Abstract
Many single-stranded, positive-sense RNA viruses regulate assembly of their infectious virions by forming multiple, cognate coat protein (CP)-genome contacts at sites termed Packaging Signals (PSs). We have determined the secondary structures of the bacteriophage MS2 ssRNA genome (gRNA) frozen in defined states using constraints from X-ray synchrotron footprinting (XRF). Comparison of the footprints from phage and transcript confirms the presence of multiple PSs in contact with CP dimers in the former. This is also true for a virus-like particle (VLP) assembled around the gRNA in vitro in the absence of the single-copy Maturation Protein (MP) found in phage. Since PS folds are present at many sites across gRNA transcripts, it appears that this genome has evolved to facilitate this mechanism of assembly regulation. There are striking differences between the gRNA-CP contacts seen in phage and the VLP, suggesting that the latter are inappropriate surrogates for aspects of phage structure/function. Roughly 50% of potential PS sites in the gRNA are not in contact with the protein shell of phage. However, many of these sit adjacent to, albeit not in contact with, PS-binding sites on CP dimers. We hypothesize that these act as PSs transiently during assembly but subsequently dissociate. Combining the XRF data with PS locations from an asymmetric cryo-EM reconstruction suggests that the genome positions of such dissociations are non-random and may facilitate infection. The loss of many PS-CP interactions towards the 3′ end of the gRNA would allow this part of the genome to transit more easily through the narrow basal body of the pilus extruding machinery. This is the known first step in phage infection. In addition, each PS-CP dissociation event leaves the protein partner trapped in a non-lowest free-energy conformation. This destabilizes the protein shell which must disassemble during infection, further facilitating this stage of the life-cycle.
| Original language | English |
|---|---|
| Article number | 167797 |
| Number of pages | 18 |
| Journal | Journal of Molecular Biology |
| Volume | 434 |
| Issue number | 20 |
| Early online date | 9 Sept 2022 |
| DOIs | |
| Publication status | Published - 30 Oct 2022 |
Bibliographical note
© 2022 The Authors.Funding Information:
Portions of this work used the XFP (17-BM) beamline at NSLS-II. Development of XFP was made possible by the US National Science Foundation, Division of Biological Infrastructure (grant No. 1228549), while operations support of XFP was provided by the US National Institutes of Health (grant No. P30-EB-009998). NSLS-II, a US Department of Energy (DOE) Office of Science User Facility and is operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. We also thank DNA Sequencing & Services (MRC I PPU, School of Life Sciences, University of Dundee, Scotland, www.dnaseq.co.uk ) for DNA sequencing.
Funding Information:
PGS & RT thank The Wellcome Trust (Joint Investigator Award Nos. 110145 & 110146 to PGS & RT, respectively) for funding. We also acknowledge the financial support of The Trust of infrastructure and equipment in the Astbury Centre, University of Leeds (089311/Z/09/Z; 090932/Z/09/Z & 106692), and for their additional support, together with The University of Leeds, of the Astbury Biostructure Facility. RT acknowledges additional funding via an EPSRC Established Career Fellowship (EP/R023204/1) and a Royal Society Wolfson Fellowship (RSWF\R1\180009).
Funding Information:
We are grateful to Professor Sarah Woodson, Johns Hopkins University, for her encouragement and support in the use of XRF. Portions of this work used the XFP (17-BM) beamline at NSLS-II. Development of XFP was made possible by the US National Science Foundation, Division of Biological Infrastructure (grant No. 1228549), while operations support of XFP was provided by the US National Institutes of Health (grant No. P30-EB-009998). NSLS-II, a US Department of Energy (DOE) Office of Science User Facility and is operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. We also thank DNA Sequencing & Services (MRC I PPU, School of Life Sciences, University of Dundee, Scotland, www.dnaseq.co.uk) for DNA sequencing. PGS & RT thank The Wellcome Trust (Joint Investigator Award Nos. 110145 & 110146 to PGS & RT, respectively) for funding. We also acknowledge the financial support of The Trust of infrastructure and equipment in the Astbury Centre, University of Leeds (089311/Z/09/Z; 090932/Z/09/Z & 106692), and for their additional support, together with The University of Leeds, of the Astbury Biostructure Facility. RT acknowledges additional funding via an EPSRC Established Career Fellowship (EP/R023204/1) and a Royal Society Wolfson Fellowship (RSWF\R1\180009). Authors declare that they have no competing interests. The processed data from the capillary electrophoresis data analysis is available as a collection on Figshare (https://doi.org/10.6084/m9.figshare.c.5395302). All other data are available from the corresponding author on reasonable request. The software package used to analyse the capillary electrophoresis (BoXFP), as well as the Supplementary Movie, and larger scale images of the MS2 secondary structures in virio as well as a transcript are available to download from GitHub at https://github.com/MathematicalComputationalVirology/XRFanalysis.
Keywords
- bacteriophage MS2
- molecular frustration
- phage infection
- RNA PS-mediated virion assembly
- RNA X-ray footprinting