Portal fusion protein constraints on function in DNA packaging of bacteriophage T4

Abstract

Architecturally conserved viral portal dodecamers are central to capsid assembly and DNA packaging. To examine bacteriophage T4 portal functions, we constructed, expressed and assembled portal gene 20 fusion proteins. C-terminally fused (gp20-GFP, gp20-HOC) and N-terminally fused (GFP-gp20 and HOC-gp20) portal fusion proteins assembled in vivo into active phage. Phage assembled C-terminal fusion proteins were inaccessible to trypsin whereas assembled N-terminal fusions were accessible to trypsin, consistent with locations inside and outside the capsid respectively. Both N- and C-terminal fusions required coassembly into portals with approximately 50% wild-type (WT) or near WT-sized 20am truncated portal proteins to yield active phage. Trypsin digestion of HOC-gp20 portal fusion phage showed comparable protection of the HOC and gp20 portions of the proteolysed HOC-gp20 fusion, suggesting both proteins occupy protected capsid positions, at both the portal and the proximal HOC capsid-binding sites. The external portal location of the HOC portion of the HOC-gp20 fusion phage was confirmed by anti-HOC immuno-gold labelling studies that showed a gold 'necklace' around the phage capsid portal. Analysis of HOC-gp20-containing proheads showed increased HOC protein protection from trypsin degradation only after prohead expansion, indicating incorporation of HOC-gp20 portal fusion protein to protective proximal HOC-binding sites following this maturation. These proheads also showed no DNA packaging defect in vitro as compared with WT. Retention of function of phage and prohead portals with bulky internal (C-terminal) and external (N-terminal) fusion protein extensions, particularly of apparently capsid tethered portals, challenges the portal rotation requirement of some hypothetical DNA packaging mechanisms.

Insights into the Structure and Assembly of the Bacteriophage ϕ29 Double-Stranded DNA Packaging Motor

“…These bases either are part of, or flank, the intermolecular pseudoknot. Additionally, the ability to simultaneously align both connectors and pRNAs when one is averaging over thousands of particles indicates that the relative orientations of these two components are fixed and that thus, the connector has a preferred orientation and is not free to rotate, in agreement with the findings of biochemical and single-particle experiments (51,52). If this were not the case, it would be impossible to simultaneously align both components unless different angular orientations were applied to subimage areas corresponding to connectors and pRNAs during image reconstruction (see reference 20 for further discussion).…”

Visualization of uncorrelated, tandem symmetry mismatches in the internal genome packaging apparatus of bacteriophage T7

“…The precession, if unidirectional, would produce less twist than would be produced by the previously proposed portal rotation that tracks the DNA double helix (34,39). Portal rotation is now known not to occur (40,41). Finally, the core stack precession could be a response to evolutionary selection against tangling of DNA as it was packaged.…”

Molecular architecture of tailed double-stranded DNA phages

“…In the early models of genome packaging, the DNA translocation was assumed to be coupled with the portal rotation. 59,61 However later single-molecule fluorescence spectroscopy of the φ29 portal 62 and studies of the T4 connector non-covalently cross-linked to the capsid shell 63 showed that the portal does not rotate during the DNA translocation.…”