Regulated HIV-2 RNA dimerization by means of alternative RNA conformations

Abstract

The dimer initiation site (DIS) hairpin of the HIV-2 untranslated leader RNA mediates in vitro dimerization through 'loop-loop kissing' of a loop-exposed palindrome sequence. Premature RNA dimerization must be prevented during the retroviral life cycle. A regulatory mechanism has been proposed for the HIV-1 leader RNA that can adopt an alternative conformation in which the DIS motif is effectively masked by long-distance base pairing with upstream leader sequences. We now report that HIV-2 RNA dimerization is also regulated. Sequestering of the DIS motif by base pairing interactions with downstream leader sequences mediates a switch to a dimerization-impaired conformation. The existence of two alternative conformations of the HIV-2 leader RNA is supported by UV melting experiments. Furthermore, the equilibrium between the two conformations can be shifted by annealing of antisense oligonucleotides or by deletion of certain leader regions. These measures have a profound impact on the dimerization properties of the transcript, demonstrating a mutual exclusivity between the alternative conformation and dimerization, similar to what has been described for the HIV-1 leader. The overall resemblance in regulation of HIV-1 and HIV-2 RNA dimerization suggests that a similar mechanism may be operating in other lentiviruses and perhaps all retroviridae.

Figure 1

HIV-2 leader RNA secondary structure model. The model of the 5′ untranslated leader RNA of the HIV-2 isolate ROD is adapted from Berkhout (31). The hairpin structures are named according to their putative function in HIV-1 replication, and several critical sequence elements are highlighted in gray (the polyadenylation signal, the PBS and the DIS palindrome). The Gag start codon at position 545 is boxed. Positions used in this study as 5′/3′ transcript ends are marked.

Figure 2

Characterization of dimerization requirements for transcripts 397/444 and 1/444. (A) Temperature dependency was assayed by incubation of the RNA samples for 10 min at 23, 37, 50 and 65°C in buffer with 1 mM (left) or 5 mM (right) MgCl₂. The RNA was slowly cooled to room temperature and analyzed on a non-denaturing TBE gel. The dimerization efficiency was calculated by phosphor imager quantification and is illustrated in bar graphs. (B) Mg²⁺ requirement was measured in a 10 min incubation in buffer with 0, 0.1, 1, 2.5 and 5 mM MgCl₂, and samples were analyzed as described for (A). (C) Dimer melting curves. RNA dimers were formed at optimal dimerization conditions (10 min at 65°C in 5 mM MgCl₂ and slow cooling). After dimer formation, the sample was diluted 10-fold in water to avoid reassociation of melted RNA, and incubated for 15 min at 23, 37, 53, 65, 70, 75 and 82°C. The samples were analyzed on a TBE gel, phosphor imager analysis was performed and the percentage of remaining dimer was calculated. Black and gray coloring represents transcript 397/444 and 1/444, respectively.

Figure 3

Mapping of sequences 3′ of the HIV-2 DIS that inhibit dimerization. (A) Transcripts 1/444 and 1/544 were analyzed for their ability to dimerize at varying RNA concentrations. We incubated 10 ng (lanes 1 and 6), 50 ng (lanes 2 and 7), 250 ng (lanes 3 and 8), 500 ng (lanes 4 and 9) and 1000 ng (lanes 5 and 10) transcript at optimal dimerization conditions (65°C, 5 mM MgCl₂), followed by gel electrophoresis on a non-denaturing 4% TBE gel and autoradiography. The monomer (M) and dimer (D) bands of both transcripts are marked. (B) Approximately 2 pmol of transcripts 1/444, 1/464, 1/484, 1/507, 1/527 and 1/544 was incubated at dimerization conditions as described in (A). Monomer and dimer bands are marked M and D. The dimerization efficiencies for the transcripts measured by phophorimager quantification are 43.6, 41.8, 44.8, 40.2, 25.0 and 17.9%, respectively. (C) Approximately 2 pmol of radiolabeled transcript 1/544 was incubated at optimal dimerization conditions (65°C, 5 mM MgCl₂) in buffer containing 0, 1, 2.5, 5, 7.5 and 10 mM MgCl₂.

Figure 4

Both 5′and 3′ leader sequences are required for inhibition of dimerization. Approximately 3 pmol of transcripts starting at position 1, 126, 185 and 397 and ending at position 444 or 544 were incubated at optimal dimerization conditions as described in the legend to Figure 2. Samples were loaded on a non-denaturing TBE gel (A) and a non-denaturing TBM gel (B). After gel drying, phosphor imager analysis was performed to calculate the dimerization efficiencies that are plotted in bar graphs. Black and white bars represent dimerization of transcripts terminating at position 444 and 544.

Figure 5

Antisense oligonucleotide scanning of the HIV-2 leader RNA. (A) Approximately 2 pmol of radiolabeled transcript 1/544 was incubated with a 50-fold molar excess of antisense DNA oligonucleotide at optimal dimerization conditions (see Fig. 2 legend) and subjected to non-denaturing gel electrophoresis. The annealing position of the antisense oligonucleotides and the domains of the HIV-2 transcript are indicated on top of the autoradiogram. (B) Excerpt of an antisense oligonucleotide scan of transcript 1/444 shown in Dirac et al. (19), which was performed as described in panel (A). The positions of dimer (D), monomer (M) and fast-migrating monomer (LDI) conformations are indicated.

Figure 6

The effect of NC protein on the HIV-2 leader RNA conformation. Approximately 2 pmol of radiolabeled transcript 1/544 was incubated either alone (lanes 1–2) or with a 50-fold molar excess of the antisense DNA oligonucleotide 418/399 (lanes 3–4) at optimal dimerization conditions (see Fig. 2 legend). After slow cooling to room temperature, 1.2 µg synthetic HIV-1 NCp7 protein was added (lanes 2 and 4) and all samples were incubated for 2 h at 37°C, followed by treatment with SDS and a phenol extraction. The positions of dimer (D), monomer (M) and fast-migrating monomer (LDI) conformations are indicated. Lanes 3 and 4 are shown at two intensities.

Figure 7

Thermal melting profiles of HIV-2 leader RNAs. Melting was monitored by UV absorption at 260 nm. The curves show the first order derivative (δA/δT) to highlight the melting transitions. We analyzed transcripts 1/124 (A), 126/544 (B), 1/444 (C) and 1/544 (D). The melting temperature T_m is indicated at the top of the peaks.

Figure 8

An RNA switch regulates dimerization in HIV-1 and HIV-2 RNA. The different structural elements within the leaders are color coded: TAR bulges and loops (green), polyadenylation signals (orange), PBS (blue) and DIS palindrome (pink). The linear presentation is rearranged to show the equivalent BMH structure. A structural BMH-to-LDI rearrangement is proposed that results in disappearance of the DIS hairpin. In HIV-2, the DIS region is proposed to base pair with 3′ sequences, and the stem of the PBS domain is extended by base pairing of sequences that are located far upstream and downstream of the DIS (sequence elements are marked in purple). Details of the DIS hairpin in the BMH conformation are shown in Figure 1. The HIV-2 LDI model is based on several experimental findings, but we were unable to unequivocally assign the new base pairing scheme (see Discussion). Many more details are available for the HIV-1 RNA, in which both the DIS and the upstream poly(A) hairpin are opened to facilitate the extension of the PBS stem to form the LDI. In both viral systems, the RNA switch is proposed to provide a mechanism for the regulated dimerization of the viral genomes.