R H Nøkling | PubMed Health

PubMed2000

In vitro assembly of bacteriophage P4 procapsids from purified capsid and scaffolding proteins.

Bacteriophage P4 is a satellite virus of bacteriophage P2, which has acquired the ability to utilize the structural gene products of P2 to assemble its own capsid. The normal P2 capsid has a T = 7 icosahedral structure comprised of the gpN-derived capsid protein, whereas the capsid produced under the control of P4 has a smaller, T = 4 structure. The protein responsible for this size determination is the P4-coded gene product Sid, which forms an external scaffold on the P4 procapsid. Using an in vitro assembly system, we show that gpN and Sid can coassemble into procapsid-like particles, indistinguishable from those produced in vivo, in the absence of any other gene products. The fidelity of the assembly reaction is enhanced by the inclusion of PEG and has a pH optimum between 8.0 and 8.5. Analysis of the assembly properties of truncated versions of Sid and gpN suggests that the amino-terminal part of Sid is involved in gpN binding, while the carboxyl-terminal part forms trimeric Sid-Sid interactions, and that the first 31 amino acids of gpN are required for binding to Sid as well as for size determination.

Bacteriophages↗

PubMed1995

The capsid size-determining protein Sid forms an external scaffold on phage P4 procapsids.

Although the phages P2 and P4 build their capsids from the same precursor, the product of the P2 N gene, the two capsids differ in size: P2 builds a 60 nm, T = 7 capsid from 420 subunits, whereas P4 makes a 45 nm, T = 4 capsid from 240 subunits. This difference leads to substantial changes in shell geometry and subunit interactions. Previous results have demonstrated that the P4 sid gene is responsible for the assembly of P4-sized shells. We have used cryo-electron microscopy and image reconstruction to determine the structure of a putative assembly intermediate of P4 capsids, produced in vivo from cloned genes. We demonstrate that Sid forms a P4-specific scaffold with icosahedral symmetry on the outside of the procapsid-like particles. The Sid molecules (60 or 120 copies) form lofty arches that interact with the gpN hexamers on the icosahedral 2-fold axes, and connect as trimers over the 3-fold axes, forming a continuous dodecahedrally shaped outer cage. The gpN shell inside the Sid cage is approximately 40 nm wide, consistent with the previously suggested maturational expansion. The main difference with respect to the mature P4 capsids is found in the hexamers, which appear strongly elongated and more protruding than in the mature shell. These and previous results are discussed in the light of a model for regulation of capsid size.

Amino Acid Sequence↗