mRNA molecules containing murine leukemia virus packaging signals are encapsidated as dimers

Abstract

Prior work by others has shown that insertion of psi (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3' untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV psi or with psi sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the psi region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Psi(+) mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops.

FIG. 1.

MLV ψ region. (A) Sequence of nucleotides 202 to 377, showing predicted stem-loops (SLs). (B) Chimeric RNAs analyzed in the present study. The gray portion of each line denotes the viral insert placed in the 3′ UTR of the hph mRNA.

FIG. 2.

Packaging levels and efficiencies of chimeric and viral RNAs. (A) Packaging of hph mRNA by ψ⁻ MLV as a function of the viral insert. Cells that had been stably transfected with hph constructs were transiently transfected with ψ⁻ MLV. The amount of hph RNA in virus particles was then measured and normalized to that in hph 215-739. The results shown are averages from several experiments. (B) Packaging levels and efficiencies of hph RNAs. The amounts of hph RNA in virions and in cell lysates were measured after transfection as in A; efficiency represents the ratio of the amount in virions to the amount in the lysate. Results were normalized to that for hph 215-739. Data are averages of multiple measurements from a single experiment but are representative of several experiments. (C) Packaging of hph and MLV RNAs. Cells that had been stably transfected with hph plasmids (indicated in the hph line below the figure) were transiently transfected with ψ⁺ (wild type), ψ⁻, or no (mock) MLV as indicated in the MLV line below the figure. The amounts of hph and MLV RNA in viral pellets from these cells were measured. The results shown are averages of several experiments. (D) Packaging levels and efficiencies of pLXSH genomic RNA, hph RNAs, and MLV genomic RNA. Cells were transiently cotransfected with a mixture of ψ⁻ MLV DNA and either pLXSH, hph 215-1006, hph MSV, or wild-type MLV DNA. Two days later, supernatants were collected and cells were lysed. In samples containing pLXSH, hph 215-1006, or hph MSV, the amounts of hph RNA in virions and cells were measured; in the sample with MLV, MLV RNA was measured. The results are normalized to pLXSH. In all cases (panels A to D), the values have been corrected for minor differences in the CA protein content of different samples.

FIG. 3.

Dissociation of dimeric RNAs of MLV and of hph (−151)-1560 mRNA following extraction from virions. (A) Nondenaturing Northern analysis, with MLV-specific (lanes 1 to 14) or hph-specific (lanes 15 to 28) probes. Lanes 1 to 7, PR⁻ MLV; 8 to 14, wild-type MLV; 15 to 21, hph mRNA packaged by PR⁻ ψ⁻ MLV; 22 to 28, hph mRNA packaged by ψ⁻ MLV. RNA preparations were heated to the indicated temperatures before electrophoresis. DD, diffuse dimers; D, dimers; M, monomers. (B) Graph of dissociation data from panel A.

FIG. 4.

Dissociation of dimeric RNAs of hph RNAs compared with genomic RNAs of HaSV, MLV, and pLXSH. (A) Graph of dissociation of HaSV RNA and of hph HaSV mRNA packaged by ψ⁻ MLV or ψ⁻ PR⁻ MLV. (B) Graph of dissociation of hph HaSV mRNA and of hph (−151)-1560 mRNA packaged by ψ⁻ MLV or ψ⁻ PR⁻ MLV. (C) T_ms of MLV genomic RNA and hph mRNAs containing MLV-derived inserts; HaSV genomic RNA and hph HaSV RNA; and pLXSH genomic RNA and hph MSV RNA (which contains the same MLV-MSV chimeric ψ region as pLXSH). Gray bars, RNAs from PR⁻ virions; black bars, RNAs from PR⁺ virions. Values shown in panel C are averages from several experiments.

FIG. 5.

Dissociation of dimeric RNAs of hph mRNAs containing truncated MLV-derived inserts. (A) Dissociation of dimeric RNAs of hph (−151)-725 (lanes 1 to 5) and hph 215-739 (lanes 6 to 10) mRNAs packaged by ψ⁻ PR⁻ MLV, monitored by nondenaturing Northern analysis. (B) T_ms of hph mRNAs containing truncated MLV inserts, packaged by ψ⁻ MLV or ψ⁻ PR⁻ MLV. The figure also shows the T_ms of PR⁻ and wild-type MLV genomic RNAs. Gray bars, RNAs from PR⁻virions; black bars, RNAs from PR⁺ virions. Values shown in panel B are averages from several experiments.

FIG. 6.

Dissociation of dimers of hph 215-507 with or without a deletion of nucleotides 290 to 305, following packaging by ψ⁻ MLV or ψ⁻ PR⁻ MLV.

FIG. 7.

Regions of possible extended base-pairing in MLV, HaSV, and MSV.

FIG. 8.

Alignments between MLV sequences and (A) MSV sequences and (B) HaSV sequences. In both panels A and B, the top line is the MLV sequence. The GACG loops in stem-loops C and D are indicated by horizontal lines above the sequence.