scholarly journals Structural transitions during the scaffolding-driven assembly of a viral capsid

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Athanasios Ignatiou ◽  
Sandrine Brasilès ◽  
Mehdi El Sadek Fadel ◽  
Jörg Bürger ◽  
Thorsten Mielke ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Major Capsid Protein ◽  
Dna Packaging ◽  
Viral Capsid ◽  
Scaffolding Proteins ◽  
Structural Rearrangements ◽  
Binding Interface ◽  
N Terminus ◽  
Auxiliary Protein ◽  
Capsid Maturation

Abstract Assembly of tailed bacteriophages and herpesviruses starts with formation of procapsids (virion precursors without DNA). Scaffolding proteins (SP) drive assembly by chaperoning the major capsid protein (MCP) to build an icosahedral lattice. Here we report near-atomic resolution cryo-EM structures of the bacteriophage SPP1 procapsid, the intermediate expanded procapsid with partially released SPs, and the mature capsid with DNA. In the intermediate state, SPs are bound only to MCP pentons and to adjacent subunits from hexons. SP departure results in the expanded state associated with unfolding of the MCP N-terminus and straightening of E-loops. The newly formed extensive inter-capsomere bonding appears to compensate for release of SPs that clasp MCP capsomeres together. Subsequent DNA packaging instigates bending of MCP A domain loops outwards, closing the hexons central opening and creating the capsid auxiliary protein binding interface. These findings provide a molecular basis for the sequential structural rearrangements during viral capsid maturation.

UL25 capsid binding facilitates mechanical maturation of the Herpesvirus capsid and allows retention of pressurized DNA

2021 ◽  
Author(s):  
Krista G. Freeman ◽  
Jamie B. Huffman ◽  
Fred L. Homa ◽  
Alex Evilevitch
Keyword(s):  
Dna Packaging ◽  
Maturation Process ◽  
Virion Assembly ◽  
Force Microscopy ◽  
Atomic Force ◽  
Auxiliary Protein ◽  
Capsid Maturation ◽  
Simplex Virus ◽  
Hsv 1

The maturation process that occurs in most viruses is evolutionarily driven as it resolves several conflicting virion assembly requirements. During herpesvirus assembly in a host cell nucleus, micron-long double-stranded herpes DNA is packaged into a nanometer-sized procapsid. This leads to strong confinement of the viral genome with resulting tens of atmospheres of intra-capsid DNA pressure. Yet, the procapsid is unstable due to weak, reversible interactions between its protein subunits, which ensures free energy minimization and reduces assembly errors. In this work we show that herpesviruses resolve these contradictory capsid requirements through a mechanical capsid maturation process facilitated by multi-functional auxiliary protein UL25. Through mechanical interrogation of herpes simplex virus type 1 (HSV-1) capsid with atomic force microscopy nano-indentation, we show that UL25 binding at capsid vertices post-assembly provides the critical capsid reinforcement required for stable DNA encapsidation; the absence of UL25 binding leads to capsid rupture. Furthermore, we demonstrate that gradual capsid reinforcement is a feasible maturation mechanism facilitated by progressive UL25 capsid binding, which is likely correlated with DNA packaging progression. This work provides insight into elegantly programmed viral assembly machinery where targeting of capsid assembly mechanics presents a new antiviral strategy that is resilient to development of drug resistance. Importance: Most viruses undergo a maturation process from a weakly assembled particle to a stable virion. Herpesvirus capsid undergoes mechanical maturation to withstand tens of atmospheres of DNA pressure. We demonstrate that this mechanical capsid maturation is mainly facilitated through binding of auxiliary protein UL25 in HSV-1 capsid vertices. We show that UL25 binding provides the critical capsid reinforcement required for stable DNA encapsidation. Our data also suggests that gradual capsid reinforcement by progressive UL25 binding is a feasible capsid maturation mechanism, correlated with DNA packaging progression.

scholarly journals Cryo-EM structure of the bacteriophage T4 isometric head at 3.3-Å resolution and its relevance to the assembly of icosahedral viruses

2017 ◽  
Vol 114 (39) ◽  
pp. E8184-E8193 ◽  
Author(s):  
Zhenguo Chen ◽  
Lei Sun ◽  
Zhihong Zhang ◽  
Andrei Fokine ◽  
Victor Padilla-Sanchez ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Bacteriophage T4 ◽  
Hexagonal Lattice ◽  
Scaffolding Proteins ◽  
Outer Capsid Protein ◽  
Head Assembly ◽  
Icosahedral Viruses ◽  
End Caps ◽  
Outer Capsid

The 3.3-Å cryo-EM structure of the 860-Å-diameter isometric mutant bacteriophage T4 capsid has been determined. WT T4 has a prolate capsid characterized by triangulation numbers (T numbers) Tend= 13 for end caps and Tmid= 20 for midsection. A mutation in the major capsid protein, gp23, produced T=13 icosahedral capsids. The capsid is stabilized by 660 copies of the outer capsid protein, Soc, which clamp adjacent gp23 hexamers. The occupancies of Soc molecules are proportional to the size of the angle between the planes of adjacent hexameric capsomers. The angle between adjacent hexameric capsomers is greatest around the fivefold vertices, where there is the largest deviation from a planar hexagonal array. Thus, the Soc molecules reinforce the structure where there is the greatest strain in the gp23 hexagonal lattice. Mutations that change the angles between adjacent capsomers affect the positions of the pentameric vertices, resulting in different triangulation numbers in bacteriophage T4. The analysis of the T4 mutant head assembly gives guidance to how other icosahedral viruses reproducibly assemble into capsids with a predetermined T number, although the influence of scaffolding proteins is also important.

scholarly journals Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

2017 ◽  
Author(s):  
Eric A. Iverson ◽  
David A. Goodman ◽  
Madeline E. Gorchels ◽  
Kenneth M. Stedman
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Vp1 Gene ◽  
Capsid Protein Gene ◽  
Vp1 Protein ◽  
Transposon Insertion ◽  
N Terminus ◽  
Capsid Protein Vp1

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein gene vp3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential for infectivity. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

scholarly journals Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

2017 ◽  
Author(s):  
Eric A. Iverson ◽  
David A. Goodman ◽  
Madeline E. Gorchels ◽  
Kenneth M. STEDMAN
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Vp1 Gene ◽  
Vp1 Protein ◽  
Transposon Insertion ◽  
N Terminus ◽  
Capsid Protein Vp1 ◽  
Virion Formation

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein VP3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

scholarly journals Human Foamy Virus Capsid Formation Requires an Interaction Domain in the N Terminus of Gag

2001 ◽  
Vol 75 (9) ◽  
pp. 4367-4375 ◽  
Author(s):  
Joelle Tobaly-Tapiero ◽  
Patricia Bittoun ◽  
Marie-Lou Giron ◽  
Manuel Neves ◽  
Marcel Koken ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Foamy Virus ◽  
Coiled Coil ◽  
Capsid Assembly ◽  
Human Foamy Virus ◽  
Viral Production ◽  
Reporter Protein ◽  
Interaction Domain ◽  
Capsid Formation ◽  
N Terminus

ABSTRACT Retroviral Gag expression is sufficient for capsid assembly, which occurs through interaction between distinct Gag domains. Human foamy virus (HFV) capsids assemble within the cytoplasm, although their budding, which mainly occurs in the endoplasmic reticulum, requires the presence of homologous Env. Yet little is known about the molecular basis of HFV Gag precursor assembly. Using fusions between HFV Gag and a nuclear reporter protein, we have identified a strong interaction domain in the N terminus of HFV Gag which is predicted to contain a conserved coiled-coil motif. Deletion within this region in an HFV provirus abolishes viral production through inhibition of capsid assembly.

scholarly journals Sequential Autoprocessing of Marek's Disease Herpesvirus Protease Differs from That of Other Herpesviruses

2007 ◽  
Vol 81 (11) ◽  
pp. 6117-6121 ◽  
Author(s):  
S. Laurent ◽  
C. Blondeau ◽  
M. Belghazi ◽  
S. Remy ◽  
E. Esnault ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Consensus Sequence ◽  
Marek's Disease ◽  
Site Directed Mutagenesis ◽  
Viral Capsid ◽  
Directed Mutagenesis ◽  
Marek’S Disease ◽  
Polar Amino Acid ◽  
Kinetics Studies ◽  
Capsid Maturation

ABSTRACT Herpesviruses encode a unique serine protease essential for viral capsid maturation. This protease undergoes autoprocessing at two sites, R and M, at the consensus sequence (V, L, I)P3-XP2-AP1/SP1′ (where X is a polar amino acid). We observed complete autoprocessing at the R and M sites of Marek's disease virus (MDV) protease following production of the polyprotein in Escherichia coli. Site-directed mutagenesis confirmed the predicted sequence of the R and M sites, with the M site sequence being nonconsensual: MP3-NP2-AP1/SP1′. Mutagenesis and expression kinetics studies suggested that the atypical MDV M site was cleaved exclusively by the processed short protease, a feature making MDV unique among herpesviruses.

scholarly journals Quinine-dependent, platelet-reactive monoclonals mimic antibodies found in patients with quinine-induced immune thrombocytopenia

Blood ◽  
2009 ◽  
Vol 113 (5) ◽  
pp. 1105-1111 ◽  
Author(s):  
Daniel W. Bougie ◽  
Jessica Birenbaum ◽  
Mark Rasmussen ◽  
Mortimer Poncz ◽  
Richard H. Aster
Keyword(s):  
Molecular Basis ◽  
Immune Thrombocytopenia ◽  
Antibody Binding ◽  
Membrane Glycoproteins ◽  
Binding Studies ◽  
Platelet Destruction ◽  
Drug Induced ◽  
N Terminus ◽  
Sequencing Studies ◽  
Drug Dependent

Abstract Drug-induced immune thrombocytopenia (DITP) is caused by drug-dependent antibodies (DDAbs) that are nonreactive in themselves but bind tightly to specific platelet membrane glycoproteins (GP) when soluble drug is present at pharmacologic concentrations. This reaction takes place without covalent linkage of drug to the target, indicating that drug does not function as a classical hapten to promote antibody binding. Studies to define other mechanism(s) responsible for this interaction have been frustrated by the polyclonal nature of human DDAbs and limited quantities of antibody usually available. We produced 2 monoclonal antibodies (mAbs), 314.1 and 314.3, from a mouse immunized with purified human GPIIb/IIIa and quinine that recognize the N terminus of the GPIIb β propeller domain only when soluble quinine is present. Both monoclonals closely mimic the behavior of antibodies from patients with quinine-induced immune thrombo-cytopenia in their reactions at various concentrations of quinine and quinine congeners. Sequencing studies showed that the 2 mAbs are closely related structurally and that mAb 314.3 probably evolved from mAb 314.1 in the course of the immune response. These monoclonal reagents are the first of their kind and should facilitate studies to define the molecular basis for drug-dependent antibody binding and platelet destruction in DITP.

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