Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography

Miyazaki, Yasuaki; Irobalieva, Rossitza N.; Tolbert, Blanton S.; Smalls-Mantey, Adjoa; Iyalla, Kilali; Loeliger, Kelsey; D'Souza, Ventris M.; Khant, Htet; Schmid, Michael F.; Garcia, Eric L.; Telesnitsky, Alice; Chiu, Wah; Summers, Michael F.

doi:10.1016/j.jmb.2010.09.009

Cited by 63 publications

(71 citation statements)

References 119 publications

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“…Instead, when Gag expression was constant and gRNA expression in cells increased, the amount of gRNA per particle increased proportionately until an upper limit was reached, beyond which a 10-fold further increase in vector expression neither increased the amount packaged per virion nor decreased the level of virus production. These observations of saturable gRNA packaging and no impairment of virion release in the presence of excess gRNA support the hypothesis that recruitment of the conserved copy number of gRNAs per MoMLV particle-presumably a single dimer-triggers an assembly transition that prevents the inclusion of excess gRNA (53,54).…”

Section: Discussionsupporting

confidence: 71%

Determinants of Moloney Murine Leukemia Virus Gag-Pol and Genomic RNA Proportions

Johnson

Collins

D'Souza

et al. 2014

J Virol

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The Moloney murine leukemia virus (MoMLV) ribonucleoprotein complex is composed of an approximately 20:1 mixture of Gag and Gag-Pol polyproteins plus a single genomic RNA (gRNA) dimer. The mechanisms that regulate these proportions are unknown. Here, we examined whether virion proportions of Gag, Gag-Pol, and gRNA were determined by sampling (that is, if they reflected expression ratios or intracellular concentrations) or more specific recruitment. To this end, MoMLV Gag, Gag-Pol, and gRNA were expressed separately or together in various ratios. Varying the expression ratios of Gag and Gag-Pol revealed that Gag-Pol incorporation was stochastic and that the conserved 20:1 Gag/Gag-Pol ratio coincided with maximal particle production. When skewed expression ratios resulted in excess Gag-Pol, the released virions maintained the intracellular Gag/GagPol ratios and the infectivity per virion was largely maintained, but virion production decreased sharply with high levels of GagPol. The determinants of gRNA proportions were addressed by manipulating the amounts and contexts of functional nucleocapsid (NC) and the ratios of Gag to gRNA. The results showed that the NC domain of either Gag or Gag-Pol could provide gRNA packaging functions equally well. Unlike Gag-Pol, gRNA incorporation was saturable. An upper limit of gRNA incorporation was observed, and particle production was not disrupted by excess gRNA expression. These results indicate that the determinants of Gag/Gag-Pol proportions differ from those for Gag/gRNA. On the basis of the assumption that MoMLV evolved to produce virion components in optimal proportions, these data provide a means of estimating the proportion of unspliced MoMLV RNA that serves as genomic RNA. IMPORTANCEViruses assemble their progeny from within the cells that they parasitize, where they must sort through a rich milieu of host proteins and nucleic acids to gather together their own building blocks, which are also proteins and nucleic acids. The research described here addresses whether or not the proportions of viral proteins and nucleic acids that are brought together to form a retroviral particle are determined by random sampling from the cell-and thus dictated by the components' availabilities within the cell-or if the amounts of each molecule are specified by the virus replication process. The results indicated that protein components of the murine retrovirus studied here are recruited by chance but that a specific counting mechanism defines the amount of nucleic acid incorporated into each progeny virion.

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Section: Discussionsupporting

confidence: 71%

Determinants of Moloney Murine Leukemia Virus Gag-Pol and Genomic RNA Proportions

Johnson

Collins

D'Souza

et al. 2014

J Virol

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“…Experiments on selectively labeled J236 RNAs were particularly valuable for analysis of NMR data given that J236 is larger than 60 nt. In fact, only a few NMR structures are available for such larger RNAs (Lukavsky et al 2003;D'Souza et al 2004;Miyazaki et al 2010;Burke et al 2012;Miller et al 2014;Keane et al 2015).…”

Section: Resultsmentioning

confidence: 99%

The NMR structure of the II–III–VI three-way junction from the Neurospora VS ribozyme reveals a critical tertiary interaction and provides new insights into the global ribozyme structure

Bonneau

Girard

Lemieux

et al. 2015

RNA

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As part of an effort to structurally characterize the complete Neurospora VS ribozyme, NMR solution structures of several subdomains have been previously determined, including the internal loops of domains I and VI, the I/V kissing-loop interaction and the III-IV-V junction. Here, we expand this work by determining the NMR structure of a 62-nucleotide RNA (J236) that encompasses the VS ribozyme II-III-VI three-way junction and its adjoining stems. In addition, we localize Mg 2+ -binding sites within this structure using Mn 2+ -induced paramagnetic relaxation enhancement. The NMR structure of the J236 RNA displays a family C topology with a compact core stabilized by continuous stacking of stems II and III, a cis WC/WC G•A base pair, two base triples and two Mg 2+ ions. Moreover, it reveals a remote tertiary interaction between the adenine bulges of stems II and VI. Additional NMR studies demonstrate that both this bulge-bulge interaction and Mg 2+ ions are critical for the stable folding of the II-III-VI junction. The NMR structure of the J236 RNA is consistent with biochemical studies on the complete VS ribozyme, but not with biophysical studies performed with a minimal II-III-VI junction that does not contain the II-VI bulge-bulge interaction. Together with previous NMR studies, our findings provide important new insights into the threedimensional architecture of this unique ribozyme.

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“…Studies of MLV packaging showed that the monomeric and dimeric RNAs have very different secondary structures (10,15,21). Furthermore, dimerization of MLV RNAs exposes highaffinity binding sites for nucleocapsids, which is critical for RNA packaging (16,22). It is thought that exposure of these sites allows Gag binding, leading to the incorporation of RNA into virus particles.…”

Section: Discussionmentioning

confidence: 99%

Determining the Frequency and Mechanisms of HIV-1 and HIV-2 RNA Copackaging by Single-Virion Analysis

Dilley

Nikolaitchik

et al. 2011

J Virol

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HIV-1 and HIV-2 are derived from two distinct primate viruses and share only limited sequence identity. Despite this, HIV-1 and HIV-2 Gag polyproteins can coassemble into the same particle and their genomes can undergo recombination, albeit at an extremely low frequency, implying that HIV-1 and HIV-2 RNA can be copackaged into the same particle. To determine the frequency of HIV-1 and HIV-2 RNA copackaging and to dissect the mechanisms that allow the heterologous RNA copackaging, we directly visualized the RNA content of each particle by using RNA-binding proteins tagged with fluorescent proteins to label the viral genomes. We found that when HIV-1 and HIV-2 RNA are present in viral particles at similar ratios, ϳ10% of the viral particles encapsidate both HIV-1 and HIV-2 RNAs. Furthermore, heterologous RNA copackaging can be promoted by mutating the 6-nucleotide (6-nt) dimer initiation signal (DIS) to discourage RNA homodimerization or to encourage RNA heterodimerization, indicating that HIV-1 and HIV-2 RNA can heterodimerize prior to packaging using the DIS sequences. We also observed that the coassembly of HIV-1 and HIV-2 Gag proteins is not required for the heterologous RNA copackaging; HIV-1 Gag proteins are capable of mediating HIV-1 and HIV-2 RNA copackaging. These results define the cis-and trans-acting elements required for and affecting the heterologous RNA copackaging, a prerequisite for the generation of chimeric viruses by recombination, and also shed light on the mechanisms of RNA-Gag recognition essential for RNA encapsidation.

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Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography

Cited by 63 publications

References 119 publications

Determinants of Moloney Murine Leukemia Virus Gag-Pol and Genomic RNA Proportions

Determinants of Moloney Murine Leukemia Virus Gag-Pol and Genomic RNA Proportions

The NMR structure of the II–III–VI three-way junction from the Neurospora VS ribozyme reveals a critical tertiary interaction and provides new insights into the global ribozyme structure

Determining the Frequency and Mechanisms of HIV-1 and HIV-2 RNA Copackaging by Single-Virion Analysis

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