Identification of the virion proteins of Cp-1 by SDS-PAGE and mass spectrometry. (A) Size marker (5- to 250-kDa prestained protein ladder; NEB). The numbers to the left of the gels indicate molecular masses in kilodaltons. (B) Migration of the structural Cp-1 proteins on a 4 to 15% precast gradient SDS-polyacrylamide gel. The arrows indicate the bands extracted for LC-MS-MS analysis. (C) Identification and characterization of the structural Cp-1 proteins detected by mass spectrometry. The predicted (pred.) and observed (obs.) molecular (Mol.) masses of the proteins are indicated.

The genomic organization of protein interactions reveals a modular organization of the Cp-1 interactome. The gene order within the Cp-1 genome is plotted against the protein-protein interactions (PPIs). Detected PPIs are indicated by black boxes, and operon borders are indicated by dashed lines. The longest transcriptional units that have been experimentally validated were considered and are indicated below the matrix (11). Expression timing of the transcripts is indicated as follows: e, early; l, late; u, unknown. The generalized function of the operons is given. orfA to -F are localized on the reverse genome strand. For clarification of the matrix, all ORFs have been illustrated in a sequential order. Because of the differences between the previous published genome annotation (11) and the current GenBank annotation, the orf and protein nomenclature may differ in certain cases (e.g., orf14 corresponds to the p15 gene product; see NCBI reference sequence file NC_001825.1). A list of ORF and protein identifications (IDs) is provided in Table S1 in the supplemental material.

A virion model of Cp-1. (A) The 12 Cp-1 gene products that were identified as structural proteins by LC-MS-MS are shown in red, while the proteins in blue undergo binary protein interactions with structural proteins. Gene products shown in blue were not identified by MS. The rectangle in the first column highlights gene products encoded by the structural operon. Protein annotations were adopted from Martin et al. (10–12). (B) Electron micrograph of a mature Cp-1 virion particle. A lateral view (top image) and top down view (bottom image) are shown. (C) A schematic model of the Cp-1 virion. Proteins are indicated by symbols or by the three-dimensional (3D) structure of φ29 homologs. PDB entries are given in Table S1 in the supplemental material. The numbers are the gene product number. The left section indicates interactions that presumably occur during prohead formation; the right section illustrates additional interactions that may play a role in the mature virion collar and tail. PPIs that were not detected in the Y2H screens but that are expected to take place in vivo are indicated by dashed arrows. pRNA, packaging RNA.