To Be or Not To Be T4: Evidence of a Complex Evolutionary Pathway of Head Structure and Assembly in Giant Salmonella Virus SPN3US

Abstract

Giant Salmonella phage SPN3US has a 240-kb dsDNA genome and a large complex virion composed of many proteins for which the functions of most are undefined. We recently determined that SPN3US shares a core set of genes with related giant phages and sequenced and characterized 18 amber mutants to facilitate its use as a genetic model system. Notably, SPN3US and related giant phages contain a bolus of ejection proteins within their heads, including a multi-subunit virion RNA polymerase (vRNAP), that enter the host cell with the DNA during infection. In this study, we characterized the SPN3US virion using mass spectrometry to gain insight into its head composition and the features that its head shares with those of related giant phages and with T4 phage. SPN3US has only homologs to the T4 proteins critical for prohead shell formation, the portal and major capsid proteins, as well as to the major enzymes essential for head maturation, the prohead protease and large terminase subunit. Eight of ~50 SPN3US head proteins were found to undergo proteolytic processing at a cleavage motif by the prohead protease gp245. Gp245 undergoes auto-cleavage of its C-terminus, suggesting this is a conserved activation and/or maturation feature of related phage proteases. Analyses of essential head gene mutants showed that the five subunits of the vRNAP must be assembled for any subunit to be incorporated into the prohead, although the assembled vRNAP must then undergo subsequent major conformational rearrangements in the DNA packed capsid to allow ejection through the ~30 Å diameter tail tube for transcription from the injected DNA. In addition, ejection protein candidate gp243 was found to play a critical role in head assembly. Our analyses of the vRNAP and gp243 mutants highlighted an unexpected dichotomy in giant phage head maturation: while all analyzed giant phages have a homologous protease that processes major capsid and portal proteins, processing of ejection proteins is not always a stable/defining feature. Our identification in SPN3US, and related phages, of a diverged paralog to the prohead protease further hints toward a complicated evolutionary pathway for giant phage head structure and assembly.

Keywords: CTS (capsid targeting sequence); Salmonella; ejection protein; giant phage; mass spectrometry; myovirus; prohead protease; virion RNA polymerase (vRNAP).

Figure 1

SDS-PAGE gel of purified SPN3US virions. Individual gel slices that underwent mass spectrometric analyses are indicated by red lines. The most abundant SPN3US proteins identified in each gel slice are indicated with gene product (gp) numbers, with the most abundant protein in each slice, or gel band, listed first. Proteins that are components of the head structure are indicated (blue).

Figure 2

Proteolytic processing of SPN3US head proteins. (A) Sequence logo representing 9 cleavage sites confirmed by the identification of semi-tryptic peptides, (B) Peptide coverage of the major capsid protein, gp75 (C) Peptide coverage of the mature form of the portal protein, gp81 identified in slice 7 (Figure 1), and (D) peptide coverage of the cleaved N-terminal region of gp81 in slice 1 (Figure 1). Red arrow indicates maturation cleavage site, green arrow indicates inferred maturation cleavage site.

Figure 3

Proteolytic processing of T4 head proteins. (A) Sequence logo representing 13 cleavage sites in 9 head proteins confirmed by N-terminal sequencing (Black et al., 1994) and/or semi-tryptic peptides (this study). (B) Peptide coverage of the major capsid protein, gp23, (C) Peptide coverage of the portal protein, gp20, which is not processed, and (D). Peptide coverage of the capsid vertex protein, gp24. Red arrow indicates maturation cleavage site.

Figure 4

Mass spectral identification of SPN3US ejection protein candidate, gp47. (A) Peptide coverage of the precursor propeptide of gp47 identified in the wild-type phage detected in a low molecular mass SDS-PAGE gel slice (slice 1, Figure 1). Red arrow indicates the prohead protease cleavage site (AVE-79) identified via a semi-tryptic peptide; black square indicates newly identified start methionine, (B) Peptide coverage of the mature polypeptide of gp47 identified in the wild-type phage detected in a higher molecular mass SDS-PAGE gel slice (slice 5, Figure 1). Green arrow indicates expected protease maturation cleavage site at ALE-111; blue box indicates predicted transmembrane helix (TMH), and (C) Peptide coverage of gp47 in the mutant 47(am1) propagated under non-permissive conditions causing gp47 to be truncated at Q482 (red cross). Note the prohead protease cleavage site from (A) is only partially cleaved in this mutant as demonstrated by the identification of both a semi-tryptic peptide that illustrates processing and a tryptic peptide that illustrates no processing at the cleavage motif, 100% refers to the peptide identification probability.

Figure 5

Auto-proteolytic processing of the SPN3US prohead protease, gp245. (A) Peptide coverage of gp245 in purified virions (slice 2, Figure 1), (B) SDS-PAGE gel of gp245 expressed from the full length gene with an N-terminal 6-histidine tag, (C) Peptide coverage of recombinant gp245 with an N-terminal 6-Histidine tag, and (D) Peptide coverage of gp245 in purified virions of 218 that had been propagated under non-permissive conditions. Red arrow indicates the prohead protease cleavage site (AQE-203) identified by a semi-tryptic peptide.

Figure 6

Transmission electron microscopy of negatively stained SPN3US amber mutants (A) vRNAP minus mutant [218(am101)] and (B) ejection protein gp47 minus mutant [47(am1)] after propagation on nonpermissive S. enterica serovar Typhimurium (strain TT9079). Space bar represents 100 nm.

Figure 7

The SPN3US gene region flanking the gene encoding the essential head ejection protein gp47 and corresponding gene regions in related giant phages. Genes in the same gene location as SPN3US_0047 in related giant phages are enclosed in a red box and those shaded the same shade blue were determined to have sequence similarity by Psi-Blast. Other genes with homologs in related phages as determined by Psi-Blast searches are shaded green. SPN3US head or tail genes are indicated. Red arrowhead indicates processed by the prohead protease.

Figure 8

SDS-PAGE profile and morphology of SPN3US mutant 243(am114). (A) Transmission electron microscopy of 243(am114) after propagation on non-permissive S. enterica serovar Typhimurium (strain TT9079) and concentration by differential centrifugation. White arrows indicate disformed, DNA-empty capsid structure, black arrows indicate contracted tails, (B) SDS-PAGE gel showing profiles of wild-type (WT) SPN3US and amber mutant phages 243(am214) and 218(am101) indicated by their mutated gene names, 243 and 218, respectively, after propagation on the non-permissive strain of S. enterica serovar Typhimurium (TT9079). D, indicates particles concentrated by differential centrifugation; P, indicates particles purified by step and buoyant density CsCl ultracentrifugation; Blue arrows indicate the high abundance ejection proteins gp53 and gp54; Red arrows highlight the absence of the high abundance ejection proteins gp53 and gp54 in 243(am114).

Figure 9

Phylogenetic trees of phage head maturation enzymes in SPN3US and related giant phages. (A) Phylogentic tree of the large terminase subunit (TerL), and (B) phylogenetic tree of the prohead protease. T4 and the T4-related phage Syn9 proteins were used to root the trees. Phage hosts are indicated.

Figure 10

Comparison of proteolytic processing in homologous SPN3US, ϕKZ and 201ϕ2-1 proteins (A) Gene region encoding four essential head proteins including the vRNAP βN subunit in SPN3US and corresponding regions in ϕKZ and 201ϕ2-1. Red arrows indicate genes encoding virion proteins processed by the prohead protease, blue arrows indicate genes encoding virion proteins not processed by the prohead protease. Gray arrows indicate non-virion genes. Plot of total mass spectra detected in SDS-PAGE gel slices by mass spectrometry for the RNAP βN subunit of (B) SPN3US, (C) ϕKZ, and (D) 201ϕ2-1.