Initial location of the RNA-dependent RNA polymerase in the bacteriophage Phi6 procapsid determined by cryo-electron microscopy.
The RNA-dependent RNA polymerases (RdRPs) of Cystoviridae bacteriophages, like those of eukaryotic viruses of the Reoviridae, function inside the inner capsid shell in both replication and transcription. In bacteriophage Phi6, this inner shell is first assembled as an icosahedral procapsid with recessed 5-fold vertices that subsequently undergoes major structural changes during maturation. The tripartite genome is packaged as single-stranded RNA molecules via channels on the 5-fold vertices, and transcripts probably exit the mature capsid by the same route. The RdRP (protein P2) is assembled within the procapsid, and it was thought that it should be located on the 5-fold axes near the RNA entry and exit channels. To determine the initial location of the RdRP inside the procapsid of bacteriophage Phi6, we performed cryo-electron microscopy of wild type and mutant procapsids and complemented these data with biochemical determinations of copy numbers. We observe ring-like densities on the 3-fold axes that are strong in a mutant that has approximately 10 copies of P2 per particle; faint in wild type, reflecting the lower copy number of approximately 3; and completely absent in a P2-null mutant. The dimensions and shapes of these densities match those of the known crystal structure of the P2 monomer. We propose that, during maturation, the P2 molecules rotate to occupy positions closer to adjacent 5-fold vertices where they conduct replication and transcription.
Sen
Anindito
A
Laboratory of Structural Biology Research, NIAMS, National Institutes of Health, Bethesda, Maryland 20892, USA.
Heymann
J Bernard
JB
Cheng
Naiqian
N
Qiao
Jian
J
Mindich
Leonard
L
Steven
Alasdair C
AC
D017126:Bacteriophage phi 6 / Q000201:enzymology* / Q000648:ultrastructure*; D002213:Capsid / Q000201:enzymology* / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D008958:Models, Molecular; D012324:RNA-Dependent RNA Polymerase / Q000648:ultrastructure*; D014764:Viral Proteins / Q000737:chemistry / Q000648:ultrastructure; D019065:Virus Assembly
D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase