Quantitative Analysis of Multi-component Spherical Virus Assembly: Scaffolding Protein Contributes to the Global Stability of Phage P22 Procapsids

2006 ◽  
Vol 359 (4) ◽  
pp. 1097-1106 ◽  
Author(s):  
Kristin N. Parent ◽  
Adam Zlotnick ◽  
Carolyn M. Teschke
Keyword(s):  
Quantitative Analysis ◽  
Global Stability ◽  
Virus Assembly ◽  
Scaffolding Protein ◽  
Phage P22

Control of the synthesis of phage p22 scaffolding protein is coupled to capsid assembly

Cell ◽  
1978 ◽  
Vol 15 (2) ◽  
pp. 551-560 ◽  
Author(s):  
Jonathan King ◽  
Carol Hall ◽  
Sherwood Casjens
Keyword(s):  
Scaffolding Protein ◽  
Capsid Assembly ◽  
Phage P22

scholarly journals Conformational Switch-Defective ϕX174 Internal Scaffolding Proteins Kinetically Trap Assembly Intermediates before Procapsid Formation

2012 ◽  
Vol 86 (18) ◽  
pp. 9911-9918 ◽  
Author(s):  
Emile B. Gordon ◽  
Christopher J. Knuff ◽  
Bentley A. Fane
Keyword(s):  
Coat Protein ◽  
Protein Function ◽  
Biochemical Characterization ◽  
Virus Assembly ◽  
Aromatic Amino Acids ◽  
Model Systems ◽  
Scaffolding Protein ◽  
Conformational Switching ◽  
Conformational Switch ◽  
Viral Coat

Conformational switching is an overarching paradigm in which to describe scaffolding protein-mediated virus assembly. However, rapid morphogenesis with small assembly subunits hinders the isolation of early morphogenetic intermediates in most model systems. Consequently, conformational switches are often defined by comparing the structures of virions, procapsids and aberrantly assembled particles. In contrast, ϕX174 morphogenesis proceeds through at least three preprocapsid intermediates, which can be biochemically isolated. This affords a detailed analysis of early morphogenesis and internal scaffolding protein function. Amino acid substitutions were generated for the six C-terminal, aromatic amino acids that mediate most coat-internal scaffolding protein contacts. The biochemical characterization of mutant assembly pathways revealed two classes of molecular defects, protein binding and conformational switching, a novel phenotype. The conformational switch mutations kinetically trapped assembly intermediates before procapsid formation. Although mutations trapped different particles, they shared common second-site suppressors located in the viral coat protein. This suggests a fluid assembly pathway, one in which the scaffolding protein induces a single, coat protein conformational switch and not a series of sequential reactions. In this model, an incomplete or improper switch would kinetically trap intermediates.

Structure and assembly of the capsid of bacteriophage P22

1976 ◽  
Vol 276 (943) ◽  
pp. 37-49 ◽  
Keyword(s):  
Coat Protein ◽  
Protein Gene ◽  
Scaffolding Protein ◽  
Bacteriophage P22 ◽  
Particle Assembly ◽  
Thin Sections ◽  
X Ray Scattering ◽  
Phage P22 ◽  
Infected Cells ◽  
Protein Shell

Identification of the genes and proteins involved in phage P22 formation has permitted a detailed analysis of particle assembly, revealing some unexpected aspects. The polymerization of the major coat protein (gene 5 product) into an organized capsid is directed by a scaffolding protein (gene 8 product) which is absent from mature phage. The resulting capsid structure (prohead) is the precursor for DNA encapsidation. All of the scaffolding protein exits from the prohead in association with DNA packaging. These molecules then recycle, directing further rounds of prohead assembly. The structure of the prohead has been studied by electron microscopy of thin sections of phage infected cells, and by low angle X-ray scattering of concentrated particles. The results show that the prohead is a double shell structure, or a ball within a shell. The inner ball or shell is composed of the scaffolding protein while the outer shell is composed of coat protein. The conversion from prohead to mature capsid is associated with an expansion of the coat protein shell. It is possible that the scaffolding protein molecules exit through the capsid lattice. When DNA encapsidation within infected cells is blocked by mutation, scaffolding protein is trapped in proheads and cannot recycle. Under these conditions, the rate of synthesis of gp8 increases, so that normal proheads continue to form. These results suggest that free scaffolding protein negatively regulates its own further synthesis, providing a coupling between protein synthesis and protein assembly.

scholarly journals NMR Mapping of Disordered Segments from a Viral Scaffolding Protein Encapsulated in a 23 MDa Procapsid Complex

2019 ◽  
Author(s):  
Richard D. Whitehead ◽  
Carolyn M. Teschke ◽  
Andrei T. Alexandrescu
Keyword(s):  
Structural Information ◽  
Scaffolding Protein ◽  
Coat Proteins ◽  
Scaffolding Proteins ◽  
Genome Packaging ◽  
C Terminus ◽  
Capsid Shell ◽  
N Terminus ◽  
Phage P22 ◽  
Nmr Signals

SummaryScaffolding proteins are requisite for the capsid shell assembly of many tailed dsDNA bacteriophages, some archaeal viruses, herpesviruses, and adenoviruses. Despite their importance, no high-resolution structural information is available for scaffolding proteins within capsids. Here we use the inherent size limit of NMR to identify mobile segments of the phage P22 scaffolding protein in solution and when incorporated into a ~23 MDa procapsid complex. Free scaffolding protein gives NMR signals from both the N and C-terminus. When scaffolding protein is incorporated into P22 procapsids, NMR signals from the C-terminal helix-turn-helix (HTH) domain disappear due to binding to the procapsid interior. Signals from the N-terminal domain persist, indicating this segment retains flexibility when bound to procapsids. The unstructured character of the N-terminus coupled with its high content of negative charges, is likely important for the dissociation and release of scaffolding protein, during the genome packaging step accompanying phage maturation.Graphical AbstractScaffolding protein (SP) nucleates the assembly of phage P22 coat proteins into an icosahedral capsid structure that envelops the viral genome. NMR spectra of free SP show signals from the N-terminus (red) and a helix-turn-helix domain at the C-terminus (blue). When SP is incorporated into empty phage P22 procapsids to form a 23 MDa complex, the subset of signals from the N-terminal 40 residues persist indicating this segment is disordered. The unfolded nature of the N-terminus coupled with its negatively charged character, is important for the functional requirement of SP to exit the capsid as it becomes packaged with its genome.

scholarly journals Architect of Virus Assembly: the Portal Protein Nucleates Procapsid Assembly in Bacteriophage P22

2019 ◽  
Vol 93 (9) ◽  
Author(s):  
Tina Motwani ◽  
Carolyn M. Teschke
Keyword(s):  
Virus Assembly ◽  
Genetic Material ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Bacteriophage P22 ◽  
Particle Assembly ◽  
Portal Protein ◽  
Double Stranded Dna ◽  
Dsdna Viruses

ABSTRACTTailed double-stranded DNA (dsDNA) bacteriophages, herpesviruses, and adenoviruses package their genetic material into a precursor capsid through a dodecameric ring complex called the portal protein, which is located at a unique 5-fold vertex. In several phages and viruses, including T4, Φ29, and herpes simplex virus 1 (HSV-1), the portal forms a nucleation complex with scaffolding proteins (SPs) to initiate procapsid (PC) assembly, thereby ensuring incorporation of only one portal ring per capsid. However, for bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is unclear. We have developed anin vitroP22 assembly assay where portal protein is coassembled into procapsid-like particles (PLPs). Scaffolding protein also catalyzes oligomerization of monomeric portal protein into dodecameric rings, possibly forming a scaffolding protein-portal protein nucleation complex that results in one portal ring per P22 procapsid. Here, we present evidence substantiating that the P22 portal protein, similarly to those of other dsDNA viruses, can act as an assembly nucleator. The presence of the P22 portal protein is shown to increase the rate of particle assembly and contribute to proper morphology of the assembled particles. Our results highlight a key function of portal protein as an assembly initiator, a feature that is likely conserved among these classes of dsDNA viruses.IMPORTANCEThe existence of a single portal ring is essential to the formation of infectious virions in the tailed double-stranded DNA (dsDNA) phages, herpesviruses, and adenoviruses and, as such, is a viable antiviral therapeutic target. How only one portal is selectively incorporated at a unique vertex is unclear. In many dsDNA viruses and phages, the portal protein acts as an assembly nucleator. However, early work on phage P22 assemblyin vivoindicated that the portal protein did not function as a nucleator for procapsid (PC) assembly, leading to the suggestion that P22 uses a unique mechanism for portal incorporation. Here, we show that portal protein nucleates assembly of P22 procapsid-like particles (PLPs). Addition of portal rings to an assembly reaction increases the rate of formation and yield of particles and corrects improper particle morphology. Our data suggest that procapsid assembly may universally initiate with a nucleation complex composed minimally of portal and scaffolding proteins (SPs).

scholarly journals The architect of virus assembly: the portal protein complex nucleates procapsid assembly in bacteriophage P22

2019 ◽  
Author(s):  
Tina Motwani ◽  
Carolyn M. Teschke
Keyword(s):  
Protein Complex ◽  
Virus Assembly ◽  
Genetic Material ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Bacteriophage P22 ◽  
Particle Assembly ◽  
Portal Protein ◽  
Dsdna Viruses

AbstractThe genetic material of tailed dsDNA bacteriophages, herpesviruses and adenoviruses is packaged into a precursor capsid through a 12-mer ring-shaped protein complex called the portal protein, located at a unique 5-fold vertex. In several phages and viruses, including T4, Φ29, and HSV-1, the dodecameric portal protein forms a nucleation complex with scaffolding proteins to initiate procapsid assembly, thereby ensuring incorporation of only one portal complex per capsid. However, for bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is unclear. We recently developed anin vitroP22 assembly assay where portal protein is co-assembled into procapsid-like particles. We also showed that scaffolding protein catalyzes oligomerization of monomeric portal protein into 12-mer rings, and possibly forming a scaffolding-protein nucleation complex that results in one portal complex per P22 procapsid. Here, we present evidence substantiating that P22 portal protein, similar to the other dsDNA viruses, can act as an assembly nucleator. We find that the presence of P22 portal protein is able to increase the rate of particle assembly. Additionally, we show that P22 portal protein proper contributes to proper morphology of the assembled particles. Our results highlight a key function of portal protein as an assembly initiator, a feature likely conserved among these classes of dsDNA viruses.

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