Packaging accessory protein P7 and polymerase P2 have mutually occluding binding sites inside the bacteriophage 6 procapsid.
Bacteriophage 6 is a double-stranded RNA (dsRNA) virus whose genome is packaged sequentially as three single-stranded RNA (ssRNA) segments into an icosahedral procapsid which serves as a compartment for genome replication and transcription. The procapsid shell consists of 60 copies each of P1(A) and P1(B), two nonequivalent conformers of the P1 protein. Hexamers of the packaging ATPase P4 are mounted over the 5-fold vertices, and monomers of the RNA-dependent RNA polymerase (P2) attach to the inner surface, near the 3-fold axes. A fourth protein, P7, is needed for packaging and also promotes assembly. We used cryo-electron microscopy to localize P7 by difference mapping of procapsids with different protein compositions. We found that P7 resides on the interior surface of the P1 shell and appears to be monomeric. Its binding sites are arranged around the 3-fold axes, straddling the interface between two P1(A) subunits. Thus, P7 may promote assembly by stabilizing an initiation complex. Only about 20% of the 60 P7 binding sites were occupied in our preparations. P7 density overlaps P2 density similarly mapped, implying mutual occlusion. The known structure of the 12 homolog fits snugly into the P7 density. Both termini-which have been implicated in RNA binding-are oriented toward the adjacent 5-fold vertex, the entry pathway of ssRNA segments. Thus, P7 may promote packaging either by interacting directly with incoming RNA or by modulating the structure of the translocation pore.
Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Daniel
D
Nemecek
Jian
J
Qiao
Leonard
L
Mindich
Alasdair C
AC
Steven
J Bernard
JB
Heymann
D017126:Bacteriophage phi 6 / Q000502:physiology* / Q000648:ultrastructure; D001665:Binding Sites; D020285:Cryoelectron Microscopy; D046911:Macromolecular Substances / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D019065:Virus Assembly; D014779:Virus Replication
D046911:Macromolecular Substances; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins