The M13 Phage Assembly Machine Has a Membrane-Spanning Oligomeric Ring Structure

Abstract

Bacteriophage M13 assembles its progeny particles in the inner membrane of the host. The major component of the assembly machine is G1p and together with G11p it generates an oligomeric structure with a pore-like inner cavity and an ATP hydrolysing domain. This allows the formation of the phage filament, which assembles multiple copies of the membrane-inserted major coat protein G8p around the extruding single-stranded circular DNA. The phage filament then passes through the G4p secretin that is localized in the outer membrane. Presumably, the inner membrane G1p/G11p and the outer G4p form a common complex. To unravel the structural details of the M13 assembly machine, we purified G1p from infected E. coli cells. The protein was overproduced together with G11p and solubilized from the membrane as a multimeric complex with a size of about 320 kDa. The complex revealed a pore-like structure with an outer diameter of about 12 nm, matching the dimensions of the outer membrane G4p secretin. The function of the M13 assembly machine for phage generation and secretion is discussed.

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

“…Other protein factors may also restrict or regulate the number of interactions in which viral nucleic acids and coat proteins are involved, such as in the formation of the “initiation complex” proposed previously (Feng et al, 1999). Once this checkpoint is satisfied, PSB15-phage DNA is released from the priming site by Trx, and phage DNA is loaded on the pI export complex (Haase et al, 2022). Finally, other coat proteins (pVII, pVIII and pIII/pVI) are added during egress to generate mature viral particles.…”

“…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). On the phage assembly site, pIV forms a large, gated channel within the outer membrane (OM), while pI and pXI form the IM component of an equimolar, multimeric trans-membrane complex (Ploss and Kuhn, 2010;Gibaud , 2021).…”

Structural mechanisms of Tad pilus assembly and its interaction with an RNA virus

“…This unique feature enhances adaptability and precision in phage display, especially in the later stages when proteins are displayed in lower quantities to improve the affinity between the displayed protein and the target. In contrast to the M13-based DNA phage display, which depends on the secretion of filamentous virions bearing displayed proteins through a specific channel in the E. coli cell envelope ( 29 ), thereby limiting the variety of displayable proteins ( 30 ), ssRNA phage capsid proteins, including Mat, directly assemble around the genomic RNA, eliminating the necessity for secretion to allow more flexible display of proteins. In addition, ssRNA phages can be produced in vitro ( 31 ), offering the possibility of cell-free display, which removes the limitations related to transformation efficiency in cell-based systems ( 32 ).…”

Cryo-electron microscopy of the f1 filamentous phage reveals a new paradigm in viral infection and assembly

“…CC-BY-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 4, 2022. ; https://doi.org/10.1101/2022.11.03.514279 doi: bioRxiv preprint pV targets the DNA-protein complex to the pI / pXI ring-shaped multimer in the cytoplasmic membrane 55 where the packaging signal hairpin is thought to act as a signal for replacement of pV by the pVII / pIX round cap (Fig. 6C).…”

“…pV targets the DNA-protein complex to the pI / pXI ring-shaped multimer in the cytoplasmic membrane 55 where the packaging signal hairpin is thought to act as a signal for replacement of pV by the pVII / pIX round cap (Fig. 6C).…”