DNA packaging intermediates of bacteriophage φX174
Like many viruses, bacteriophage phi X174 packages its DNA genome into a procapsid that is assembled from structural intermediates and scaffolding proteins. The procapsid contains the structural proteins F, G and H, as well as the scaffolding proteins B and D. Provirions are formed by packaging of DNA together with the small internal J proteins, while losing at least some of the B scaffolding proteins. Eventually, loss of the D scaffolding proteins and the remaining B proteins leads to the formation of mature virions.



phi X174 108S 'procapsids' have been purified in milligram quantities by removing 114S (mature virion) and 70S (abortive capsid) particles from crude lysates by differential precipitation with polyethylene glycol. 132S 'provirions' were purified on sucrose gradients in the presence of EDTA. Cryo-electron microscopy (cryo-EM) was used to obtain reconstructions of procapsids and provirions. Although these are very similar to each other, their structures differ greatly from that of the virion. The F and G proteins, whose atomic structures in virions were previously determined from X-ray crystallography, were fitted into the cryo-EM reconstructions. This showed that the pentamer of G proteins on each five-fold vertex changes its conformation only slightly during DNA packaging and maturation, whereas major tertiary and quaternary structural changes occur in the F protein. The procapsids and provirions were found to contain 120 copies of the D protein arranged as tetramers on the two-fold axes. DNA might enter procapsids through one of the 30 A diameter holes on the icosahedral three-fold axes.



Combining cryo-EM image reconstruction and X-ray crystallography has revealed the major conformational changes that can occur in viral assembly. The function of the scaffolding proteins may be, in part, to support weak interactions between the structural proteins in the procapsids and to cover surfaces that are subsequently required for subunit-subunit interaction in the virion. The structures presented here are, therefore, analogous to chaperone proteins complexed with folding intermediates of a substrate.
Ilag
Leodevico L
LL
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
Olson
Norman H
NH
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
Dokland
Terje
T
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
Music
Cynthia L
CL
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
Cheng
R Holland
RH
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
Bowen
Zorina
Z
Department of Microbiology and Immunology, Center for the Health Sciences, University of Tennessee, 858 Madison Avenue, Memphis, TN 38163, USA.
McKenna
Robert
R
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
Rossmann
Michael G
MG
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
Baker
Timothy S
TS
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
Incardona
Nino L
NL
Department of Microbiology and Immunology, Center for the Health Sciences, University of Tennessee, 858 Madison Avenue, Memphis, TN 38163, USA.
D010584:Bacteriophage phi X 174 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism; D002213:Capsid / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D018360:Crystallography, X-Ray; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D009024:Morphogenesis; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D015678:Viral Structural Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D018832:Molecular Chaperones; D015678:Viral Structural Proteins; D002118:Calcium