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. 2013 Aug 16;288(33):24140-50.
doi: 10.1074/jbc.M113.477265. Epub 2013 Jul 9.

Mechanism of HIV-1 RNA dimerization in the central region of the genome and significance for viral evolution

Dorota Piekna-Przybylska  1 , Gaurav Sharma, Robert A Bambara
Affiliations

Mechanism of HIV-1 RNA dimerization in the central region of the genome and significance for viral evolution

Dorota Piekna-Przybylska et al. J Biol Chem. .

Abstract

The genome of HIV-1 consists of two identical or nearly identical RNA molecules. The RNA genomes are held in the same, parallel orientation by interactions at the dimer initiation site (DIS). Previous studies showed that in addition to interactions at DIS, sequences located 100 nucleotides downstream from the 5' splice site can dimerize in vitro through an intermolecular G-quartet structure. Here we report that the highly conserved G-rich sequence in the middle portion of the HIV-1 genome near the central polypurine tract (cPPT) dimerizes spontaneously under high ionic strength in the absence of protein. The antisense RNA does not dimerize, strongly indicating that RNA dimerization does not exclusively involve A:U and G:C base pairing. The cation-dependent reverse transcriptase pausing profile, CD spectra profile, and cation-dependent association and thermal dissociation characteristics indicate G-quartet structures. Different forms of G-quartets are formed including monomers and, significantly, intermolecular dimers. Our results indicate that RNA genome dimerization and parallel alignment initiated through interactions at DIS may be greatly expanded and stabilized by formation of an intermolecular G-quartet at a distant site near the cPPT. It is likely that formation of G-quartet structure near the cPPT in vivo keeps the RNA genomes in proximity over a long range, promoting genetic recombination in numerous hot spots.

Keywords: Genome Structure; HIV-1; Homologous Recombination; RNA Structure; Virus.

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Figures

FIGURE 1.
FIGURE 1.
The guanine-rich sequence of the HIV-1 cPPT region may fold into a G-quartet. According to QGRS Mapper, a run of G residues in the cPPT (shaded nucleotides) and two other downstream G-rich elements are capable of forming a G-quartet. The structure can be polymorphic and can fold into several different conformations either as a single-molecule quadruplex (monomer) or intermolecular quadruplex (dimer) with parallel (b and d) and antiparallel configuration (a, c, and e). Examples of three different dimers are shown. Two-molecule G-quartets are composed of two sequences that interact in different orientations with only intermolecular hydrogen bonding (c) or with a combination of intra- and intermolecular hydrogen bonding (d and e) (46, 47).
FIGURE 2.
FIGURE 2.
Reverse transcription assay determining G-quartet formation in the cPPT region. Cation-dependent pausing of reverse transcription at the guanine-rich elements in the cPPT region was analyzed with wild-type (WT) and mutant (MT) RNA templates. A fragment of the RNA template is shown (top) with G-rich elements (G-1, G-2, G-3) and mutations (nucleotides on gray background) introduced into the second and third G-rich elements. A, strong pauses of the RT at G-rich elements were observed in the presence of 50 mm KCl, but not LiCl, indicating that three runs of G residues are involved in the formation of different types of G-quartet structure. B, shown is a pausing profile of wild-type and mutant RNA templates folded in the presence of 200 mm KCl, NaCl, or LiCl and then diluted to 50 mm salt before initiation of reverse transcription. The profile reveals common G-quartet folding features, consistent with both the expected order of cation preference to form a stable structure and an influence of salt concentration on G-quartet formation. A cation-dependent pausing of RT at G-3 was not observed with mutant RNA templates. F, full-length extension products; P, DNA primer; M, DNA marker; K, KCl; Na, NaCl; L, LiCl.
FIGURE 3.
FIGURE 3.
CD spectral analysis of the G-rich RNA sequence near the cPPT in HIV-1. A, shown are CD spectra of the G-quadruplex-forming sequence in different salts at concentrations of 10 and 50 mm. The effects of K+, Na+, and Li+ on the ellipticity signal are compared with the signal for RNA incubated in the absence of salt. Salt concentrations are 50 mm KCl (red), 10 mm KCl (orange), 50 mm NaCl (dark blue), 10 mm NaCl (light blue), 50 mm LiCl (dark green), and 10 mm LiCl (light green) and no salt (gray). B, shown are normalized CD melting temperature curves for G-quadruplexes formed in 50 mm KCl (red), 50 mm NaCl (blue), 50 mm LiCl (green), and 10 mm KCl (orange).
FIGURE 4.
FIGURE 4.
Affinity selection of RNAs interacting through intermolecular G-quartet structure. Poly(A)-tagged RNA templates were elongated at the 3′ end with an A25 polymer to select them with oligo(dT)25 magnetic beads. To determine whether RNA molecules can interact, the poly(A)-tagged RNA was mixed with corresponding non-tagged RNA and incubated under dimerization conditions for 2 h. The oligo(dT)25 magnetic beads were added to select the poly(A)-tagged RNAs, and after three rounds of washes, the samples were eluted. The selected poly(A) RNAs and interacting partners were analyzed in a denaturing gel stained with ethidium bromide. A, a control experiment shows that the non-tagged RNA with DIS (RNA genomic sequence 1–520) can be selected with oligo(dT)25 magnetic beads only in the presence of poly(A)-tagged RNA with DIS (183–520). No RNA was selected in the absence of a poly(A)-tagged partner (line C). B, the RNAs of the gag region (RNA genomic sequence 303–415 of the MAL isolate and 290–403 of the NL4-3) and cPPT region (4309–4396 of the NL4-3) could be isolated with the corresponding poly(A)-tagged RNA partners after affinity selection with oligo(dT)25 magnetic beads. AS, affinity selection; T, poly(A)-tagged RNA; P, non-tagged RNA partner; C, control.
FIGURE 5.
FIGURE 5.
Dimerization of the HIV-1 cPPT region. The RNA templates of sense (WT) and antisense (As) strands were incubated under dimerization conditions for 2 h in the presence of 1 m KCl or LiCl. Formation of monomers (M), dimers (D), and tetramers (T) was analyzed on a non-denaturing gel run at 4 °C in the presence of 10 mm KCl and 0.5 × Tris borate-EDTA. Li, lithium; K, potassium; U, unfolded RNA.
FIGURE 6.
FIGURE 6.
Cation-dependent association and thermal stability of the RNA dimer formed in the cPPT region. The RNA dimers were formed in parallel at a concentration of 4 μm in buffers containing 1 m KCl, NaCl, or LiCl. 1 volume of 1 × Tris-EDTA buffer was added, and 15-μl aliquots were incubated at the indicated temperatures for 8 min. Thermal stabilities were measured by analyzing samples in non-denaturing gels run at 4 °C. M, monomer; D, dimers.
FIGURE 7.
FIGURE 7.
Antisense oligonucleotide binding assay. Dimerization of the cPPT region was performed in the presence of a 5 m excess of different antisense DNA oligomers (a--g) binding to specific cPPT region sequences.
FIGURE 8.
FIGURE 8.
G-quartet formation in the cPPT region facilitates RT template switching during reverse transcription. A, G-quartet formation promoted minus-strand DNA transfer (gray line) through pausing of the RT (blue oval), which increased RNA cleavages (blue triangle) and allowed interaction with the second RNA template. The transfers were also promoted through an RNA template proximity effect resulting from template dimerization through a G-quartet dimer. B, shown is a reconstituted system to analyze the influence of G-rich elements on strand transfer during HIV-1 minus-strand DNA synthesis in vitro. Donor and acceptor RNA templates represent two copies of the viral RNA genome in which reverse transcription is initiated from a 32P-labeled DNA primer (P) annealed to donor RNA. The transfer reactions onto acceptor RNA will result in synthesis of TP distinguished in a denaturing gel from the DE by its greater length. The acceptor RNA does not share a homology (green dot) with two nucleotides at the 5′ end of the donor RNA. These alterations prevent end transfers of donor extension products; thus, transfer products should only originate internally. C, shown is a time course of strand transfer reactions performed in the presence of potassium and lithium ions. Samples were collected at 1, 5, 15, and 30 min after the reaction was initiated. Formation of a G-quartet in the RNA templates paused the RT during minus-strand DNA synthesis and influenced the yield of the final products. The transfer efficiency was 2.6-fold higher in reactions in which the templates could form a G-quartet than for reactions with substrates that could not form the structure.
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