Cryo-electron microscopy of the f1 filamentous phage reveals insights into viral infection and assembly

Abstract

Phages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ffs have seen an extraordinary range of applications, yet the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. In this work, we use cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods to determine a structure of a filamentous virus including the tips. We show that structure combined with mutagenesis can identify phage domains that are important in bacterial attack and for release of new progeny, allowing new models to be proposed for the phage lifecycle.

M13-Phage-Based Star-Shaped Particles with Internal Flexibility

“…These glycine-rich linkers result in the spreading of p3 away from the virus body and the presence of knobs as seen in electron micrographs, and they provide to the protein its ability for conformational change. Moreover, the elastic and structural flexibility of p3 has been recently shown to play a major role in the infection of the bacterial host . In its standard configuration, as used here, the spatial extension of the five p3 tip proteins far exceeds the 7 nm of the virus diameter and has been found to be around l ∼ 20 nm, enabling a high accessibility. , The corresponding footprint of the p3 proteins can then be estimated by π­( l /2) 2 and be compared with the area available on the Au NP surface (neglecting curvature effect): π d Au 2 with d Au = 51 nm.…”

“…Moreover, the elastic and structural flexibility of p3 has been recently shown to play a major role in the infection of the bacterial host . In its standard configuration, as used here, the spatial extension of the five p3 tip proteins far exceeds the 7 nm of the virus diameter and has been found to be around l ∼ 20 nm, enabling a high accessibility. , The corresponding footprint of the p3 proteins can then be estimated by π­( l /2) 2 and be compared with the area available on the Au NP surface (neglecting curvature effect): π d Au 2 with d Au = 51 nm. The ratio between these two areas provides an estimation of the highest number of viral arms that can be grafted to the nanoparticle surface, n max ≃ 26, very close to the average valency n ≃ 23 found experimentally (Figure and Figure S1).…”

Diversification of Phage-Displayed Peptide Libraries with Noncanonical Amino Acid Mutagenesis and Chemical Modification

“…22 Through recent Cryo-EM studies, it was determined that the C-terminal domain is primarily composed of alpha helices for insertion within the virion (Figure 2D). 34 The Cterminal end of the protein also contains a transmembrane region that serves to anchor the protein in the host membrane during assembly. 35…”

“…N1 and N2 are similarly folded with each containing two antiparallel β-sheets and an α-helix. , Infection into host cells is mediated through the N1 and N2 domains of pIII, while the C domain appears to be involved with interactions to pVI to terminate viral assembly . Through recent Cryo-EM studies, it was determined that the C-terminal domain is primarily composed of alpha helices for insertion within the virion (Figure D) . The C-terminal end of the protein also contains a transmembrane region that serves to anchor the protein in the host membrane during assembly …”

A packaging signal-binding protein regulates the assembly checkpoint of integrative filamentous phages

“…PS is a cis-acting DNA element that distinguishes viral genomes from cellular DNAs or RNAs (Tate and Peterson, 1974;Shen et al, 1979;Webster et al, 1981;Dotto et al, 1981). During encapsidation, directionality of Ff particles matters as phage particles are structurally asymmetric at their leading and trailing end (Wickner and Killick, 1977;Conners et al, 2023;Jia and Xiang, 2023). At the leading end, pVII and pIX protein complex interact with the PS hairpin of pV-coated phage ssDNA (Russel and Model, 1989;Russel, 1993;Haase et al, 2022).…”