The Beauty of Bacteriophage T4 Research: Lindsay W. Black and the T4 Head Assembly

Abstract

Viruses are biochemically complex structures and mainly consist of folded proteins that contain nucleic acids. Bacteriophage T4 is one of most prominent examples, having a tail structure that contracts during the infection process. Intracellular phage multiplication leads to separate self-directed assembly reactions of proheads, tails and tail fibers. The proheads are packaged with concatemeric DNA produced by tandem replication reactions of the parental DNA molecule. Once DNA packaging is completed, the head is joined with the tail and six long fibers are attached. The mature particles are then released from the cell via lysis, another tightly regulated process. These processes have been studied in molecular detail leading to a fascinating view of the protein-folding dynamics that direct the structural interplay of assembled complexes. Lindsay W. Black dedicated his career to identifying and defining the molecular events required to form the T4 virion. He leaves us with rich insights into the astonishingly precise molecular clockwork that co-ordinates all of the players in T4 assembly, both viral and cellular. Here, we summarize Lindsay's key research contributions that are certain to stimulate our future science for many years to come.

Keywords: DNA packaging; T4 bacteriophage; giant phage; head assembly; internal proteins.

Figure 1

Overview of the major steps in T4 phage head assembly. (A,B) Assembly initiates via the formation of a protein core anchored to the E. coli inner membrane via the portal, and around which the major capsid protein concomitantly assembles. Only in the absence of gp23 can naked cores be observed. (A) Electron microscopy of proheads produced by 21- mutants in vivo (upper, thin section) and in vitro (lower). White arrow indicates a central hole in proheads assembled in the absence of the prohead protease (adapted from van Driel, Traub and Showe [6]). (C,D) Proteolytic maturation involves cleavage and removal of scaffold/core proteins as well as the propeptides of the internal and shell proteins, release of the prohead from the inner membrane and semi-expansion of the shell. (E,F) Packaging of the genome into the prohead occurs via the action of the main packaging proteins, TerS (gp16) and TerL (gp17). (C,E). Electron microscopy of thin sections of wild-type T4-infected E. coli (reproduced from Black and Thomas [7]). (F) Scheme of how DNA packaging in vivo is integrated with late transcription, and DNA replication (reproduced from Black and Peng [8]). (G) Recombinant Δhoc phage particles after immuno-gold labelling with an anti-Hoc antibody (inset WT phage particle). The visualization of the “gold necklace” provided evidence that the portal structure contained fusion proteins (gp20-Hoc). Confirming the recombinant phenotype was important, as these particles had a central role in refuting the rotary portal packaging model (reproduced from Baumann, Mullaney and Black [9]). (H) Cryo-electron micrograph of a T4 Alt mutant imaged after the eighth exposure of a dose series of 16.5 el/Ų per exposure). The bubbles are generated from the internal proteins, which are inferred to be randomly positioned within the DNA, but excluded from a zone of about 100–110 Å directly under the outer shell (reproduced from Wu et al. [10]).

Figure 2

Packaging of concatemeric DNA into the T4 head. One end of the DNA is held at the gp20 portal, whereas the free strand is funneled through the portal as a loop structure (A) by sequential movements of the TerL subunits and hydrolysis of ATP (B).

Figure 3

Proteolytic cleavage of head proteins by the prohead proteases of T4 and giant phages. Mass spectral protein sequence coverage of T4 head proteins (A) gp23, (B) gp24 and (C) Alt, with their respective gp21 cleavage sites indicated by red arrows. Giant phages ϕKZ and SPN3US have diverged homologies to T4 gp21 and also cleave their head proteins C-terminal to a glutamyl residue after a short sequence motif. Protein sequence logos of the regions flanking the prohead protease cleavage sites in head proteins in (D) T4, (E) ϕKZ and (F) SPN3US. Sequence logos were created using WebLogo (https://weblogo.berkeley.edu/logo.cgi, accessed on 12 February 2022).

Figure 4

Delivery of β-galactosidase into E. coli via the T4 expression-packaging-processing system. (A) Scheme of the incorporation of β-galactosidase produced by an expression plasmid into the T4 head via its fusion to the T4 internal protein IPIII and (B) subsequent infection of the progeny “blue”-T4Δ phage and ejection of β-galactosidase into an infected cell. The T4Δ genotype is Δe-ΔIPIII-ΔIPII-alt−s12 [32,52]. Light microscopy of E. coli cells infected with (C) T4Δ, (D) T4Δ and “blue”-T4Δ and (E) “blue”-T4Δ after incubation with X-Gal. (B–E) adapted from Hong and Black (1993).

Figure 5

The first mutant of Salmonella phage SPN3US, 47(am1)—isolated by Lindsay Black—has an amber mutation in a low copy number head ejection protein gp47. (A) Transmission electron micrograph of 47(am1) grown under non-permissive conditions shows particles with an apparent wild-type phenotype, but are non-viable (reversion rate < 1 × 10⁻⁵). Space bar represents 100 nm. (B) The gp47 gene is located in cluster of head genes conserved in related giant phages, including Erwinia phage PhiEaH2, Cronobacter phage CR5 and Pseudomonas phages ϕKZ, 201ϕ2-1 and ϕPA3. Red arrowhead indicates a gene product undergoes cleavage by the prohead protease. Images adapted from Ali et al. [54].

Figure 6

Proteolytic cleavage of head proteins by the prohead protease is essential for head maturation in Salmonella phage SPN3US. (A) Scheme of the SPN3US prohead protease gp245 showing the locations of catalytic residues. (B) Cross-plating of SPN3US protease mutants spotted on a lawn of a non-permissive strain seeded with mutant am59, showing complementation (intragenic recombination) with am66 and am255. (C–E) Electron microscopy of thin sections of a non-permissive strain of Salmonella infected with (C) the wild-type phage at 10 min post-infection and (D,E) am59 at 90 min post-infection. Examples of proheads attached to the host inner membrane are indicated with white arrows. (E) In the absence of proteolysis, proheads were observed still attached to the inner membrane after cell lysis (black arrow), which was delayed relative to normal. Space bar represents 200 nm (C,D) and (E) 1 µm.