and Laughrea, 2010; Ohishi et al.,
2011; Grohman et al., 2014). Interestingly, the different
stabilities observed in immature and mature virions are
very similar to those observed in vitro for loose and tight
RNA dimers, respectively, suggesting these conformations
may reflect the maturation process of gRNA into viral
particles.
In this review, we will focus on our current understanding
of the mechanisms and molecular factors involved in gRNA
dimerization for different retrovirus families both in vitro and
in cellula, its role during the retroviral life cycle, and finally
its potential targeting by molecules aimed at inhibiting viral
replication.
ALPHA-RETROVIRUSES GENOME
DIMERIZATION
Even though the first evidence for retroviral RNA dimerization
came from sedimentation and electron microscopy studies
of alpha-retroviruses such as RSV (Duesberg, 1968; Canaani
et al., 1973; Mangel et al., 1974), the precise mechanisms
underlying this process remains surprisingly poorly defined in
comparison with other model retroviruses such as HIV-1 and
MuLV. Strikingly, while most DLS/DIS are found within the
5 -untranslated region (UTR) of gRNA, electron micrographs
of gRNA dimers extracted from RSV virions located the DLS
within the gag gene, around position 480–540 from the 5 end
(Figure 2A) (Murti et al., 1981). One other peculiarity lies
in the fact that RSV DLS contains an imperfect palindrome
that was first proposed to contribute to RNA dimerization
(Schwartz et al., 1983). In rather good agreement with these
findings, in vitro analyses of the dimerization of the first 634
nucleotides (nts) of RSV gRNA suggested that the DLS would
be located between positions 544–564 (Bieth et al., 1990; Lear
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Dubois et al. RNA Dimerization in Retroviruses
et al., 1995) and would likely involve Watson–Crick basepairing
of the palindrome mentioned above (Lear et al., 1995).
However, analysis of gRNA dimerization of the avian sarcomaleucosis
virus (ASLV), another alpha-retrovirus, relies on the
L3 element, a conserved 19-nts hairpin harboring a perfect
palindrome sequence in its apical loop and located upstream
of the major Splice Donor (SD) site (Figure 2B) (Fossé et al.,
1996). Interestingly, in the absence of NC, viral RNA fragments
of 626 nts in length form loose dimers at 37◦C, involving
loop-loop interactions through the L3 element (Figure 2C,
left) (Polge et al., 2000). However, when incubated at 60◦C,
these fragments formed tight dimers, and heterodimerization
of L3 stem mutants supported the notion that in vitro
ASLV gRNA tight dimers are characterized by the formation
of an extended duplex (Figure 2C, right) (Polge et al.,
2000).
BETA-RETROVIRUSES GENOME
DIMERIZATION
Recently, Rizvi and collaborators provided the first in vitro
analyses of structure and dimerization mechanisms of the 5 end
region of the mouse mammary tumor virus (MMTV)
and Mason-Pfizer monkey virus (MPMV) gRNAs (Aktar et al.,
2013, 2014; Kalloush et al., 2016). Selective 2 -hydroxyl acylation
analyzed by primer extension (SHAPE) and in vitro dimerization
assays of MMTV gRNA fragments revealed that loose dimer
formation is potentially mediated by two palindromic sequences,
respectively, within the primer binding site (PBS-Pal) and in a
bifurcated stem-loop structure (SL4) located between the PBS
and the translation initiation codon of gag (Pal II) (Figure 3A).
However, Pal II is the main DIS since its mutation had a greater
impact on RNA dimerization than mutation of the PBS-Pal
(Figure 3B) (Aktar et al., 2014). In MPMV, the palindromic
sequence that functions as the gRNA DIS folds into a short
hairpin (Pal SL) (Figures 3C,D) (Aktar et al., 2013). These motifs
are highly conserved in MMTV and MPMV strains and their
mutation leads to severe gRNA packaging and viral replication
defects (Jaballah et al., 2010; Aktar et al., 2014). Interestingly,
secondary structures of the 5 -end of both MPMV and MMTV
gRNAs present long-range interactions (LRI) involving the 5
unique (U5) region and a region spanning the gag translation
initiation codon (Figures 3A,C) (Aktar et al., 2014; Kalloush
et al., 2016), similarly to what has been observed for lentiviruses,
for which such interaction was proposed to promote gRNA
dimerization (see below). In MPMV, these LRIs are required for
gRNA packaging and viral propagation, even though mutations
destabilizing LRIs have only modest effects on RNA dimerization
(Kalloush et al., 2016).
GAMMA-RETROVIRUSES GENOME
DIMERIZATION
A general feature of gamma-retroviruses RNA dimerization is
the presence of several palindromes within stem-loops that
contribute to dimer formation at different degrees. Within this
family, murine leukemia virus (MuLV) is the most studied model,
and its minimal DLS corresponds to a 170-nts long region
containing four stem-loops (SL-A to SL-D) each contributing
to the dimerization process (Prats et al., 1990; Roy et al.,
1990; Tounekti et al., 1992; Mougel et al., 1993; Torrent et al.,
1994; Girard et al., 1995; De Tapia et al., 1998; Badorrek and
Weeks, 2005) (Figure 4A). However, comparison of the chemical
reactivity between monomer and dimer also suggested that
gag region could contribute to dimerization in a full-length
genome context (Mougel et al., 1993). The first two stemloops,
SL-A and SL-B, contain palindromes of 10- and 16-nts,
respectively, both critical for the in vitro dimerization process
(Figures 4B,C) (Tounekti et al., 1992; Girard et al., 1995, 1996;
Oroudjev et al., 1999; Ly and Parslow, 2002). Additionally,
NMR and SHAPE analyses of MuLV DLS indicated that SLC
and SL-D, both presenting a highly conserved GACG apical
tetraloop (Konings et al., 1992) (Figure 4A), form canonical
heterologous loop–loop interactions that significantly contribute
to dimer stability (De Tapia et al., 1998; Kim and Tinoco,
2000; D’Souza et al., 2004; Badorrek et al., 2006; Gherghe
and Weeks, 2006; Miyazaki et al., 2010b) (Figure 4B). While
MuLV initial loose dimer involves only few intermolecular
base-pairs in the apical loops of SL-A and SL-B, in vitro
analysis indicated that, when RNA fragments containing MuLV
DLS are incubated at 60◦C or at 37◦C in the presence of
NC, both contacts undergo structural rearrangements, and
expand to the whole SL-A and SL-B palindromes (Figure 4C,
right) (Roy et al., 1990; Girard et al., 1996; Ly and Parslow,
2002; Badorrek and Weeks, 2006). Interestingly, formation of
the extended duplex exposes UCUG quartets that are basepaired
in the monomeric structure (compare Figures 4A with
4C), and that correspond to high affinity binding sites for
NC protein. Furthermore, electron tomography and hydroxyl
footprinting data both indicated that MuLV DLS adopts a
compact structure upon dimerization (Badorrek et al., 2006;
Badorrek and Weeks, 2006; Miyazaki et al., 2010b) suggesting
that the stability of tight RNA dimers is also mediated by
stacked helices, in addition to formation of the extended
duplex.
In viro SHAPE analyses on extracted gRNA from mature
MuLV particles also revealed that the gRNA dimer adopts the
SL-A/SL-A and SL-B/SL-B extended duplexes conformation
(Gherghe et al., 2010) (Figure 4C, right). Interestingly, recent
chemical probing data on extracted gRNA dimers from
immature MuLV particles indicated that the SL-B element would
not be involved in the inter-molecular duplex (Figure 4C,
left) (Grohman et al., 2014), thus supporting the notion
of a conformational switch between immature and mature
RNA dimer forms. This structural switch is likely to occur
through the chaperone activity of MuLV NC, and is further
facilitated by the stabilization of the duplex by SL-C and
SL-D, as previously suggested (D’Souza et al., 2004; Badorrek
et al., 2006; Miyazaki et al., 2010b). These data are in
good agreement with findings showing that deletion of SL-A
significantly decreased MuLV gRNA packaging and affected
replication kinetics in cell culture, while mutation/deletion of
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Dubois et al. RNA Dimerization in Retroviruses
FIGURE 3 | Secondary structure of the 5 -end of MMTV and MPMV genomic RNAs and dimerization models. (A) MMTV gRNA secondary structure, the different
palindromic sequence (pal I, II, III, and PBS-pal) and the long-range interaction (LRI) between U5 and the beginning of gag are indicated. The different stem-loops
(SL) are numbered as proposed by (Aktar et al., 2014). SL4 comprises both pal II which is the proposed DIS, and the major SD site. The R and PBS regions are also
represented. (B) MMTV pal II switch models from kissing-loop complex to extended duplex. The nucleotide positions are represented and the hexanucleotide
palindrome is highlighted in red. (C) MPMV gRNA secondary structure, the different stem-loops (SLs) are numbered as proposed by (Aktar et al., 2013), the LRI
between U5 and the beginning of gag is also represented. The palindromic sequence folded in a short hairpin (Pal SL) which is the proposed DIS is highlighted in
red. (D) MPMV Pal SL switch models from the kissing-loop complex to the extended duplex. The nucleotide positions are represented and the hexanucleotide
palindromic sequence is highlighted in red.
SL-B only presented moderate effect regarding these processes
(Aagaard et al., 2004). Taken together, these results suggest
that only the 10-nts palindromic sequence within SL-A is
crucial to form the immature gRNA dimer (Figure 4C,
left).
Finally, in vitro analysis of the gRNA dimer of feline
endogenous RD-114, another gamma-retrovirus, revealed the
presence of several palindromic stem-loops (psl-1 to psl-5)
leading to RNA dimer formation (Kharytonchyk and Pedersen,
2010). Interestingly, and similarly to MuLV, the RD-114 DLS
also contains two GACG tetraloops that are close to the crucial
DIS elements and contribute to in vitro RNA dimerization
(Kharytonchyk and Pedersen, 2010).
DELTA-RETROVIRUSES GENOME
DIMERIZATION
Site-directed mutagenesis, antisense oligonucleotides mapping
experiments, as well as structural predictions identified the DLS
of bovine leukemia virus (BLV) in the 5 -region of the genome,
in a region overlapping U5, the PBS and 30 bases downstream
of this latter (Katoh et al., 1993). Interestingly, the DLS of
BLV also overlaps the Psi (packaging signal), similarly to other
retroviruses, since deletion of this region completely abrogated
genome encapsidation and viral replication (Mansky et al., 1995).
However, a particular feature of BLV is the fact that in vitro
dimerization of gRNA is promoted by the viral matrix protein,
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Dubois et al. RNA Dimerization in Retroviruses
FIGURE 4 | Secondary structures and dimerization of MuLV genomic RNA. (A) Secondary structure of the 5 -end of MuLV gRNA containing the four stem-loops
SL-A to SL-D. The nucleotide positions are indicated. (B) In vitro model of the kissing-loop/loose dimer complex. SL-A and SL-B contain a palindromic sequence of
10 and 16-nts long, respectively (in red), that promote the initiation of RNA dimerization. SL-C (purple) and SL-D (blue) both present a GACG tetraloop involved in
heterologous non-canonical loop-loop interactions that stabilize the duplex. In this conformation, several UCUG quartets (green), which constitute high affinity
binding sites for the viral NC protein, are trapped within SL-A and SL-B. (C) Secondary structure model of gRNA dimer maturation steps derived from SHAPE data
(Grohman et al., 2014): the extracted ex viro immature form (left) is converted by NC into the mature 5 -end RNA dimer (right). In the immature conformation, only
SL-A and SL-A are paired and adopt an extended conformation while the SL-B elements are unpaired. This conformation exposes the UCUG quartets. In the
mature conformation, both SL-A/SL-A and SL-B/SL-B loop–loop interactions adopt an extended duplex structure, thus increasing dimer stability (Gherghe et al.,
2010). This model also exposes the UCUG quartets. The mature dimer is thought to be to be similar to in vitro 5 -end tight dimer structure.
and not by NC, contrary to what is observed for other retroviruses
(Katoh et al., 1991, 1993).
Similar experimental approaches revealed that a 32-nts
sequence just upstream the PBS mediates in vitro dimerization of
human T-cell leukemia virus type-1 (HTLV-1) gRNA (Greatorex
et al., 1996). Within this region, a conserved 14-nts palindromic
stem-loop sequence was identified as the DIS (Greatorex et al.,
1996; Monie et al., 2001), since deletion of this motif abrogated
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Dubois et al. RNA Dimerization in Retroviruses
FIGURE 5 | HTLV-I DIS model. Contrary to what is observed for other
retroviruses, the DIS of HTLV-I presents a peculiar secondary structure
harboring a single A residue in the apical loop (Monie et al., 2004). The
palindrome is highlighted in red and the nucleotide positions are indicated.
The proposed dimer interaction is also represented (Monie et al., 2001).
in vitro RNA dimerization. Since HTLV-1 RNA dimers present
a very high thermal stability (Tm ∼ 70–80◦C), it was proposed
that Watson–Crick base-pairings could not be solely responsible
for RNA dimerization, and that other non-canonical or tertiary
interactions would further contribute to the in vitro RNA
dimer stability (Greatorex et al., 1996). Interestingly, biochemical
structural analysis of this motif also indicated that, unlike other
retroviruses displaying DIS larger loops, the HTLV-1 DIS forms
a hairpin with a single A residue in the apical loop closed by
non-canonical C-synG base-pairs (Figure 5) (Monie et al., 2004).
However, in contrast to other retroviruses, deletion of the 37-nts
DLS only caused a modest decrease in viral infectivity (20–25%)
(Le Blanc et al., 2000). Therefore, further studies will be necessary
to identify all the determinants involved in these processes.
LENTIVIRUSES GENOME DIMERIZATION
The dimerization of lentiviral RNA genomes is the best
characterized retroviral dimerization process, and several
important features for gRNA dimerization are conserved in
the genus. Like in all retroviruses, the DLS is located at the 5
region of the viral genome. The DIS of primate lentiviruses such
as HIV-1/2 or simian immunodeficiency virus (SIV) is located
within the 5 -UTR, downstream of the PBS (Skripkin et al., 1994;
Dirac et al., 2001; Whitney and Wainberg, 2006) (Figure 6A).
Contrary to gamma-retroviruses, the lentiviral DIS consists
in a single palindromic sequence, typically located within the
apical loop of a hairpin structure and forming homologous
intermolecular canonical Watson–Crick base-pairs (Figure 6B).
This initial intermolecular contact generally expands to a larger
sequence during the stabilization of the tight dimer (Figure 6).
Interestingly, the DIS of feline immunodeficiency virus (FIV)
seems to be located within the gag coding region (Kenyon
et al., 2011), although a structural study proposed an alternative
hairpin harboring a less conserved palindromic sequence
upstream of gag as a potential DIS (James and Sargueil, 2008).
Another conserved feature in lentiviruses is the existence of
a conformational switch regulating gRNA dimerization. Indeed,
gRNA fragments encompassing the 5 region of the genome
adopt alternative structures involving long-range interactions
(LRI) between the R/U5 elements and regions overlapping the gag
translation initiation codon (see below for HIV-1) to expose the
DIS and promote RNA dimerization (Figure 7), or alternatively
to prevent it (Figure 8) (Huthoff and Berkhout, 2001; Abbink and
Berkhout, 2003; Lanchy et al., 2003a,b; Whitney and Wainberg,
2006; Kenyon et al., 2008, 2011; Lu et al., 2011; Tran et al., 2015).
Human Immunodeficiency Virus Type 1
gRNA Dimerization
The HIV-1 DIS is located in the 5 region of the gRNA,
within the packaging signal, Psi, which is composed of four
stem-loops (SL1–SL4) (Figure 6A) (Lever et al., 1989; Aldovini
and Young, 1990; Clavel and Orenstein, 1990; Clever et al.,
1995; Mailler et al., 2016), although SL4, encompassing gag
initiation codon, seems to be poorly stable and would be most
likely in equilibrium with the U5 region as discussed below
(Abbink and Berkhout, 2003; Lu et al., 2011). Importantly, SL1
contains in its apical loop a 6-nts palindromic sequence that have
been shown by interference of chemical modifications and sitedirected
mutagenesis analyses to be responsible for the in vitro
gRNA dimerization through formation of a loop-loop kissing
complex (Figure 6B, up) SL4 (Marquet et al., 1994; Paillart et al.,
1994; Skripkin et al., 1994) and is important for viral replication
(Berkhout and van Wamel, 1996; Paillart et al., 1996a; Clever
and Parslow, 1997; Laughrea et al., 1997, 1999). The DIS of
HIV-1 is submitted to strong selection pressure, and its diversity
is rather limited in vivo, with a strong prevalence for three
GC-rich palindromes (GCGCGC, GUGCAC, and GUGCGC)
(Skripkin et al., 1994; Berkhout, 1996; Paillart et al., 1996b;
Hussein et al., 2010). Accordingly, in vitro selection experiments
from randomized DIS mutants indicated that selected sequences
contain the central two G-C base-pairs that are critical for
kissing complex stability, while mutants presenting more than
one A-U base-pair were poorly replicating (Clever et al., 1996;
Laughrea et al., 1999; Lodmell et al., 2000, 2001; Hussein et al.,
2010).
In HIV-1, the conformational switch from the kissingloop
complex to the extended duplex was extensively studied
in vitro using 23-mer (corresponding to SL1 upper part) and
35-mer (corresponding to the whole SL1 hairpin) SL1 RNA
fragments (Figure 6B). Both 3D structures were solved by NMR
(Kieken et al., 2006; Ulyanov et al., 2006) (Figure 6C) and
X-ray crystallography (Ennifar et al., 1999, 2001) (Figure 6D).
Crystal structures of SL1 dimers revealed that Mg2+ ions
promote exposure of two conserved unpaired purine residues
flanking the DIS (Ennifar et al., 1999) (Figure 6D) that play an
important role in the rate of NC-catalyzed duplex formation and
were proposed to be involved in non-canonical intermolecular
interactions (Paillart et al., 1997; Mihailescu and Marino, 2004;
Mundigala et al., 2014). Interestingly, only the HIV-1 DIS
containing the flanking purines can replace the cognate betaretroviruses
palindromic sequence (Aktar et al., 2013, 2014),
further supporting the importance of these purine residues in
HIV-1 palindrome dimerization, even in a heterologous context.
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Dubois et al. RNA Dimerization in Retroviruses
FIGURE 6 | Mechanism of HIV-1 RNA dimerization. (A) Schematic secondary structure model of the 5 -end region of the HIV-1 gRNA. TAR, trans-activation
response element; Poly-A, stem-loop containing the 5 -copy of the polyadenylation signal in the apical loop; U5, unique in 5 ; PBS, primer binding site; DIS,
dimerization initiation site; SL1–4: stem-loops 1–4 containing the Dimerization Initiation Site (DIS), the major Splice Donor (SD) site, the historical packaging signal,
and the gag AUG initiation codon, respectively. The packaging signal (Psi) region, spanning SL1-SL4 elements, is indicated. (B) Model of the SL1 switch from the
kissing-loop complex to the extended duplex conformation. The nucleotide positions are indicated. (C) Solution structures of SL1 23-mer kissing-loop complex (KC)
(up) (Kieken et al., 2006) and of SL1 35-mer extended duplex (ED) (down) (Ulyanov et al., 2006) as determined by NMR. The DIS palindromic sequences are
highlighted in red, and the purines flanking the DIS are in orange. Structures were drawn using the coordinates deposited on the PDB (PDB ID: 2F4X – KC – and
2GM0 – ED). (D) X-ray crystal structures of SL1 23-mer both in KC (up) (Ennifar et al., 2001) and ED (down) (Ennifar et al., 1999) conformations. The DIS palindromic
sequences are highlighted in red. One Mg2+ ion found in the ED crystal structure exposing the highly conserved purines flanking the DIS (in orange) is drawn in
purple. Structures were drawn using the coordinates deposited on the PDB (PDB ID: 2B8R – KC – and 2F4X – ED).
Although it has been proposed, based on the crystal structure
of the SL1 23-mer dimer (Figure 6D), that the extended
duplex formation may be the result of an intermolecular transesterification
reaction (Ennifar et al., 2001), NMR and UVmelting
analyses on SL1 35-mers indicated that the transition
from loose to tight dimers involves melting of the SL1 upper stem,
without melting the kissing-loop duplex interface, followed by
interstrand exchange as refolding of the stem occurs (Mundigala
et al., 2014). This model is supported by several studies showing
that the SL1 internal loop destabilizes the SL1 upper stem
(Takahashi et al., 2000; Greatorex et al., 2002; Baig et al., 2007;
Mujeeb et al., 2007) and constitutes a NC binding site that was
shown to promote NC-mediated transition from the kissingloop
complex to the extended duplex (Rist and Marino, 2002;
Hagan and Fabris, 2007). Interestingly, the SL1 internal loop was
recently found to constitute a major binding site for Pr55Gag
(Abd El-Wahab et al., 2014; Smyth et al., 2015; Bernacchi et al.,
2017), suggesting that formation of the extended duplex could be
promoted by the NC domain within Pr55Gag as well. Moreover,
Mg2+ ions strongly impact the rate at which the duplex is formed
(Bernacchi et al., 2005) and were observed to bind the SL1
internal loop, possibly preventing premature transition from the
kissing-loop to the extended duplex (Sun et al., 2007). Therefore,
one could speculate on a model where Mg2+ and Pr55Gag (or NC)
compete for binding to the SL1 internal loop, thus stabilizing or
destabilizing the upper stem. Consistent with this notion, the SL1
internal loop was shown to be important for RNA dimerization,
packaging and viral infectivity in cell cultures (Clever et al., 1996;
Paillart et al., 1996a; Clever and Parslow, 1997; Harrison et al.,
1998; Laughrea et al., 1999; Shen et al., 2000) and stabilization
of the SL1 hairpin resulted in replication defects (van Bel et al.,
2014).
The existence of an extended duplex in large HIV-1 RNA
fragments encompassing the whole DLS constitutes a question
of debate and remains to be clearly demonstrated in the
viral context. First, probing techniques cannot discriminate
intramolecular from intermolecular base-pairings, thus making
difficult the discrimination of both conformations. Second, SL1
trans-complementary mutants that are not able to form the
extended duplex efficiently dimerized in vitro and were found
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Dubois et al. RNA Dimerization in Retroviruses
FIGURE 7 | Secondary structure models of HIV-1 genomic RNA. (A) HIV-1 long-distance interaction model (LDI) as proposed by Huthoff and Berkhout (2001), in
which SL1 DIS is base-paired together with the poly-A element. (B) HIV-1 U5:DIS model proposed by Lu et al. (2011), in which SL1 DIS is base-paired with the U5
region. This model was proposed to promote the translation of unspliced gRNAs by repressing dimerization (Lu et al., 2011). (C,D) Dimerization-competent
structural models of HIV-1 5 -end. (C) The branched multiple hairpin (BMH)/U5:AUG models (Huthoff and Berkhout, 2001; Lu et al., 2011) in which SL1 DIS is
exposed while U5 base-pairs with the region overlapping gag translation initiation codon, which promotes dimerization and was proposed to repress translation
(Lu et al., 2011). (D) Keane et al. (2015) recently proposed a putative three-way junction structure of the extended duplex conformation in which the whole region
downstream of SL1 is exchanged. This conformation was proposed to be achieved through NC chaperone activity.
to be as stable as wild-type RNA fragments (Paillart et al.,
1996c). Besides, the conformational switch from the kissingloop
complex to the extended duplex in full-length gRNA or
even in complete 5 -UTR context presents huge topological
and steric constraints. Therefore, it is reasonable to consider
that HIV-1 RNA tight dimer formation would rather be the
result of conformational rearrangements at a larger scale, with
the formation of additional contacts (see below). The notion
of additional inter-genomic contacts outside SL1/DIS is also
supported by electron microscopy studies on full-length gRNA
dimers extracted from virions and atomic force microscopy
(AFM) analysis on gRNA fragments encompassing the first
744 nts of the HIV-1 genome showing that HIV-1 gRNA
dimers are not Y-shaped, like in other retroviruses, but rather
formed a loop toward the genome 5 -end (Höglund et al.,
1997; Andersen et al., 2004; Pallesen, 2011). Besides the DIS,
the most extensively described gRNA motif potentially having
a role in the dimerization process is the TAR element at the
5 extremity of the genome (Figure 6A). Although HIV-1 TAR
contains a 10-nts palindromic sequence in its apical loop that
mediates dimerization of a 57-nts long TAR fragment (Andersen
et al., 2004), it seems this link with gRNA dimerization is
rather indirect. Indeed, even though electrophoretic mobility
analyses of mutant TAR RNA dimers extracted from virions
presented dimerization defects (Song et al., 2008; Jalalirad et al.,
2012), compensatory mutations failed to restore the putative
intermolecular TAR-TAR interaction (Jalalirad et al., 2012).
Additionally, other in cellula and in vitro studies indicated that
TAR has a moderate effect on RNA dimerization (Das et al.,
2007; Lu et al., 2011; Heng et al., 2012), suggesting that TAR
destabilization impacts the overall gRNA structure, thus inducing
aberrant gRNA dimerization and packaging (Ooms et al., 2004;
Vrolijk et al., 2008; Das et al., 2012). Therefore, the intermolecular
TAR-TAR interactions would promote gRNA structure stability
rather than RNA dimerization.
HIV-1 gRNA can alternatively serve as a template for the
synthesis of Gag precursors, and/or be selected for encapsidation
within virions as dimers. Consistent with this notion, it was
suggested that the regulation of HIV-1 gRNA translation or
dimerization and packaging would be due to RNA structural
switches. Using biochemical and chemical probing, Huthoff
and Berkhout (2001), Berkhout et al. (2002), Abbink and
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Dubois et al. RNA Dimerization in Retroviruses
FIGURE 8 | Secondary structure models of HIV-2 genomic RNA. (A) Schematic representation of the secondary structure model of the 5 - region of the HIV-2
gRNA. The red line delimits the packaging signal (Psi) region containing the five stem-loops SL1, 1 to 3 and SD. Similar to HIV-1, SL1 contains in its apical loop a
hexanucleotide palindromic sequence constituting the DIS. SL1 also contains in its basal part a 10-nts palindromic sequence partially entrapped (PAL). The gag
translation initiation codon is located within a G-rich region (G-box). Upstream of Psi, are found the TAR, the poly-A, a C-rich region important for RNA dimerization
(C-box) and the PBS. (B) In the CGI dimer structural conformation, the C-box and G-Box are base-paired. This conformation restricts HIV-2 RNA dimerization and is
adopted by loose dimers when the DIS and TAR hairpin III are involved in kissing-loop interactions (Lanchy et al., 2003a,b). (C) The RNA switch model from loose to
tight dimer, as proposed by Purzycka et al. (2011). HIV-2 SL1 stem B is melted through Gag/NC chaperone activity, freeing the PAL region that can thus forms
additional intermolecular base-pairings to stabilize the RNA dimer.
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Dubois et al. RNA Dimerization in Retroviruses
Berkhout (2003), Abbink et al. (2005) proposed an RNA-switch
mechanism from a long-distance interaction (LDI) to a branched
multiple hairpin (BMH) conformation that would regulate
gRNA dimerization and translation (Figures 7A,B). In the LDI
conformation, SL1 is disrupted and base-paired with the polyA
element, thus sequestrating the DIS sequence and potentially
promoting translation (Figure 7A). Alternatively, in the BMH
conformation, the gag AUG initiation codon together with the
U5 region forms the so-called U5:AUG interaction, displaying
SL1-SL3 to fold hairpin structures (Figure 7B) and promoting
gRNA dimerization. Importantly, if the U5:AUG interaction is
supported by phylogenic studies showing its conservation and
co-variation throughout HIV-1 isolates (Abbink and Berkhout,
2003; Russell et al., 2003; Damgaard et al., 2004; Song et al., 2008;
Wilkinson et al., 2008; Lu et al., 2011; Sakuragi et al., 2012;
Keane et al., 2015, 2016; Tran et al., 2015; Mueller et al.,
2016) and other related lentiviruses such as SIV (Whitney and
Wainberg, 2006; Tran et al., 2015) or HIV-2 (see below), the LDI
conformation has never been characterized in vivo, and RNA
mutants aiming to disrupt LDI failed to affect HIV-1 unspliced
RNA translation (Abbink et al., 2005), thus questioning the
biological relevance of the LDI conformation. Also, mutational
analysis of two highly conserved sequences, upstream of SL1
and downstream of SL4, suggested that these are part of
a pseudoknot-like conformation contributing to HIV-1 DLS
structure and promoting dimerization (Sakuragi et al., 2012).
An alternative in vitro RNA conformational switch model
derived from the previous one was proposed by the group of
Michael Summers by using a new NMR approach (long-range
probing by adenosine interaction detection or lr-AID) which
is based on selective isotopic labeling of each RNA molecule
in the dimer thus allowing the discrimination between intraand
inter-molecular base-pairs (Lu et al., 2011). In this model,
contrary to the LDI-BMH conformational switch, the DIS and gag
initiation codon are alternatively base-paired with the U5 region
(Figures 7B,C), promoting translation or RNA dimerization,
respectively. Even though the dimerization competent U5:AUG
RNA structure appears rather similar to the BMH conformation
(Lu et al., 2011) (Figure 7B), a striking difference corresponds
to the three-way junction RNA structure at the base of SL1, in
which SL2 base-pairs with the bottom region of the PBS from
the other gRNA molecule (Figure 7D) (Keane et al., 2015, 2016).
However, this latter conformation was very recently challenged
by a phylogenic study supporting the existence of SL2 hairpin
structure (Mueller et al., 2016).
Human Immunodeficiency Virus Type 2
gRNA Dimerization
The secondary structure and in vitro dimerization properties of
HIV-2 gRNA present similarities to HIV-1 gRNA (Figure 8A).
First, loose dimer formation involves several contacts including
the SL1 region of Psi. HIV-2 SL1 also contains a 6-nts palindromic
sequence within the apical loop driving the dimerization of
5 RNA fragments (Dirac et al., 2001). Second, the HIV-2
TAR hairpin III (Figures 8A,B) palindromic sequence was also
shown to contribute to the stabilization of the kissing-complex
by forming homologous intermolecular base-pairs (Figure 8B)
(Purzycka et al., 2011). Interestingly, in the context of RNA
fragments encompassing the whole 5 -UTR (up to position
∼560), another palindromic sequence in the PBS was also
described to be required for RNA dimerization (Jossinet et al.,
2001; Lanchy and Lodmell, 2002; Lanchy et al., 2003b).
However, in vitro tight dimers of HIV-2 RNA fragments
seem to be quite different from HIV-1. Indeed, RNA fragments
encompassing the whole 5 -UTR, in the absence of HIV-2 NC
protein and at physiological temperature, were shown to form
only loose dimers through the PBS palindrome (Jossinet et al.,
2001; Lanchy and Lodmell, 2002; Lanchy et al., 2003b), while
shorter transcripts (up to position ∼440, i.e., ending immediately
3 of SL1) formed tight dimers through the SL1 apical loop (Dirac
et al., 2001; Lanchy et al., 2003a). A possible explanation relies
on the fact that HIV-2 RNA secondary structure forms a LRI
between a C-rich region upstream of the PBS and a G-rich region
overlapping the gag translation initiation codon termed C boxG
box interaction (CGI), homologous to the HIV-1 U5:AUG
interaction (compare Figures 7B, 8B). Interestingly, while this
latter was proposed to promote HIV-1 RNA dimerization
through SL1 (Dirac et al., 2001; Lanchy et al., 2003a), the CGI
seems to prevent formation of tight HIV-2 RNA dimers since
targeting the CGI with oligonucleotides restores SL1-dependent
dimerization in vitro (Lanchy et al., 2003a,b). Moreover,
incubation of RNA fragments either at high temperature (>50◦C)
or in presence of HIV-2 NC has been shown to destabilize
the CGI and restore SL1-dependent tight dimerization in vitro
(Lanchy and Lodmell, 2002; Lanchy et al., 2003b). Interestingly,
in addition to the apical loop of SL1, an upstream 10 nt-long
conserved palindromic sequence (termed PAL) also contributes
to in vitro RNA tight dimer formation (Figure 8C). Indeed, by
using antisense oligonucleotides targeting the SL1 apical DIS or
PAL, this latter was shown to serve as an alternative stabilization
element when the apical DIS is repressed (Lanchy et al., 2003a).
Importantly, in vitro SHAPE and biochemical analyses revealed
that stem B of SL1 (Figure 8C), formed by base-pairing between
the 3 -end of PAL and a region downstream of SL1 stem A
(Figure 8C), regulates HIV-2 RNA dimerization (Baig et al., 2007;
Purzycka et al., 2011). Indeed, in the proposed model, upon
opening of SL1 stem B through the chaperone activity of Gag/NC,
the PAL region becomes available to build interstrand contacts.
Interestingly, a comparative study revealed that the dimerization
properties of HIV-1, HIV-2 and SIV RNAs are regulated through
the stability of SL1 stem B (Baig et al., 2008).
Consistently with these in vitro results, mutations in the
PAL region strongly impaired HIV-2 RNA dimerization and
infectivity in cellula, while mutations in the SL1 apical loop
had limited impact (L’Hernault et al., 2007, 2012). Similarly,
the integrity of SL1 stem B was shown to be crucial for HIV-
2 gRNA packaging (Lanchy and Lodmell, 2007). Finally, it was
also proposed that, in cellula, translation of HIV-2 gRNA by the
ribosome may disrupt the CGI to allow RNA dimerization, a
notion consistent with a study proposing that HIV-2 genome
is packaged co-translationally (Griffin et al., 2001). However,
this cis-packaging model was recently challenged and it was
elegantly shown by visualization HIV-2 RNA in individual
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Dubois et al. RNA Dimerization in Retroviruses
particles that trans-packaging is the major mechanism of HIV-2
RNA packaging, as it was observed for HIV-1 (Ni et al., 2011).
SPUMAVIRUSES GENOME
DIMERIZATION
Spumaretroviruses, or foamy viruses (FVs), have the largest
genome in the Retroviridae family (∼12 kb) and are singularly
different from other retroviruses. Amongst these differences,
FV Gag proteins do not present the NC Cys-His zinc fingers,
a hallmark of other retroviral Gag proteins, but have instead
Gly-Arg rich domains binding nucleic acids with equal affinity
and required for viral replication (Yu et al., 1996; Linial,
1999). Furthermore, reverse transcription is a late event in FV
replication cycle and infectious viral particles contain DNA
genomes (Linial, 1999; Yu et al., 1999). Despite these differences,
three different sites (SI, SII, and SIII) were shown to contribute
to in vitro dimerization of RNA fragments corresponding to the
5 region of FV gRNA (Erlwein et al., 1997). Amongst these
domains, SII maps to positions 391–410 and contains a highly
conserved 10-nts UCCUAGGA palindrome that is crucial for
in vitro RNA dimerization since antisense oligonucleotides or
mutations targeting this motif abolish RNA dimer formation
(Erlwein et al., 1997; Cain et al., 2001). SI and SIII also
contribute to RNA dimerization, but to a lesser extent since
antisense oligonucleotide targeting of these elements alone failed
to completely abrogate in vitro RNA dimerization of primate
FV 5 -RNA fragments (Erlwein et al., 1997; Cain et al., 2001).
However, targeting bovine FV (BFV) SI element by antisense
oligonucleotides inhibited in vitro RNA dimerization similarly to
what was observed when targeting the SII palindrome (Yu et al.,
2007).
Importantly, mutation of the SII palindrome in cell culture
resulted in a strong viral replication defect, even though genome
packaging was not affected (Park and Mergia, 2000; Cain et al.,
2001), differently from other retroviruses such as HIV-1 which
showed defects in gRNA packaging when mutation of the DIS
occurred (Berkhout and van Wamel, 1996; Paillart et al., 1996a;
Laughrea et al., 1997, 1999; Houzet et al., 2007). Since it is well
known that RNA dimerization plays an important role in reverse
transcription (Paillart et al., 1996a; Berkhout et al., 1998; Parent
et al., 2000), it seems likely that this replication defect may come
from reverse transcription defects (Park and Mergia, 2000).
IN CELLULA GENOME DIMERIZATION:
WHERE AND WHEN?
Retroviruses select specifically two copies of their genome from
the pool of cellular and viral spliced RNAs for packaging.
Since aberrant gRNA dimerization may interfere with different
steps in the retroviral life cycle, including viral assembly, the
timing and localization of this process must be finely regulated
within the host cell. An important question has been whether
two monomers are packaged and dimerize during/after viral
assembly, or if a pre-formed RNA dimer is selected and packaged
into viral particles. However, since gRNA dimerization and
packaging are interconnected events (Aagaard et al., 2004; Houzet
et al., 2007; Moore et al., 2007; Miyazaki et al., 2010a; Mailler
et al., 2016), gRNA dimerization was supposed to initiate upon
gRNA recognition by the Gag precursor. In the following section,
we will review the current understanding of the spatio-temporal
regulation of retroviral RNA dimerization.
Various studies using protease-deficient (PR−) virions or
different Gag mutants suggested that HIV-1 gRNA is packaged
as two monomers (Shehu-Xhilaga et al., 2001; Song et al., 2007).
It is, however, possible that gRNA extraction protocols may
disrupt immature loose dimers, especially when the stability
of RNA mutants is reduced (Höglund et al., 1997). Although
it has been reported that, in some cell types, HIV-1 gRNA
DIS mutants presenting packaging defects can still be dimeric
(Laughrea et al., 1997; Hill et al., 2003), a majority of studies
showed that disruption of SL1 kissing-loop interface impaired
or affected gRNA packaging (Berkhout and van Wamel, 1996;
Paillart et al., 1996a; Houzet et al., 2007; Moore et al., 2007).
Moreover, duplication of HIV-1 DLS leads to production of
partially monomeric genomes, due to gRNA circularization by
self-dimerization (Sakuragi et al., 2001, 2007), contradicting the
notion that HIV-1 packages its genome as RNA monomers.
The comparison of HIV-1 gRNA structure in cellula and in viro
revealed little structural rearrangements between these two
conditions (Paillart et al., 2004), and recombination analyses
supported the notion that gRNA dimerization initiates in the
cytoplasm (Moore et al., 2009).
An extensive set of analyses support the idea that gRNA
dimerization occurs prior budding of viral particle (Moore
et al., 2007; Jouvenet et al., 2009; Moore and Hu, 2009; Sardo
et al., 2015; Ferrer et al., 2016). Because retroviral assembly
involves only two copies of gRNA, detecting RNA dimerization
in cells is challenging. Several bio-imaging approaches based
on fluorescence have been used to address this issue. To
allow specific gRNA observation in cellula, the viral genome is
labeled by incorporating numerous stem-loops that specifically
bind the coat protein of MS2 bacteriophage (Bertrand et al.,
1998; Fusco et al., 2003), or the phage lambda protein λN22
(Daigle and Ellenberg, 2007) fused to fluorescent protein(s).
Although it was suggested that HIV-1 gRNA migrates at the
plasma membrane as a pre-formed dimer (Jouvenet et al., 2009;
Chen et al., 2016), further ex vivo analyses showed that HIV-
1 gRNA dimer reaches the plasma membrane in association
with low-order multimers of Gag precursor (Jouvenet et al.,
2008, 2009; Kutluay and Bieniasz, 2010; Kutluay et al., 2014).
Importantly, Ferrer et al. (2016) recently combined Fluorescence
In Situ Hybridization (FISH), TIRF-M, 3D-super-resolution
microscopy and Fluorescence Cross-Correlation Spectroscopy
(FCCS) to show that HIV-1 gRNA dimerization already occurs
in the cytosol but that RNA dimers are more easily detected
at the plasma membrane. One possible explanation could be
the concentration of Gag at the plasma membrane, which
might promote gRNA dimer stabilization through the chaperone
activity of its NC domain (Ferrer et al., 2016). In this context,
Hu and co-workers used a similar approach and proposed
that HIV-1 gRNA dimerization would occur preferentially at
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Dubois et al. RNA Dimerization in Retroviruses
the plasma membrane (Chen et al., 2016). Their comparison
between TIRF-M and in silico simulations showed that RNA
heterodimerization would occur at frequencies similar to those
observed in Ferrer et al. (2016) study (∼10–15%). Since the colocalization
frequencies increased at the plasma membrane in a
Gag-dependent manner, they thus proposed that HIV-1 genome
diffuses into the cytoplasm and reaches the plasma membrane as a
monomer. However, a common point for both studies resides in
the differences between heterodimers frequencies at the plasma
membrane (∼10–15% co-localization) and in virions (∼40–50%)
(Chen et al., 2009; Ferrer et al., 2016), suggestive of an RNA dimer
stabilization step at the plasma membrane prior to encapsidation.
It is, however, possible that technical difficulties for the detection
of RNA molecules in a highly dynamic environment, such as
the cytosol and the plasma membrane, compared to the stable
environment within viral particles, could affect these estimations.
Further analyses are definitively needed to clarify spatiotemporal
HIV-1 gRNA dimerization.
Finally, studies suggest that MuLV gRNA dimerization, unlike
HIV-1, occurs in the nucleus. Indeed, heterodimerization is
enhanced when two proviruses are spatially close, suggesting
that MuLV gRNA dimerization is coupled to transcription and
splicing processes (Maurel and Mougel, 2010). Consistent with
these observations, MuLV RNAs transcribed from the same locus
form dimers at high frequencies (Flynn et al., 2004; Kharytonchyk
et al., 2005; Rasmussen and Pedersen, 2006; Maurel et al.,
2007). Interestingly, mutations of RNA elements involved in
dimerization or packaging processes impact the intracellular
transport of viral genome and result in aberrant accumulation in
the nucleus or in the cytoplasm (Basyuk et al., 2005; Smagulova
et al., 2005).
RNA DIMERIZATION AND REVERSE
TRANSCRIPTION/RECOMBINATION
Reverse transcription is initiated at the PBS immediately 3 to
the 5 copy of the R (repeat) region of the genome by the
viral RT enzyme and generates the complete viral cDNA with
duplicated LTR. In order to achieve cDNA synthesis, the negative
strong-stop DNA must be translocated from the 5 to the 3 end
of the gRNA during the first strand transfer occurring in an
intra- or inter-molecular manner (Panganiban and Fiore, 1988;
Hu and Temin, 1990; Berkhout and van Wamel, 1996). During
reverse transcription, recombination occurs as the RT enzyme
switches between the two RNA templates. This process is due
to RNA sequence homology and promoted by particular RNA
structures, nicks in the viral genome or RT pausing (Galetto
and Negroni, 2005). Importantly, recombination provides a
mechanism for genome repair or, in the case of a heterodiploid
genome, a way to increase genetic diversity by genomes shuffling.
This leads to immune escape and drug resistance, especially
in the case of HIV-1 (Morris et al., 1999; Rambaut et al.,
2004), for which it has previously been shown that gRNA
heterodimerization rate can reach ∼40–50% in viro (Chen
et al., 2009; Ferrer et al., 2016), and that recombination occurs
at least three times during a single viral replication cycle
(Zhuang et al., 2002; Schlub et al., 2010, 2014; Smyth et al.,
2014).
Genomic RNA dimerization may play an important role
during reverse transcription since RSV mutant particles
containing monomeric genomes display a 100-fold decrease
in cDNA synthesis (Parent et al., 2000) and heat-induced
dissociation of HIV-1 gRNA dimers inhibits the first strand
transfer (Berkhout et al., 1998). In MuLV, template switching
occurs preferentially at direct RNA–RNA interactions mediated
by palindromic sequences (Mikkelsen et al., 1998a,b, 2000). In
the case of a co-infection by two genetically different viruses, the
subsequent packaging of a heterozygous dimer increases the viral
diversity by enabling the production of new recombinant strains
(Mikkelsen et al., 2000). In HIV-1, trans-complementary DIS
mutants showed that the recombination rate is enhanced when
two palindromic mutants can form Watson–Crick base-pairs
(from 50 to 90% depending on the mutants) (Moore et al., 2009).
Similarly, both the in vitro and in cellula HIV-1 recombination
rates are directly linked to the dimeric state of the viral genome
(Balakrishnan et al., 2001, 2003; Sakuragi et al., 2015, 2016).
Taken together, these findings highlight the central role of gRNA
dimerization in the reverse transcription and recombination
processes.
RNA DIMERIZATION AS A POTENTIAL
ANTIRETROVIRAL TARGET
While the current antiretroviral treatments have proved
successful in extending the life expectancy of infected patients,
the increasing number of multi-drug resistant HIV-1 mutants
highlights the need for the discovery of new antiretroviral
drugs. In this context, gRNA dimerization is an attractive target
since it is crucial for several key steps in the retrovirus life
cycle. Interestingly, the crystal structure of the HIV-1 kissingloop
complex revealed strong structural similarities with the
16S ribosomal A site, a natural aminoglycoside binding site
(Ennifar et al., 2003). Indeed, some aminoglycosides, such as
neomycin and lividomycin, bind the HIV-1 kissing-loop complex
in vitro with high affinity and strongly stabilize it, preventing
its conversion to an extended (Ennifar et al., 2003, 2006, 2007;
Bernacchi et al., 2007). Interestingly, footprint analysis also
revealed that the DIS of HIV-1 gRNA is also protected by
aminoglycosides in cells and in virions (Ennifar et al., 2006,
2007), thus providing an interesting basis for the development of
new antiretroviral strategies aiming at targeting retroviral gRNA
dimerization (Ennifar et al., 2007, 2013; Blond et al., 2014).
CONCLUDING REMARKS
Retroviral gRNA dimerization is a highly conserved mechanism
in retroviruses, and plays a critical role in several key steps of
their replicative cycle. RNA dimerization, which is a pre-requisite
for packaging, plays a critical role in the generation of multidrug
resistant recombinant viruses during reverse transcription.
In this context, a general feature of retroviruses is the existence
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Dubois et al. RNA Dimerization in Retroviruses
of complex structural switches regulating the transition
between a loose kissing-loop complex to a stable extended
duplex conformation, and the equilibrium between gRNA
translation and dimerization for packaging. Nevertheless, in vitro
structural models most likely do not exactly reflect the in vivo
situation and, despite recent progresses, much remains to
be unraveled to reach complete understanding of the RNA
dimerization process. This understanding could also allow the
development of new antiretroviral strategies that are essential to
counterbalance the increasing prevalence of multi-drug resistant
viruses.
AUTHOR CONTRIBUTIONS
ND, RM, J-CP, and SB conceived the review topic. ND drafted
the manuscript and generated the figures. SB, J-CP, and RM
corrected and edited the manuscript. All authors read and
approved the final version of the manuscript.
FUNDING
This work was supported by the CNRS and grants from
SIDACTION (RM) and the Agence Nationale de Recherches sur
le SIDA et les Hépatites Virales “ANRS” (SB and J-CP) and a
doctoral fellowship of the French Ministry of Higher Education
and Research to ND.
ACKNOWLEDGMENTS
We are grateful to D. Ferhadian for his help with drawing the
HIV-1 SL1 duplex structures with PyMOL.
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Frontiers in Microbiology | www.frontiersin.org 19 March 2018 | Volume 9 | Article 527