Bacteriophage MS2 displays unreported capsid variability assembling T = 4 and mixed capsids

Abstract

Bacteriophage MS2 is a positive-sense, singlestranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, in which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study, we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 Å and 6 Å respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.Abbreviations: CP, coat protein; IAU, icosahedral asymmetric unit; MP, maturation protein; VLP, virus-like particle.

A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics

“…We determined the VLP size distribution using dynamic light scattering (DLS) to be~27 nm ( Fig. 1c), which matched well with a previously characterized MS2 VLP construct 13 . Next, we employed reverse-transcriptase droplet digital PCR (RT-ddPCR) to obtain absolute quantities of three serial dilutions of VLPs.…”

“…Recombinant MS2 bacteriophage VLPs carrying the SARS-CoV-2 N-gene were produced in Escherichia coli from an expression plasmid using protocols described previously and modified to transcribe and package the RNA for the SARS-CoV-2 N protein (Fig. 1a ) 13 , 14 . The assembled MS2-SARS-CoV-2 VLPs were purified and treated with DNase and RNase, to ensure the preparation was free from template DNA and RNA contamination.…”

Direct transfer of electron microscopy samples to wetted carbon and graphene films via a support floatation block

“…This minimises dissociation, which can occur during centrifugal separation, while providing a quenched, and therefore stable, final sample. An illustration of the application of this protocol to MS2 virions ( de Martín Garrido et al, 2019 ) is shown in Fig. 4 a.…”

A new role for Biofoundries in rapid prototyping, development, and validation of automated clinical diagnostic tests for SARS-CoV-2

“…Recombinant MS2 bacteriophage VLPs carrying the SARS-CoV-2 N gene were produced in E. coli from an expression plasmid using protocols described previously and modified to transcribe and package the primer-probe target sites for the CoV-2 N protein (Fig 1a) [12]. The assembled MS2-SARS-CoV-2 VLPs were purified and treated with DNase and RNase to ensure the preparation was free from template DNA and RNA contamination.…”

“…The N gene was cloned into a previously described plasmid backbone (Addgene #128233) using Type IIs assembly. The sequence verified (Eurofins Genomics) The protein purification workflow is based on previously described work [12], [30]. All protein purification steps were performed at 4 °C.…”