Biology subjects

Neil, C.

Publications and source records attributed to Neil, C..

3 recordsLinked to original sources

bioRxiv2024

The pseudoknot region and poly-(C) tract comprise an essential RNA packaging signal for assembly of foot-and-mouth disease virus

Virus assembly is a crucial step for the completion of the viral replication cycle. In addition to ensuring efficient incorporation of viral genomes into nascent virions, high specificity is required to prevent incorporation of host nucleic acids. For picornaviruses, including FMDV, the mechanisms required to fulfil these requirements are not well understood. However, recent evidence has suggested that specific RNA sequences dispersed throughout picornavirus genomes are involved in packaging. Here, we have shown that such sequences are essential for FMDV RNA packaging and have demonstrated roles for both the pseudoknot (PK) region and the poly-(C) tract in this process, where the length of the poly-(C) tract was found to influence the efficiency of RNA encapsidation. Sub-genomic replicons containing longer poly-(C) tracts were packaged with greater efficiency in trans, and viruses recovered from transcripts containing short poly-(C) tracts were found to have greatly extended poly-(C) tracts after only a single passage in cells, suggesting that maintaining a long poly-(C) tract provides a selective advantage. We also characterised a critical packaging signal (PS) located in the pseudoknot (PK) region, adjacent to the poly-(C) tract, as well as several other non-essential but beneficial PSs elsewhere in the genome. Collectively, these PSs greatly enhanced encapsidation efficiency, with the poly-(C) tract possibly facilitating nearby PSs to adopt the correct conformation. Using these data, we have proposed a model where interactions with capsid precursors control a transition between two RNA conformations, directing the fate of nascent genomes to either be packaged or alternatively to act as templates for replication and/or for protein translation. Author summaryGenome packaging, whereby viral RNA is incorporated into protective protein capsids to produce more virus particles, is a crucial step in RNA virus life cycles. It is a stringent process as only viral RNA is encapsidated, while cellular RNA is excluded. This study reveals the essential role of packaging signals in FMDV RNA packaging, specifically those in the pseudoknot region and in a region that can contain >100 cytosines, termed the poly-(C) tract. We demonstrate that the length of the poly-(C) tract significantly affects packaging efficiency; genomes containing longer poly-(C) tracts are favoured. This is the first role that has been identified for the poly-(C) tract in FMDV. We have also found an essential packaging signal in the pseudoknot region, which is assisted by other packaging signals located throughout the genome, that together facilitate encapsidation of FMDV RNA. These results provide compelling evidence for the involvement of RNA packaging signals in FMDV assembly. Based on this, we propose a simple model for FMDV RNA packaging, which involves a transition from genome replication to genome packaging and is controlled by packaging signals. This knowledge could pave the way for future research and development of novel antiviral strategies targeting FMDV and other picornaviruses.

microbiology↗

bioRxiv2020

Mechanisms of scaffold-mediated microcompartment assembly and size-control

This article describes a theoretical and computational study of the dynamical assembly of a protein shell around a complex consisting of many cargo molecules and long flexible scaffold molecules. Our study is motivated by bacterial microcompartments, which are proteinaceous organelles that assemble around a condensed droplet of enzymes and reactants. As in many examples of cytoplasmic liquid-liquid phase separation, condensation of the microcompartment interior cargo is driven by long flexible scaffold proteins that have weak multivalent interactions with the cargo. We describe a minimal model for the thermodynamics and dynamics of assembly of a protein shell around cargo and scaffold molecules, with scaffold-mediated cargo coalescence and encapsulation. Our results predict that the shell size, amount of encapsulated cargo, and assembly pathways depend sensitively on properties of the scaffold, including its length and valency of scaffold-cargo interactions. Moreover, the ability of self-assembling protein shells to change their size to accommodate scaffold molecules of different lengths depends crucially on whether the spontaneous curvature radius of the protein shell is smaller or larger than a characteristic elastic length scale of the shell. Beyond natural microcompartments, these results have important implications for synthetic biology efforts to target new molecules for encapsulation by microcompartments or viral shells. More broadly, the results elucidate how cells exploit coupling between self-assembly and liquid-liquid phase separation to organize their interiors.

biophysics↗

bioRxiv2020

The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus.

The positive stranded RNA genomes of picornaviruses comprise a single large open reading frame flanked by 5' and 3' untranslated regions (UTRs). Foot-and-mouth disease virus (FMDV) has an unusually large 5' UTR (1.3 kb) containing five structural domains. These include the internal ribosome entry site (IRES), which facilitates initiation of translation, and the cis-acting replication element (cre). Less well characterised structures are a 5' terminal 360 nucleotide stem-loop, a variable length poly-C-tract of approximately 100-200 nucleotides and a series of two to four tandemly repeated pseudoknots (PKs). We investigated the structures of the PKs by selective 2' hydroxyl acetylation analysed by primer extension (SHAPE) analysis and determined their contribution to genome replication by mutation and deletion experiments. SHAPE and mutation experiments confirmed the importance of the previously predicted PK structures for their function. Deletion experiments showed that although PKs are not essential for replication, they provide genomes with a competitive advantage. However, although replicons and full-length genomes lacking all PKs were replication competent, no infectious virus was rescued from genomes containing less than one PK copy. This is consistent with our earlier report describing the presence of putative packaging signals in the PK region.

molecular biology↗