DNA packaging intermediates of bacteriophage φX174

Leodevico L. Ilag, Norman H. Olson, Terje Dokland, Cynthia L. Music, R. Holland Cheng, Zorina Bowen, Robert McKenna, Michael G. Rossmann, Timothy S. Baker, Nino L. Incardona

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

Background: Like many viruses, bacteriophage φX174 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. Results φX174 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 å diameter holes on the icosahedral three-fold axes. Conclusion 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.

Original languageEnglish (US)
Pages (from-to)353-363
Number of pages11
JournalStructure
Volume3
Issue number4
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

DNA Packaging
Bacteriophages
Virion
Cryoelectron Microscopy
Proteins
X Ray Crystallography
GTP-Binding Proteins
Virus Assembly
Computer-Assisted Image Processing
Capsid
DNA
Edetic Acid
Sucrose
Genome
Viruses

Keywords

  • cryo-electron microscopy
  • DNA packaging
  • icosahedral capsid
  • scaffolding proteins
  • viral assembly

ASJC Scopus subject areas

  • Molecular Biology
  • Structural Biology

Cite this

Ilag, L. L., Olson, N. H., Dokland, T., Music, C. L., Cheng, R. H., Bowen, Z., ... Incardona, N. L. (1995). DNA packaging intermediates of bacteriophage φX174. Structure, 3(4), 353-363. https://doi.org/10.1016/S0969-2126(01)00167-8

DNA packaging intermediates of bacteriophage φX174. / Ilag, Leodevico L.; Olson, Norman H.; Dokland, Terje; Music, Cynthia L.; Cheng, R. Holland; Bowen, Zorina; McKenna, Robert; Rossmann, Michael G.; Baker, Timothy S.; Incardona, Nino L.

In: Structure, Vol. 3, No. 4, 1995, p. 353-363.

Research output: Contribution to journalArticle

Ilag, LL, Olson, NH, Dokland, T, Music, CL, Cheng, RH, Bowen, Z, McKenna, R, Rossmann, MG, Baker, TS & Incardona, NL 1995, 'DNA packaging intermediates of bacteriophage φX174', Structure, vol. 3, no. 4, pp. 353-363. https://doi.org/10.1016/S0969-2126(01)00167-8
Ilag LL, Olson NH, Dokland T, Music CL, Cheng RH, Bowen Z et al. DNA packaging intermediates of bacteriophage φX174. Structure. 1995;3(4):353-363. https://doi.org/10.1016/S0969-2126(01)00167-8
Ilag, Leodevico L. ; Olson, Norman H. ; Dokland, Terje ; Music, Cynthia L. ; Cheng, R. Holland ; Bowen, Zorina ; McKenna, Robert ; Rossmann, Michael G. ; Baker, Timothy S. ; Incardona, Nino L. / DNA packaging intermediates of bacteriophage φX174. In: Structure. 1995 ; Vol. 3, No. 4. pp. 353-363.
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abstract = "Background: Like many viruses, bacteriophage φX174 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. Results φX174 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 {\aa} diameter holes on the icosahedral three-fold axes. Conclusion 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.",
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T1 - DNA packaging intermediates of bacteriophage φX174

AU - Ilag, Leodevico L.

AU - Olson, Norman H.

AU - Dokland, Terje

AU - Music, Cynthia L.

AU - Cheng, R. Holland

AU - Bowen, Zorina

AU - McKenna, Robert

AU - Rossmann, Michael G.

AU - Baker, Timothy S.

AU - Incardona, Nino L.

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N2 - Background: Like many viruses, bacteriophage φX174 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. Results φX174 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 å diameter holes on the icosahedral three-fold axes. Conclusion 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.

AB - Background: Like many viruses, bacteriophage φX174 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. Results φX174 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 å diameter holes on the icosahedral three-fold axes. Conclusion 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.

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