Mechanism of Scaffolding-Directed Virus Assembly Suggested by Comparison of Scaffolding-Containing and Scaffolding-Lacking P22 Procapsids

Thuman-Commike, Pamela A.; Greene, Barrie; Malinski, Justine A.; Burbea, Michelle; McGough, Amy; Chiu, Wah; Prevelige, Peter E.

doi:10.1016/s0006-3495(99)77479-5

Cited by 59 publications

(79 citation statements)

References 69 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These electrostatic interactions are consistent with previous biochemical studies (31,32). Note that the interactions between the scaffolding protein and coat protein have never been visualized in such fine detail and modeled in any authentic bacteriophage procapsid, although previous attempts at lower resolutions were made in herpes virus capsid (33) and P22 (17).…”

Section: Resultssupporting

confidence: 88%

“…The Cα models for the asymmetric units of the bacteriophage P22 procapsid and virion have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2XYY and 2XYZ, respectively). previously reported for the GuHCl treated procapsid (16)(17)(18)(19)(20). Each of the gp5 models consists of eight distinct domains: A domain, extra-density (ED) domain, E loop, D loop, F loop, P domain, N arm, and a long helix (Fig.…”

Section: Resultsmentioning

confidence: 87%

See 1 more Smart Citation

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Chen

Baker

Hryc

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

202

319

View full text Add to dashboard Cite

Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8-and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T ¼ 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.sDNA viruses infecting both prokaryotes and eukaryotes share a common assembly pathway proceeding from a precursor (procapsid) to an infectious virion (1-4). In addition to the coat proteins, the procapsid requires scaffolding proteins, absent from the virion, for proper assembly, and a portal for DNA packaging and subsequent DNA ejection. However, despite a half-century of research on icosahedral viruses, it remains unclear how initially identical subunits adopt both hexameric and pentameric conformations in the virus and select the correct locations needed to form closed shells of the proper size (5). Packaging of DNA through the portal is accompanied by the exit of scaffolding proteins from the procapsid and conformational changes in the coat proteins as the capsid matures (2, 6).Understanding the molecular mechanisms of dsDNA virus assembly and maturation requires knowledge of the interactions among the coat, scaffolding, and portal proteins, all of which are essential for these processes. X-ray crystallography (7-9) and electron cryomicroscopy (cryo-EM) (10-12) have yielded nearatomic to atomic resolution models of several dsDNA icosahedral viruses and provided a structural framework of interactions among their coat proteins. However, the structural details of procapsid portal incorporation, scaffolding protein bind...

show abstract

Section: Resultssupporting

confidence: 88%

Section: Resultsmentioning

confidence: 87%

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Chen

Baker

Hryc

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

202

319

View full text Add to dashboard Cite

show abstract

“…Prior to calculation of the difference maps, radial scaling and density scaling were performed (1,10,22). Both algebraic difference maps (11) and the Student t test (12,13,21) were used to identify and confirm statistically significant structural differences (19). Shown in white is the three-dimensional structure of the procapsid assembled in the absence of the scaffolding protein (20).…”

mentioning

confidence: 99%

Identification of Additional Coat-Scaffolding Interactions in a Bacteriophage P22 Mutant Defective in Maturation

Thuman-Commike

Greene

Jakana

et al. 2000

J Virol

Self Cite

View full text Add to dashboard Cite

Scaffolding proteins play a critical role in the assembly of certain viruses by directing the formation and maturation of a precursor capsid. Using electron cryomicroscopy difference mapping, we have identified an altered arrangement of a mutant scaffolding within the bacteriophage P22 procapsid. This mutant scaffolding allows us to directly visualize scaffolding density within the P22 procapsid. Based on these observations we propose a model for why the mutant prevents scaffolding release and capsid maturation.Critical stages in many viral life cycles involve large-scale conformational transitions. In certain classes of viruses, including the herpesviruses, adenoviruses, and double-stranded-DNA bacteriophages, the regulation of these transitions requires scaffolding proteins, molecules not found in the mature virion but essential for assembly (2,4,17). The assembly pathway for these viruses is epitomized by the Salmonella phage P22, a Tϭ7 icosahedral phage. Assembly of P22 requires approximately 300 scaffolding subunits in addition to 420 coat subunits (3). These coassemble into a precursor structure, the procapsid, that contains scaffolding subunits within the capsid instead of DNA and is smaller and rounder than the mature FIG. 1. Localization of the scaffolding protein within the R74C/L177I procapsid by difference maps computed between procapsids assembled in the absence of the scaffolding protein (20) and the 22-Å structure of the R74C/L177I capsids. The R74C/L177I three-dimensional structure was determined using 161 particle images obtained from purified R74C/L177I (6) capsids imaged at ϳ1.0 m underfocus with 100-kV flood beam imaging. Prior to calculation of the difference maps, radial scaling and density scaling were performed (1, 10, 22). Both algebraic difference maps (11) and the Student t test (12, 13, 21) were used to identify and confirm statistically significant structural differences (19). Shown in white is the three-dimensional structure of the procapsid assembled in the absence of the scaffolding protein (20). Shown in red is the density corresponding to the differences attributable to the scaffolding protein. No significant differences were observed on the outer procapsid surface. (A) Inner surface, with differences at the trimer tips of the b, c, d, e, f, and g subunits circled. (B) Schematic diagram depicting the procapsid icosahedral lattice inner surface view. The subunits with R74C/L177I scaffolding bound are identified by red circles.

show abstract

“…The microvirus internal scaffolding protein shares many properties and functions with the scaffolding proteins found in other viruses (7,9,10,14,15,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). However, it does not physically construct the procapsid or control morphogenetic fidelity.…”

Section: Discussionmentioning

confidence: 99%

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro

Cherwa

Tyson

Bedwell

et al. 2017

J Virol

Self Cite

View full text Add to dashboard Cite

During X174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro. In previous studies, X174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. IMPORTANCE Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo. We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During X174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.KEYWORDS bacteriophage X174, Microviridae, microvirus, scaffolding protein, virus assembly D ue to their rapid replication cycle, detailed structural information, and well developed genetic and biochemical assays, the microviruses have become an ideal model system for experimental evolution (1-4). The morphogenetic pathway can be characterized under restrictive conditions and adaptive mutations can be mapped onto X-ray structures, providing insights into evolutionary mechanisms and protein structure-function relationships (2-8).

show abstract

Mechanism of Scaffolding-Directed Virus Assembly Suggested by Comparison of Scaffolding-Containing and Scaffolding-Lacking P22 Procapsids

Cited by 59 publications

References 69 publications

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Identification of Additional Coat-Scaffolding Interactions in a Bacteriophage P22 Mutant Defective in Maturation

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro

Contact Info

Product

Resources

About