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Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts.

ABSTRACT: Picornaviruses are important viral pathogens, but despite extensive study, the assembly process of their infectious virions is still incompletely understood, preventing the development of anti-viral strategies targeting this essential part of the life cycle. We report the identification, via RNA SELEX and bioinformatics, of multiple RNA sites across the genome of a typical enterovirus, enterovirus-E (EV-E), that each have affinity for the cognate viral capsid protein (CP) capsomer. Many of these sites are evolutionarily conserved across known EV-E variants, suggesting they play essential functional roles. Cryo-electron microscopy was used to reconstruct the EV-E particle at ~2.2 Å resolution, revealing extensive density for the genomic RNA. Relaxing the imposed symmetry within the reconstructed particles reveals multiple RNA-CP contacts, a first for any picornavirus. Conservative mutagenesis of the individual RNA-contacting amino acid side chains in EV-E, many of which are conserved across the enterovirus family including poliovirus, is lethal but does not interfere with replication or translation. Anti-EV-E and anti-poliovirus aptamers share sequence similarities with sites distributed across the poliovirus genome. These data are consistent with the hypothesis that these RNA-CP contacts are RNA Packaging Signals (PSs) that play vital roles in assembly and suggest that the RNA PSs are evolutionarily conserved between pathogens within the family, augmenting the current protein-only assembly paradigm for this family of viruses.

SUBMITTER: Chandler-Bostock R

PROVIDER: S-EPMC7793301 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts.

Chandler-Bostock Rebecca R Mata Carlos P CP Bingham Richard J RJ Dykeman Eric C EC Meng Bo B Tuthill Tobias J TJ Rowlands David J DJ Ranson Neil A NA Twarock Reidun R Stockley Peter G PG

PLoS pathogens 20201228 12

Picornaviruses are important viral pathogens, but despite extensive study, the assembly process of their infectious virions is still incompletely understood, preventing the development of anti-viral strategies targeting this essential part of the life cycle. We report the identification, via RNA SELEX and bioinformatics, of multiple RNA sites across the genome of a typical enterovirus, enterovirus-E (EV-E), that each have affinity for the cognate viral capsid protein (CP) capsomer. Many of these ...[more]

PMID: 33370422

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Project description:The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ.

Project description:Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T=3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem-loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T=3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.

Dataset Information

Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts.

Publications

Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts.

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