scholarly journals Subassemblies and Asymmetry in Assembly of Herpes Simplex Virus Procapsid

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Anastasia A. Aksyuk ◽  
William W. Newcomb ◽  
Naiqian Cheng ◽  
Dennis C. Winkler ◽  
Juan Fontana ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Building Blocks ◽  
Scaffolding Protein ◽  
Icosahedral Symmetry ◽  
Large Set ◽  
Protein Subunits ◽  
Cryo Electron Microscopy ◽  
Simplex Virus

ABSTRACTThe herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expulsion of the internal scaffolding protein. Assembly also relies on an external scaffolding protein, the triplex, an α2β heterotrimer that coordinates neighboring capsomers in the procapsid and becomes a stabilizing clamp in the mature capsid. To investigate the mechanisms that regulate its assembly, we developed a novel isolation procedure for the metastable procapsid and collected a large set of cryo-electron microscopy data. In addition to procapsids, these preparations contain maturation intermediates, which were distinguished by classifying the images and calculating a three-dimensional reconstruction for each class. Appraisal of the procapsid structure led to a new model for assembly; in it, the protomer (assembly unit) consists of one triplex, surrounded by three major capsid protein (MCP) subunits. The model exploits the triplexes’ departure from 3-fold symmetry to explain the highly skewed MCP hexamers, the triplex orientations at each 3-fold site, and the T=16 architecture. These observations also yielded new insights into maturation.IMPORTANCEThis paper addresses the molecular mechanisms that govern the self-assembly of large, structurally complex, macromolecular particles, such as the capsids of double-stranded DNA viruses. Although they may consist of thousands of protein subunits of many different kinds, their assembly is precise, ranking them among the largest entities in the biosphere whose structures are uniquely defined to the atomic level. Assembly proceeds in two stages: formation of a precursor particle (procapsid) and maturation, during which major changes in structure and composition take place. Our analysis of the HSV procapsid by cryo-electron microscopy suggests a hierarchical pathway in which multisubunit “protomers” are the building blocks of the procapsid but their subunits are redistributed into different subcomplexes upon being incorporated into a nascent procapsid and are redistributed again in maturation. Assembly is a highly virus-specific process, making it a potential target for antiviral intervention.

scholarly journals Roles of Triplex and Scaffolding Proteins in Herpes Simplex Virus Type 1 Capsid Formation Suggested by Structures of Recombinant Particles

1999 ◽  
Vol 73 (8) ◽  
pp. 6821-6830 ◽  
Author(s):  
Ali Saad ◽  
Z. Hong Zhou ◽  
Joanita Jakana ◽  
Wah Chiu ◽  
Frazer J. Rixon
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Structural Features ◽  
Spherical Particles ◽  
Scaffolding Protein ◽  
Icosahedral Symmetry ◽  
Capsid Formation ◽  
A Subunit ◽  
Simplex Virus ◽  
Capsid Stability

ABSTRACT Typical herpes simplex virus (HSV) capsids contain seven proteins that form a T=16 icosahedron of 1,250-Å diameter. Infection of cells with recombinant baculoviruses expressing two of these proteins, VP5 (which forms the pentons and hexons in typical HSV capsids) and VP19C (a component of the triplexes that connect adjacent capsomeres), results in the formation of spherical particles of 880-Å diameter. Electron cryomicroscopy and computer reconstruction revealed that these particles possess a T=7 icosahedral symmetry, having 12 pentons and 60 hexons. Among the characteristic structural features of the particle are the skewed appearance of the hexons and the presence of intercapsomeric mass densities connecting the middle domain of one hexon subunit to the lower domain of a subunit in the adjacent hexon. We interpret these connecting masses as being formed by VP19C. Comparison of the connecting masses with the triplexes, which occupy equivalent positions in the T=16 capsid, reveals the probable locations of the single VP19C and two VP23 molecules that make up the triplex. Their arrangement suggests that the two triplex proteins have different roles in controlling intercapsomeric interactions and capsid stability. The nature of these particles and of other aberrant forms made in the absence of scaffold demonstrates the conformational adaptability of the capsid proteins and illustrates how VP23 and the scaffolding protein modulate the nature of the VP5-VP19C network to ensure assembly of the functional T=16 capsid.

Dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy

2003 ◽  
Vol 10 (5) ◽  
pp. 334-341 ◽  
Author(s):  
J. Bernard Heymann ◽  
Naiqian Cheng ◽  
William W. Newcomb ◽  
Benes L. Trus ◽  
Jay C. Brown ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Time Lapse ◽  
Virus Capsid ◽  
Cryo Electron Microscopy ◽  
Capsid Maturation ◽  
Simplex Virus

scholarly journals Structure and Polymorphism of the UL6 Portal Protein of Herpes Simplex Virus Type 1

2004 ◽  
Vol 78 (22) ◽  
pp. 12668-12671 ◽  
Author(s):  
Benes L. Trus ◽  
Naiqian Cheng ◽  
William W. Newcomb ◽  
Fred L. Homa ◽  
Jay C. Brown ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Portal Protein ◽  
Axial Channel ◽  
Simplex Virus

ABSTRACT By electron microscopy and image analysis, we find that baculovirus-expressed UL6 is polymorphic, consisting of rings of 11-, 12-, 13-, and 14-fold symmetry. The 12-mer is likely to be the oligomer incorporated into procapsids: at a resolution of 16 Å, it has an axial channel, peripheral flanges, and fits snugly into a vacant vertex site. Its architecture resembles those of bacteriophage portal/connector proteins.

scholarly journals Assembly of the Herpes Simplex Virus Procapsid from Purified Components and Identification of Small Complexes Containing the Major Capsid and Scaffolding Proteins

1999 ◽  
Vol 73 (5) ◽  
pp. 4239-4250 ◽  
Author(s):  
William W. Newcomb ◽  
Fred L. Homa ◽  
Darrell R. Thomsen ◽  
Benes L. Trus ◽  
Naiqian Cheng ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Capsid Protein ◽  
Major Capsid Protein ◽  
Scaffolding Protein ◽  
Oligomeric State ◽  
Cell Extracts ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT An in vitro system is described for the assembly of herpes simplex virus type 1 (HSV-1) procapsids beginning with three purified components, the major capsid protein (VP5), the triplexes (VP19C plus VP23), and a hybrid scaffolding protein. Each component was purified from insect cells expressing the relevant protein(s) from an appropriate recombinant baculovirus vector. Procapsids formed when the three purified components were mixed and incubated for 1 h at 37°C. Procapsids assembled in this way were found to be similar in morphology and in protein composition to procapsids formed in vitro from cell extracts containing HSV-1 proteins. When scaffolding and triplex proteins were present in excess in the purified system, greater than 80% of the major capsid protein was incorporated into procapsids. Sucrose density gradient ultracentrifugation studies were carried out to examine the oligomeric state of the purified assembly components. These analyses showed that (i) VP5 migrated as a monomer at all of the protein concentrations tested (0.1 to 1 mg/ml), (ii) VP19C and VP23 migrated together as a complex with the same heterotrimeric composition (VP19C1-VP232) as virus triplexes, and (iii) the scaffolding protein migrated as a heterogeneous mixture of oligomers (in the range of monomers to ∼30-mers) whose composition was strongly influenced by protein concentration. Similar sucrose gradient analyses performed with mixtures of VP5 and the scaffolding protein demonstrated the presence of complexes of the two having molecular weights in the range of 200,000 to 600,000. The complexes were interpreted to contain one or two VP5 molecules and up to six scaffolding protein molecules. The results suggest that procapsid assembly may proceed by addition of the latter complexes to regions of growing procapsid shell. They indicate further that procapsids can be formed in vitro from virus-encoded proteins only without any requirement for cell proteins.

scholarly journals pH Reduction as a Trigger for Dissociation of Herpes Simplex Virus Type 1 Scaffolds

2002 ◽  
Vol 76 (15) ◽  
pp. 7407-7417 ◽  
Author(s):  
David A. McClelland ◽  
James D. Aitken ◽  
David Bhella ◽  
David McNab ◽  
Joyce Mitchell ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Essential Feature ◽  
Scaffolding Protein ◽  
Capsid Shell ◽  
Shell Proteins ◽  
Simplex Virus

ABSTRACT Assembly of the infectious herpes simplex virus type 1 virion is a complex, multistage process that begins with the production of a procapsid, which is formed by the condensation of capsid shell proteins around an internal scaffold fashioned from multiple copies of the scaffolding protein, pre-VP22a. The ability of pre-VP22a to interact with itself is an essential feature of this process. However, this self-interaction must subsequently be reversed to allow the scaffolding proteins to exit from the capsid to make room for the viral genome to be packaged. The nature of the process by which dissociation of the scaffold is accomplished is unknown. Therefore, to investigate this process, the properties of isolated scaffold particles were investigated. Electron microscopy and gradient sedimentation studies showed that the particles could be dissociated by low concentrations of chaotropic agents and by moderate reductions in pH (from 7.2 to 5.5). Fluorescence spectroscopy and circular dichroism analyses revealed that there was relatively little change in tertiary and secondary structures under these conditions, indicating that major structural transformations are not required for the dissociation process. We suggest the possibility that dissociation of the scaffold may be triggered by a reduction in pH brought about by the entry of the viral DNA into the capsid.

scholarly journals Herpes Simplex Virus Capsid Structure: DNA Packaging Protein UL25 Is Located on the External Surface of the Capsid near the Vertices

2006 ◽  
Vol 80 (13) ◽  
pp. 6286-6294 ◽  
Author(s):  
William W. Newcomb ◽  
Fred L. Homa ◽  
Jay C. Brown
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
External Surface ◽  
Dna Packaging ◽  
Scaffolding Protein ◽  
Experimental Conditions ◽  
Simplex Virus ◽  
Quantitative Immunoblotting ◽  
Late Stages

ABSTRACT UL25 is one of seven herpes simplex virus-encoded proteins involved specifically in DNA encapsidation. Its role appears to be to stabilize the capsid so that DNA is prevented from escaping once it has entered. To clarify the function of UL25, we have examined capsids with the goal of defining where it is located. Analysis of trypsin-treated capsids showed that UL25 is sensitive to cleavage like other proteins such as the major capsid and portal proteins that are exposed on the capsid surface. Internal proteins such as the scaffolding protein and protease were not affected under the same experimental conditions. Capsids were also examined by electron microscopy after staining with gold-labeled antibody specific for UL25. Images of stained capsids demonstrated that most labeled sites (71% in C capsids) were at capsid vertices, and most stained C capsids had label at more than one vertex. A quantitative immunoblotting method showed that the capsid contents of UL25 were 56, 20, and 75 copies per capsid in A, B, and C capsids, respectively. Finally, soluble UL25 protein was found to bind in vitro to purified capsids lacking it. The amount of bound UL25 corresponded to the amount present in B capsids, and bound UL25 was found by immunoelectron microscopy to be located predominantly at the capsid vertices. The results are interpreted to suggest that five UL25 molecules are found at or near each of the capsid vertices, where they are exposed on the capsid surface. Exposure on the surface is consistent with the view that UL25 is added to the capsid as DNA is packaged or during late stages of the packaging process.

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