Bacteriophage P2 head morphogenesis: Cleavage of the major capsid protein

Lengyel, Judith A.; Goldstein, Richard; Marsh, Margaret; Sunshine, Melvin G.; Calendar, Richard

doi:10.1016/0042-6822(73)90461-3

Cited by 95 publications

(62 citation statements)

References 39 publications

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“…In addition we now know that the formation of bacteriophage heads is coupled with the maturation of the virus chromosome; cutting of a mature length chromosome from the overlength replicating DNA does not happen in solution but only in the presence of a head precursor shell (4)(5)(6)(7)(8). Furthermore, in many viruses the proteins forming the head are proteolytically cleaved during the shell formation and DNA packaging steps (9)(10)(11)(12)(13)(14)(15)(16)(17).…”

Section: Introductionmentioning

confidence: 99%

P22 morphogenesis I: Catalytic scaffolding protein in capsid assembly

1974

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About 250 molecules of the 42,000 molecular weight gene 8 product catalyze the polymerization of the major phage coat protein into a precursor shell temporarily containing both proteins. The resulting prohead appears to be a shell structure with the P8, or scaffolding protein, on the inside, and the coat protein on the outside. In concert with DNA condensation inside the shell, all 250 scaffolding molecules exit from the prohead, without proteolytic cleavage. These molecules then recycle and catalyze the formation of more proheads from newly synthesized coat protein. Such proteins, which catalyze assembly by temporarily associating with an intermediate stage, may represent a general mechanism of macromolecular assembly.

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Section: Introductionmentioning

confidence: 99%

P22 morphogenesis I: Catalytic scaffolding protein in capsid assembly

1974

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“…The results suggest that the DNA packaged in tailless P2 capsids is arranged in a way that leads to the formation of a complex knot when the ends join. In an intact phage particle, the anchoring of one terminus of the DNA to the head-proximal end of the tail [ [11][12][13][14][15][16][17][18][19][20][21][22] presumably diminishes or prevents this kind ofjoining. The novel knotted DNA can be used to assay type II DNA topoisomerases that break and rejoin DNA in a double-stranded fashion.…”

mentioning

confidence: 99%

Knotted DNA from bacteriophage capsids.

Liu¹,

Perkocha²,

Calendar³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

115

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The majority of the DNA prepared from tailless capsids of bacteriophage P2 by the phenol extraction procedure consists of monomeric rings that have their cohesive ends joined. Electron microscopic and ultracentrifugal studies indicate that these molecules have a complex structure that is topologically knotted; they have a more compact appearance and a higher sedimentation coefficient when compared with regular nicked P2 DNA rings. Linearization of these rings by thermal dissociation or repair of the cohesive ends by DNA polymerase I in the presence ofall four deoxynucleoside triphosphates gives molecules that are indistinguishable from normal P2 DNA that has been similarly treated. The knotted nature of the majority of P2 head DNA is further supported by analyzing the products when these molecules are treated with ligase and the ligase-treated molecules are subsequently nicked randomly with DNase I. The data are consistent with the notion that, if such a molecule is first converted to a form that contains only one single-chain scission per molecule, strand separation gives a linear strand and a highly knotted singlestranded ring. The results suggest that the DNA packaged in tailless P2 capsids is arranged in a way that leads to the formation of a complex knot when the ends join. In an intact phage particle, the anchoring of one terminus of the DNA to the head-proximal end of the tail [Chattoraj, D. K. & Inman, R. B. (1974) J. MoL BioL 87, 11-22] presumably diminishes or prevents this kind ofjoining. The novel knotted DNA can be used to assay type II DNA topoisomerases that break and rejoin DNA in a double-stranded fashion.Mature P2 phage DNA consists of double-stranded molecules 33 kilobases long (1, 2) with 19-base cohesive ends (3, 4) that allow the formation of circular and concatemeric molecules in solution (1, 2). P2 phage preparations typically contain equal proportions of viable phage particles and tailless DNA-containing capsids (heads) (5). The two cohesive ends of the P2 DNA in the mature phage are not hydrogen bonded and are asymmetrically oriented with the left end anchored on the headproximal end ofthe tail (6). When DNA is extracted from a concentrated preparation of mature P2 phage, a viscous solution is obtained, due to the formation of hydrogen-bonded concatemers via pairing of the cohesive ends (1). We observed, however, that DNA extracted from concentrated P2 heads is much less viscous than the corresponding DNA from mature phage particles. In this communication, we present evidence that the DNA from tailless capsids (henceforth referred to as head DNA) represents a novel class of topological isomers of hydrogenbonded monomeric circular P2 DNA. The apparent low viscosity ofhead DNA relative to that of DNA from mature phages (henceforth referred to as phage DNA) is due to the presence of complex topological knots. MATERIALS AND METHODSBacteria and Bacteriophages. Escherichia coli C strains Cla (7) and C-520 (8), carrying the amber suppressor supD, were used as hosts to grow stocks of p...

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“…Induction of sid resulted in a 500-fold decrease in helper P2 PFUs (compare Fig. 3 A (9,33). Although a processed form of the N gp accounts for the major subunit of both the P2-and P4-sized capsid, the 0 gp is not found as a structural component within either type of capsid (33,34 particles/ml).…”

Section: Methodsmentioning

confidence: 99%

“…3 A (9,33). Although a processed form of the N gp accounts for the major subunit of both the P2-and P4-sized capsid, the 0 gp is not found as a structural component within either type of capsid (33,34 particles/ml). This increase in lysate titer correlated quantitatively with the appearance of small P4-sized capsid phage particles as visualized by EM.…”

Section: Methodsmentioning

confidence: 99%

Regulation of icosahedral virion capsid size by the in vivo activity of a cloned gene product.

Agarwal

Arthur

Arbeit

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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Determination of icosahedral virion capsid size can be directly studied during helper-dependent lytic development of satellite P4 because the assembly pathway specified by the P2 helper virus is altered to yield smaller-sized capsids. Size determination (sid) mutations identify a P4-encoded function regulating this process. To determine whether the sid gene product is necessary and sufficient to redirect the assembly pathway, we (i) cloned the sid structural gene in a plasmid vector (pMA30) under the control of an inducible promoter and (i) constructed a packaging substrate (pMA1), a P4 genome-sized plasmid containing only that region of P4, the cos site, necessary for encapsidation. Superinfection by P2 of a host carrying pMA30 under induced conditions resulted in a shift from large to small capsid production. P2 superinfection of a host carrying the cos plasmid pMA1 plus pMA30 under induced conditions yielded pMAl-transducing particles of P4 capsid size. These cloning-based analyses directly demonstrate that sid protein is the only P4 gene product required for small-capsid size determination. In the absence of the P2 0 gene product no capsids of any size are assembled during solo infection by P2. Nevertheless, P2 Oam mutant superinfection of a host carrying pMA1 and pMA30 under induced conditions yielded small P4-sized transducing particles. We therefore propose that (i) the sid gene product competes with the 0 gene product to determine the assembly of small vs. large capsid sizes and (U) both gene products probably function as temporary scaffolding proteins.Regular polyhedra composed of identical subunits were proposed by Crick and Watson (1) as the basic capsid structure of simple spherical viruses. To account for both the 5-and 6-fold clustering of such subunits necessary to the formation of icosahedral shells, Casper and Klug (2) conceived of a related model involving conformational flexibility of the identical subunit proteins based on the "quasi-equivalence" of their capsid shell environments. Such flexibility proved to be not an intrinsic property but to result from specific interactions with virally encoded scaffolding proteins (3-5).Thus, rather than the simple "self-assembly" of identical subunits, in vivo construction of an icosahedral virion shell typically involves a pathway ofordered steps that provide the perturbations necessary to induce the quasi-equivalence of the capsid subunits. A striking example of this phenomenon occurs during the lytic development of satellite virus P4. Here, the assembly pathway normally followed by a helper virus-encoded major subunit capsid protein is perturbed by the activity of a P4-encoded gene product (gp). The result of this interference is a distinct class of smaller-sized functional icosahedral capsids appropriate for encapsidation of the P4 genome (6). This report focuses on the gps that shift the helper virus-encoded capsid subunits into alternate assembly pathways.Although originally described as a satellite virus (7), P4 virl is a naturally occurri...

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Bacteriophage P2 head morphogenesis: Cleavage of the major capsid protein

Cited by 95 publications

References 39 publications

P22 morphogenesis I: Catalytic scaffolding protein in capsid assembly

P22 morphogenesis I: Catalytic scaffolding protein in capsid assembly

Knotted DNA from bacteriophage capsids.

Regulation of icosahedral virion capsid size by the in vivo activity of a cloned gene product.

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