Preliminary physical mapping of RNA-RNA linkages in the genomic RNA of Moloney murine leukemia virus

Abstract

Retrovirus particles contain two copies of their genomic RNA, held together in a dimer by linkages which presumably consist of a limited number of base pairs. In an effort to localize these linkages, we digested deproteinized RNA from Moloney murine leukemia virus (MLV) particles with RNase H in the presence of oligodeoxynucleotides complementary to specific sites in viral RNA. The cleaved RNAs were then characterized by nondenaturing gel electrophoresis. We found that fragments composed of nucleotides 1 to 754 were dimeric, with a linkage as thermostable as that between dimers of intact genomic RNA. In contrast, there was no stable linkage between fragments consisting of nucleotides 755 to 8332. Thus, the most stable linkage between monomers is on the 5' side of nucleotide 754. This conclusion is in agreement with earlier electron microscopic analyses of partially denatured viral RNAs and with our study (C. S. Hibbert, J. Mirro, and A. Rein, J. Virol. 78:10927-10938, 2004) of encapsidated nonviral mRNAs containing inserts of viral sequence. We obtained similar results with RNAs from immature MLV particles, in which the dimeric linkage is different from that in mature particles and has not previously been localized. The 5' and 3' fragments of cleaved RNA are all held together by thermolabile linkages, indicating the presence of tethering interactions between bases 5' and bases 3' of the cleavage site. When RNAs from mature particles were cleaved at nucleotide 1201, we detected tethering interactions spanning the cleavage site which are intramonomeric and are as strong as the most stable linkage between the monomers.

FIG. 1.

RNase H cleavage of MLV RNA at nt 754. RNA from mature MLV virions was incubated with RNase H in the presence (lanes 1D and 2D) or absence (lanes 1 and 2) of an oligonucleotide (Oligo) complementary to MLV nt 754 to 783. It was then analyzed by nondenaturing Northern blotting before (lanes 1 and 1D; Un, unheated) or after (lanes 2 and 2D) heating to 65°C, using a probe complementary to nt 215 to 739. The presence of tethering interactions holding the fragments of the cleaved, unheated RNA together (lane 1D) is indicated in the illustration to the right.

FIG. 2.

Thermostability of RNA-RNA linkages in cleaved MLV RNA. (A) RNA from mature MLV virions was cleaved as described in the legend to Fig. 1 and incubated at different temperatures before analysis as described in the legend to Fig. 1. The temperatures are indicated at the top. Un, unheated sample. Results are shown for RNA incubated with RNase H in the absence (lanes 1 to 5) or presence (lanes 1D to 5D) of oligonucleotide (oligo). The RNA migrating as a dimer in lanes 1D and 2D has been cleaved by the RNase H but is still held together by thermolabile tethering interactions. (B) The samples shown in lanes 1D to 5D of panel A, in which the cleaved RNA had been incubated at different temperatures, were analyzed by denaturing Northern analysis without (lanes 1 to 5) or with (lanes 6 to 10) UV-psoralen cross-linking.

FIG. 3.

RNA-RNA linkages in immature MLV RNA. (A) RNA from PR⁻ MLV virions was analyzed exactly as in Fig. 2A. All samples were incubated with RNase H, but the RNA was not cleaved in the absence of oligonucleotide (oligo; lanes 1 to 5). (B) The membrane shown in panel A was stripped and then reprobed with radioactive RNA complementary to MLV nt 7830 to 8196. Un, unheated.

FIG. 4.

Linkages in MLV RNA following cleavage at nt 713 or 1201. RNA from mature MLV virions was incubated with RNase H in the presence of oligonucleotides (oligo) complementary to MLV nt 713 to 742 (lanes 1 to 8) or 1201 to 1230 (lanes 9 to 16). They were then heated at the temperatures indicated at the top before analysis as in Fig. 1. D, dimer; 5′ D, dimer of 5′ fragment; 5′ F, monomer of 5′ fragment.

FIG. 5.

Schematic depiction of linkages in MLV RNA. The figure shows that the most stable linkage between monomers in MLV dimeric RNA is on the 5′ side of nt 754 (shown as solid vertical lines). In addition, there are tethering linkages joining sequences on the 5′ side of nt 754 to sequences on the 3′ side; these are shown as dashed lines. These linkages, which are less thermostable than the most stable linkage, could be either intra- or intermonomeric, although they are depicted as intramonomeric. There are also bonds connecting sequences between nt 754 and nt 1201 with sequences to the 3′ side of nt 1201 (shown as dotted lines). These are as stable as the most stable linkage and are exclusively intramonomeric. Intervals are not drawn to scale.

FIG. 6.

Potential tethering interactions in retroviral genomes. The figure shows possible base pairings between segments of retroviral genomes. In each case, the first nucleotide shown is the nucleotide immediately following the AATAAA polyadenylation signal. (A) MLV RNAs: Moloney MLV (Mo-MLV; GenBank accession no. J02255), complementarity between nt 53 to 77 and nt 758 to 781; AKR MLV (Akv; accession no. J01998), complementarity between nt 53 to 77 and nt 776 to 798; Friend MLV (F-MLV; accession no. Z11128), complementarity between nt 53 to 80 and nt 755 to 776. Sequence changes in AKR MLV and Friend MLV relative to Moloney MLV which preserve base pairing are underlined. (B) Other gammaretroviral RNAs: gibbon ape leukemia virus (GALV; accession no. M26927), complementarity between nt 53 to 67 and nt 764 to 778; feline leukemia virus (FeLV; accession no. M18247), complementarity between nt 393 to 422 and nt 1037 to 1066; baboon endogenous virus (BaEV; accession no. d10032) 1, complementarity between nt 383 to 406 and nt 1154 to 1175; 2, complementarity between nt 383 to 406 and nt 1123 to 1141. Nucleotides 1123 to 1141 (structure 2) are in the same position in the MA coding region as shown for the other viruses in panels A and B, but structure 1 is somewhat more stable than structure 2. (C) HIV-1 Mal RNA (accession no. X04415), complementarity between nt 77 to 93 and nt 456 to 470.