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

Abstract

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...

QuShape: Rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis

“…Experiments with minor RNA-specific variations have been reported for the TPP riboswitch (Steen et al 2012), a retroviral genome signaling domain (Gherghe et al 2010;Grohman et al 2011), ribosomal RNAs McGinnis et al 2012), and long viral RNAs Watts et al 2009;Gherghe et al 2010). The key new feature is that all experiments are now resolved in two capillaries.…”

Deciphering the Role of the Gag-Pol Ribosomal Frameshift Signal in HIV-1 RNA Genome Packaging

“…These results support the hypothesis that recognition of one dimeric RNA by Gag is a key point of viral RNA encapsidation. Recent advances shed light on how Gag recognizes dimeric RNA; for example, studies of MLVs have revealed that dimerization of the viral RNA exposes high-affinity Gag binding sites to allow the initiation of RNA packaging (49)(50)(51). Studies in HIV-1 have also identified structural elements important in dimerization, Gag binding, and packaging (17,20,21).…”

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

“…A model for the switch between translation and packaging has been proposed based on NMR spectroscopy data showing that the DIS loop of SL1 folds back and interacts with an upstream region in the viral RNA, thereby preventing dimerization of SL1 and promoting translation (Lu et al 2011a). In MLV, dimerization of the gRNA exposes high-affinity binding sites for the NC domain of Gag (D'Souza and Summers 2004;Gherghe et al 2010;Miyazaki et al 2010), suggesting that packaging and Gag binding to ψ are coordinated with dimerization. In HIV-1, a 159-nt "core encapsidation signal" was recently described that binds NC with similar affinity as the full-length 5 ′ leader sequence (Heng et al 2012).…”