All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs-only four nucleotides per genomic RNA-reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.retrovirus | RNA recognition code | RNA SHAPE chemistry E xpression of a single viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. If present in the cell, the viral genomic RNA (vRNA) is selectively packaged into nascent particles; this selectivity is due to a cisacting packaging signal in the RNA, termed Ψ (1, 2). Remarkably, when no Ψ-containing RNA is present, Gag still assembles efficiently, encapsidating cellular mRNAs nonselectively in place of the vRNA (3-5).There are many indications that Ψ represents a high-affinity binding site for the Gag protein both in HIV-1 and in simpler retroviruses (6-14). However, the molecular mechanisms underlying selective encapsidation of vRNAs are incompletely understood, as are the features that enable Gag to bind preferentially to vRNA rather than to other cellular RNAs. Gag proteins contain several distinct domains, always including matrix (MA), capsid, and nucleocapsid (NC). vRNA packaging is mediated by the multidomain Gag protein, but Gag is cleaved following release of the virus from the cell. The NC domain plays a principal role in interactions with nucleic acids and is largely responsible for the specific interaction between Gag and its cognate viral RNA (12,13). This domain of Gag is highly basic and contains one or more "zinc knuckles" with a conserved spacing of Zn 2þ -coordinating cysteine and histidine residues. Mutations that abolish Zn 2þ coordination impair selective encapsidation of vRNA during virus assembly (6, 15). In addition, MA domains of many retroviral Gag proteins interact with nucleic acids (16-21) and may also contribute to specific i...

Retroviral genomes are dimeric, comprised of two sense-strand RNAs linked at their 5 ends by noncovalent base pairing and tertiary interactions. Viral maturation involves large-scale morphological changes in viral proteins and in genomic RNA dimer structures to yield infectious virions. Structural studies have largely focused on simplified in vitro models of genomic RNA dimers even though the relationship between these models and authentic viral RNA is unknown. We evaluate the secondary structure of the minimal dimerization domain in genomes isolated from Moloney murine leukemia virions using a quantitative and single nucleotide resolution RNA structure analysis technology (selective 2-hydroxyl acylation analyzed by primer extension, or SHAPE). Results are consistent with an architecture in which the RNA dimer is stabilized by four primary interactions involving two sets of intermolecular base pairs and two loop-loop interactions. The dimerization domain can independently direct its own folding since heating and refolding reproduce the same structure as visualized in genomic RNA isolated from virions. Authentic ex virio RNA has a SHAPE reactivity profile similar to that of a simplified transcript dimer generated in vitro, with the important exception of a region that appears to form a compact stem-loop only in the virion-isolated RNA. Finally, we analyze the conformational changes that accompany folding of monomers into dimers in vitro. These experiments support well-defined structural models for an authentic dimerization domain and also emphasize that many features of mature genomic RNA dimers can be reproduced in vitro using properly designed, simplified RNAs.Retroviruses, including both simple model viruses and complex viruses like human immunodeficiency virus (HIV), contain genomes in the form of two coding RNA strands, noncovalently linked at their 5Ј ends (11,19,20,26,38,45). This 5Ј linkage is termed the genomic RNA dimer. Packaging of RNA genomes into new virions is highly specific, even in the presence of a large background of cellular RNA (1,7,26). This packaging function is carried out by the Gag protein (14,30,43), which recognizes RNA sequences that overlap with the RNA dimerization domain (16,26,42,44). The specific Gagdimer interaction represents an elegant and direct mechanism by which exactly two RNA genomes are packaged into each nascent virion.The genomic RNA dimer is initially assembled into an immature and noninfectious viral particle (9,(18)(19)(20). After the immature particle buds from the host cell, it undergoes extensive morphological changes to form the mature and infectious virion (21, 51). Maturation is initiated through cleavage of the Gag polyprotein by the viral protease to yield smaller Gagderived proteins and also involves changes in the structure of the RNA dimer region. The RNA dimer structure appears to be more compact and, for many retroviruses, more thermostable in mature than in immature virions (18)(19)(20)26).The closely related Moloney murine leukemia and sarcoma viruse...

Retroviral genomes are assembled from two sense-strand RNAs by noncovalent interactions at their 5 ends, forming a dimer. The RNA dimerization domain is a potential target for antiretroviral therapy and represents a compelling RNA folding problem. The fundamental dimerization unit for the Moloney murine sarcoma gamma retrovirus spans a 170-nucleotide minimal dimerization active sequence. In the dimer, two self-complementary sequences, PAL1 and PAL2, form intermolecular duplexes, and an SL1-SL2 (stem-loop) domain forms loop-loop base pairs, mediated by GACG tetraloops, and extensive tertiary interactions. To develop a framework for assembly of the retroviral RNA dimer, we quantified the stability of and established nucleotide resolution secondary structure models for sequence variants in which each motif was compromised. Base pairing and tertiary interactions between SL1-SL2 domains contribute a large free energy increment of ؊10 kcal/mol. In contrast, even though the PAL1 and PAL2 intermolecular duplexes span 10 and 16 bp in the dimer, respectively, they contribute only ؊2.5 kcal/mol to stability, roughly equal to a single new base pair. First, these results emphasize that the energetic costs for disrupting interactions in the monomer state nearly balance the PAL1 and PAL2 base pairing interactions that form in the dimer. Second, intermolecular duplex formation plays a biological role distinct from simply stabilizing the structure of the retroviral genomic RNA dimer.Retroviruses carry their genetic information in the form of RNA and replicate through a DNA intermediate (1). The retroviral genome consists of two noncovalently linked sense-strand RNAs, forming a dimer (2-4). Formation of the genomic RNA dimer appears to be important for key stages of the retroviral replication cycle, including selective packaging of genomic RNA (4 -6), reverse transcription, and recombination (7-10).Although a retroviral RNA genome forms multiple contacts between its constituent monomers, the most stable point of contact is situated at the 5Ј end of the RNA (11-14). Our laboratory has defined and structurally characterized a minimal dimerization active sequence (MiDAS) 2 of 170 nucleotides for a representative gamma retrovirus, the Moloney murine sarcoma virus (MuSV), as the major 5Ј dimerization motif (15-17) (Fig. 1). The MiDAS is a structurally independent domain that dimerizes efficiently to yield homogeneous monomer and dimer populations in vitro. The MiDAS RNA dimerizes under biologically relevant conditions of both temperature and ionic environment, in the absence of proteins. Moreover, RNAs spanning sequences closely related to the MiDAS are sufficient to mediate RNA dimerization and encapsidation in vivo when inserted into nonviral mRNAs (3, 18). Thus, the MiDAS domain is an important system for studying retroviral RNA dimerization as it contains all of the RNA sequence elements required for the high selectivity and affinity of this process both in vitro and in vivo. Structures of the MiDAS domain for MuSV in the monomeri...

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