mRNA Molecules Containing Murine Leukemia Virus Packaging Signals Are Encapsidated as Dimers

Abstract

Prior work by others has shown that insertion of (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 or with sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the 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 ⌿ ؉ 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.Retroviral proteins package their genomic RNAs with very high selectivity. This selectivity results from the recognition by the viral Gag protein of cis-acting packaging signals (generically called ) in the viral RNA. While this recognition event is far from understood in molecular terms, all available evidence indicates that the major elements in signals are located near the 5Ј end of the genomic RNA (10).In all normal retrovirus particles, the genomic RNA is in dimeric form (10). It consists of two identical, positive-strand monomers, joined together by a thermolabile linkage. The linkage presumably consists of a limited number of base pairs. After the assembled virion is released from the cell, the viral protease (PR) is activated and cleaves the viral proteins into a series of smaller proteins (62). This set of cleavages, termed maturation of the particle, convert the particle into infectious form. The cleaved proteins in turn cause a change in the conformation of the dimeric RNA in the particle, frequently resulting in a more stable dimeric linkage than that present in immature virions (24,26,61) The change in conformation of the RNA has been called maturation of the dimers. It seems likely that dimer maturation is due to the action of the nucleocapsid (NC) protein on the RNA: this protein is known to possess nucleic acid chaperone activity (the ability to catalyze the conformational rearrangement of nucleic acids into the structure with the maximum number of base pairs), and is tightly associated with th...

Structure of an RNA switch that enforces stringent retroviral genomic RNA dimerization

“…This conclusion is strongly corroborated by recent experiments showing that the SL1-SL2 domain (in the absence of PAL2 and most of PAL1) is sufficient to direct modest packaging of a heterologous RNA, in a dimeric state, into virions (8).…”

“…Dimerization therefore must exclude noncognate interactions with the enormous background (30,31) of both retroviral and general cellular mRNAs (6)(7)(8). We suggest that the two-step mechanism (Fig.…”

“…1A). The MiDAS domain correlates closely with retroviral genomic sequences sufficient to package heterologous RNAs into virions (6,8,11,12), as dimers (8). The MiDAS domain also includes conserved sequence elements previously proposed to specify the noncovalent interactions that mediate RNA dimerization.…”

“…Mature retroviral virions contain almost exclusively retroviral genomic RNA plus a few select cellular RNAs (4,5). Many other cellular RNAs, including mRNAs (6)(7)(8), are accessible to the retroviral packaging process. Specific recognition of retroviral genomic RNA against a large background of cellular RNA thus represents a striking example of molecular recognition in biology.…”

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

“…There is strong, albeit indirect, evidence that dimerization is linked to packaging (15,(22)(23)(24)(25), so that only dimers of vRNA are selectively packaged. The selective packaging of dimers, but not monomers, of MuLV vRNA likely reflects, in part, the exposure of UCUG sequence elements that become specifically accessible in dimers (26).…”