An RNA structural switch regulates diploid genome packaging by Moloney murine leukemia virus

Abstract

Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5' untranslated region that contains all residues necessary for efficient RNA packaging (Psi(WT); residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Psi(WT)](2)) binding 12+/-2 NC molecules with high affinity (K(d)=17+/-7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Psi(M)), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (approximately 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.

Figure 1

Organization and predicted secondary structures of the MoMuLV genome. (a) Location of the packaging signal (Ψ) and the puromycin resistance expression cassette (P_SV40, puroR) that was inserted in the GPP derivatives used for in vivo RNA packaging experiments. (b) Secondary structure of the monomeric Ψ-site determined by traditional nucleotide accessibility mapping and mutagenesis experiments,,. Nucleotides shown in green, pink, red, and blue correspond to dimer-promoting palindromic elements, potential high affinity NC binding sites,, known NC binding sites identified from studies of RNA fragments, and the start codon for a glyco-gag gene product– of unknown function, respectively. (c) Secondary structure of portions of the dimeric Ψ-site predicted on the basis of mutagenesis experiments,, and NMR studies of RNA fragments. The base-paired UCUG residues of DIS-2 (pink) that were base-paired in RNA fragments could be exposed in the native UTR.

Figure 2

Constructs and NC binding results obtained for in vitro transcribed Ψ-RNAs. (a) Segment of the Ψ-site used for in vitro dimerization and NC binding studies. Mutations that stabilize the monomeric conformation (Ψ^M) are colored red. (b) Native agarose gel electrophoresis data indicating that Ψ^WT and Ψ^M form dimers and monomers, respectively. (c) Denaturing PAGE data showing relative amounts of NC retained with Ψ^WT (dimer) and Ψ^M (monomer) after successive washes with buffers containing different NaCl concentrations (shown). (d) The ratio of NC retained with Ψ^WT versus Ψ^M increases with increasing ionic strength of the wash buffer. (e) Representative ITC data obtained for NC titrations with dimeric [Ψ^WT]₂ (black) and momomeric Ψ^M (red). Top panels: Raw data obtained using 10 µl/injection titrations of NC (100 µM). Bottom panels: Binding isotherms obtained after peak integration, normalization to molar concentration and subtraction of dilution enthalpies.

Figure 3

Packaging of Ψ mutant and wild type gRNAs. (a–c) Packaging of Ψ mutant gRNAs in virus produced by transient transfection. (a) Intracellular expression of Ψ mutant RNAs. MoMuLV wild type and Ψ mutant gRNA levels were normalized to 7SL RNA by RNase protection assay (RPA) of total RNA extracted from transiently transfected cells. Lane 1: mock-transfected cell sample; lane 2: cells transfected with pGPP Ψ^WT; lane 3: cells transfected with pGPP derivative with Ψ^M ; lane 4: with Ψ^ΔCES; lane 5: with Ψ⁻; lane 6: with Ψ^Δ193; lane 7: cells transfected with pNGVL-3’, which is a helper construct that expresses MLV Gag and Pol but contains a large 5’ UTR deletion; and lane C: no RNA control. Migrations of probe fragments protected by MoMuLV wild type and mutant gRNAs, and by 7SL RNA, are indicated on the right. (b) Packaging of Ψ mutant RNAs. RPA of virion RNA extracted from media samples normalized by RT activity. The helper plasmid, pNGVL-3’-gag-pol, lacks native 5’ UTR sequences, and thus no MoMuLV-derived fragment was detected in lane 6. (c) Quantification of gRNA RNA packaging in virions produced by transient transfection. Reported as percent of wild type levels, using 7SL to normalize number of virions. Data are from three separate RPAs of viral RNA as shown in (b). (d) Packaging of Ψ mutant gRNAs under competition with co-expressed wild type gRNAs. RPA of cell and viral RNA from 293T cells transiently co-transfected with plasmids expressing test gRNAs (Ψ variant GPPs) and a wild type 5’ UTR control gRNA (pBAG 71). % WT packaging was calculated by dividing the viral Test:Control gRNA ratio by the cellular Test:Control gRNA ratio, and setting the wild type value to 100%. (e–g) Packaging of Ψ mutant gRNAs under single copy expression conditions (e) RPA of cellular RNA extracted from cells expressing MoMuLV Ψ variants as single stably integrated proviruses. (f) RPA of gRNA in virions produced by single integrants. Migration of probe fragments protected by MoMuLV gRNA and 7SL are indicated on the right. (g) Quantification of gRNA/7SL ratios from three separate RPAs of viral RNA. For all RPAs, lanes include undigested probe (P); RNA size markers (M); and digested probe alone control (C). Note that the doublet 7SL RNA bands as well as the MLV gRNA doublets observed in panel b reflect incomplete RNase T1 digestion that was reproducibly observed with the 7SL probe and the MLV RNA probe used in panel b but not with the pol-complementary probe used in panel a. That the altered mobilities of 7SL products in lane 6 of panel b and lane 4 of panel f were not reproducibly observed, and likely reflect minor variation in salts in these samples.

Figure 4

Dimerization status of encapsidated wild type and Ψ mutant RNAs. Virion RNA samples were normalized to contain roughly the same number of gRNAs per lane and were subjected to the indicated treatments as described in the text. Arrowheads at right indicate the mobilities of wild type products. That the slightly reduced mobility of the wild type dimeric RNA observed at 58°, relative to the unheated sample, is consistent with previous findings,.