PLE encodes an inhibitory protein, TcaP, which modifies ICP1’s capsid assembly process to produce small capsids

A-B) Representative transmission electron micrographs (TEMs) from 3 independent biological replicates show A) PLE virions have small, ∼50 nm wide capsids and long contractile tails while B) ICP1 virions have large, ∼80 nm wide capsids and long contractile tails. Scale bars are 200 nm.

C-D) Representative TEMs of lysates produced from ICP1 infection of V. cholerae expressing C) TcaP (ptcaP) or D) an empty vector (pEV). Arrowheads show exemplary capsids and their sizes according to the legend. Scale bars are 200 nm.

E) Efficiency of plaquing of wild type ICP1 or escape phages harboring the substitution indicated in the coat protein on V. cholerae expressing TcaP relative to an empty vector control. Each dot represents a biological replicate, bars represent the mean, and error bars show the standard deviation. The dotted line indicates an efficiency of plaquing of 1 where the expression of TcaP is not inhibitory to plaque formation. Figure 1 - Source data 1. This spreadsheet contains the data used to create Figure 1E.

TcaP is the only PLE-encoded factor necessary for directing small capsid assembly, which is required for efficient PLE transduction

A-C) Representative TEMs from 3 independent biological replicates of lysate from ICP1-infected strains of V. cholerae with wild type PLE or PLEΔtcaP, as indicated, carrying either the empty vector (EV) or a vector expressing TcaP (tcaP). Insets are enlarged regions of the images highlighting representative particles, and arrowheads indicate capsid types and sizes, as described in the legend. Scale bars are 100 nm.

D) Quantification of PLE genome transduction for the strain indicated represented as the transduction efficiency relative to wild type PLE with an empty vector (pEV). Each dot represents a biological replicate, bars represent the mean, and error bars show standard deviation. The dotted line indicates an efficiency of 100%.

Figure 2 - Source data 1. This spreadsheet contains the data used to create Figure 2D.

The size of procapsid-like-particles is determined by ICP1 and PLE scaffolds

Representative TEMs and Coomassie stained SDS-PAGE analyses of resulting affinity-purified procapsid-like-particles (PLPs) produced in the heterologous assembly platform in E. coli from 2-3 independent biological replicates following expression of A) coatwild type or B) coatR223H proteins encoded on plasmids as shown in the diagrams in the central panel (numbered 1-5). ICP1-encoded genes are shown in red and PLE-encoded genes are shown in blue. Bent arrow icons indicate Ptac promoters. 6xHis represents the tag fused to the C-terminus of the coat. Protein standards are indicated by black tick marks and a subset are marked by their sizes in kDa as indicated (standard range: 250, 150, 100, 75, 50, 37, 35, 20, 15, 10 kDa). Protein bands of interest are indicated by colored tick marks and labels (see legend for calculated molecular weights and accession numbers of these proteins, complete gene and protein information is provided in Key Resources Table). In the absence of protease inhibitors, ICP1’s scaffold appears to be cleaved and the resulting cleavage products are indicated by scaffold*. Protease inhibitors were included in all coatR223H purifications. TEM insets are enlarged sections of the images highlighting representative particles and arrowheads indicate capsid types and sizes, as described in the legend. Scale bars are 200 nm.

Cryo-EM reveals TcaP is an external scaffold

A-B) Representative micrographs from 3 independent biological replicates from A) transmission electron and B) cryo-electron microscopy of PLE PLPs produced from co-expression of ICP1’s cot and PLE’s TcaP. Scale bars are 50 nm.

C) Isosurface reconstruction of PLE PLPs, resolved to 3.4 Å, colored radially (Å) as indicated on the legend.

D) Ribbon model of the solved structure for eight coat proteins, two from the adjacent pentamers (coatA tan) and six in a hexamer (coatB coral, coatC salmon, and coatD pink), and two partial TcaP proteins (TcaPA blue and TcaPB teal). Substituted residues in coat that escape TcaP-mediated remodeling are shown as balls and sticks (magenta for R223 and red for E234) and lie along the region of the hexamer where TcaP binds. Side chains that could not be fully resolved are modeled as alanines.

E) Details of the interactions between TcaP and the coat subunits. Residues in TcaP that contact coat are shown as sticks. For TcaP, the numbers, colored according to their chain, indicate the first and last residue within the region of contact. Distance measurements between residues are shown as dashed lines and measured in Å. Side chains that could not be fully resolved are modeled as alanines.

F) Ribbon model of the solved structure of the partial TcaP dimer, oriented as in panel D and G.

E) G) Electrostatic potential surface representation of the coat with a ribbon diagram of TcaP show the negative pocket on coatC that is filled by the positively charged arginine from TcaP. Electrostatic potential is calculated using APBS and the coloring is from –5 kT/e (red) to +5 kT/e (blue).

Genetic analysis of tcaP alleles from 10 PLEs reveals PLE5’s nonfunctional TcaP variant

A) Gene graphs of tcaP and its two neighboring genes, from the 10 known unique PLEs. PLE5’s tcaP, the shortest allele, is shown in light blue, while the other, full-length tcaP alleles are shown in dark blue, and the neighboring genes are shown in gray. Lower scale bar is in nucleotides. Boxes outline regions aligned in (B).

B) Alignment of the region encoding tcaP and their translated products from PLE1 and PLE5 from the boxed regions in Panel A. Nonidentical nucleotides and amino acids are shown in red on the PLE5 sequence, gaps are shown as dashes on either sequence, and stop codons are shown as asterisks. The gray box indicates an in-frame deletion. The blue boxes indicate the notable features of the tcaPPLE5sequence: the 14-nucleotide deletion that results in the frameshift, the resulting early stop codon, and the ATG and M of the alternative, originally annotated start site, which restores the original reading frame.

C-D) Representative TEMs from 2-3 independent biological replicates of lysate from ICP1-infected strains of PLE5(+) V. cholerae C) with an empty vector (pEV), or D) expressing tcaP from PLE1 (ptcaPPLE1). Scale bars are 100 nm. Arrowheads show capsids and their sizes according to the legend.

E) Efficiency of ICP1 plaquing on V. cholerae expressing tcaP from PLE1 (ptcaPPLE1) or tcaP from PLE5 (ptcaPPLE5) (from the originally annotated start site producing the truncated allele) compared to an empty vector (pEV). Each dot represents a biological replicate, bars represent the mean, and error bars show the standard deviation. The dotted line indicates an efficiency of plaquing of 1, where the expression of TcaP is not inhibitory to plaque formation.

F) Transduction efficiency of the strain indicated relative to PLE1 with an empty vector. Each dot represents a biological replicate, bars represent the mean, and error bars show standard deviation. The dotted line indicates an efficiency of 100%.

G) Alignment of the first nucleotides of the tcaP alleles from the PLE5 variants encoding the “ancestral” (anc) sequences from before 1991, from 2016, or from 2017. The light blue box highlights the 14-nucleotide insertion in the PLE5 sequence from 2017.

Figure 5 - Source data 1. This spreadsheet contains the data used to create Figure 5E.

Figure 5 - Source data 2. This spreadsheet contains the data used to create Figure 5F.

Model of TcaP-mediated small capsid assembly

A) A cartoon model of ICP1 infecting a PLE(+) V. cholerae cell. Injection of ICP1’s genome triggers activation of PLE, then TcaP directs the assembly of coat proteins into small capsids, inhibiting the formation of large capsids. PLEs genome is then packaged into the small capsids, a process that likely triggers the removal of the external scaffold, TcaP. PLE virions are released from the cell. ICP1 components are red, PLE components are blue, and V. cholerae components are grey.

B) A model showing the impact of capsid size on genome packaging. The replicated ICP1 and PLE genomes form concatemers, from one pac site (indicated as a vertical line) to the next. Headful packaging results in ∼105-110% of the genome within the capsid. The length of the genome packaged is indicated by the small arrows for T=4 capsids and longer arrows for T=13 capsids. ICP1’s genome is partially packaged into a T=4 capsid and several copies of PLE’s genome are packaged into a T=13 capsid. ICP1’s genome is red and PLE’s is blue.

Escape mutation information

Mass spectrometry results of ICP1 PLPs

Mass spectrometry results of PLE PLPs

Cryo EM data collection parameters

Reconstruction model parameters