The Maturation of Bacteriophage ɸXl74: the Isolation and Characterization of Subviral Particles

Citation

Rohwer, Robert George (1975) The Maturation of Bacteriophage ɸXl74: the Isolation and Characterization of Subviral Particles. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/csff-mr85. https://resolver.caltech.edu/CaltechTHESIS:11052021-183302539

Abstract

A search was conducted for possible intermediates in the assembly of bacteriophage ɸXl74. Artificial lysates of ɸXl74 infected cells labeled with either amino acids or nucleotides were fractionated by velocity sedimentation and the viral DNAs and infection specific proteins analysed by recentrifugations and electrophoreses. Infection specific proteins were detected by comparing the incorporation patterns of differentially labeled infected and uninfected cells. Many experiments employed ³H, ¹⁴C and ³²P labels simultaneously and an exact solution to the discriminator ratios equations for three channel scintillation counting was derived to process these data.

A major proportion of the phage structural proteins that had not yet been incorporated into phage was observed in a very unstable 111S particle composed of ɸX proteins F, G and D all three of which are also required for ɸX SS DNA synthesis. The 111S particle does not appear to contain DNA. The D protein, present in large proportional amounts, is not found in whole virions. Depending upon the storage buffer, the 111S particle degrades into 12S (F,G protein), 9S (G protein) and 2.5S (D protein) subunits. These same particles are also observed in whole lysates. However, conditions can be found for which the 9S and 12S particles are not present suggesting that they may be 111S particle decomposition artifacts as opposed to in vivo particles. The properties of the 111S particle are those expected of a procapsid although this role in the infection has not been demonstrated. Analysis of the proteins in the 70S region of the gradient revealed that a similar D protein containing particle (distinct from the 70S lysis artifact) sediments there.

Approximately 10% of the total ɸX replicative intermediate DNA in these gradients sedimented in the 70S - 114S regions. After deproteinization this DNA resedimented at the slower velocities expected for free replicative intermediates, indicating that it may have been associated with ɸX proteins. This associated protein moiety is obscured by the much more extensively labeled pools of whole virus, 1118 particle and the 70S lysis artifact. It could, however, be the same as the 111S particle.

A radioimmunoassay was developed for ɸX viral proteins and was employed to show that replicating intermediates in SS DNA synthesis (sedimenting from 20 - 30S), but not the "resting" RF II form DNA, are tightly complexed with a ɸX viral antigen. A major peak (the 20S particle) of infection specific protein also sediments in this region. However any association of the DNA replicative intermediates with this protein peak is too fragile to account for the serum binding result. The concentration of DNA-binding antigen is below the resolving power of the amino acid labeling technique.

The infection specific 20S protein peak contains ɸX gene F protein, lesser amounts of other infection-specific components and a host protein (the major constituent); the host protein is also observed to sediment at 208 in uninfected cells. This protein has a molecular weight of about 67,000 daltons and is one of three ɸX stimulated host proteins with molecular weights greater than that of the largest ɸX coded protein, A'.

The ɸX174 literature is reviewed with an emphasis on those observations that bear upon the ɸX174 assembly process. A model of the ɸX maturation process incorporating the major features of our present knowledge is proposed as a working hypothesis for future study.

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