Structure of the Pseudorabies Virus Capsid: Comparison with Herpes Simplex Virus Type 1 and Differential Binding of Essential Minor Proteins (original) (raw)
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Structure of the herpesvirus major capsid protein
2003
Abstract Herpes simplex virus-1 (HSV-1) virions are large, complex enveloped particles containing a proteinaceous tegument layer connected to an icosahedral capsid. The major capsid protein, VP5 (149 kDa), makes up both types of capsomere, pentons and hexons. Limited trypsin digestion of VP5 identified a single stable 65 kDa fragment which represents a proposed protein folding nucleus.
Virology, 1997
The herpes simplex virus-1 (HSV-1) capsid shell has 162 capsomers arranged on a T Å 16 icosahedral lattice. The major capsid protein, VP5 (MW Å 149,075) is the structural component of the capsomers. VP5 is an unusually large viral capsid protein and has been shown to consist of multiple domains. To study the conformation of VP5 as it is folded into capsid protomers, we identified the sequence recognized by a VP5-specific monoclonal antibody and localized the epitope on the capsid surface by cryoelectron microscopy and image reconstruction. The epitope of mAb 6F10 was mapped to residues 862-880 by immunoblotting experiments performed with (1) proteolytic fragments of VP5, (2) GST-fusion proteins containing VP5 domains, and (3) synthetic VP5 peptides. As visualized in a three-dimensional density map of 6F10-precipitated capsids, the antibody was found to bind at sites on the outer surface of the capsid just inside the openings of the trans-capsomeric channels. We conclude that these sites are occupied by peptide 862-880 in the mature HSV-1 capsid.
Journal of Virology, 2001
Examination of the three-dimensional structure of intact herpes simplex virus type 1 (HSV-1) virions had revealed that the icosahedrally symmetrical interaction between the tegument and capsid involves the pentons but not the hexons (Z. 73:3210-3218, 1999). To account for this, we postulated that the presence of the small capsid protein, VP26, on top of the hexons was masking potential binding sites and preventing tegument attachment. We have now tested this hypothesis by determining the structure of virions lacking VP26. Apart from the obvious absence of VP26 from the capsids, the structures of the VP26 minus and wild-type virions were essentially identical. Notably, they showed the same tegument attachment patterns, thereby demonstrating that VP26 is not responsible for the divergent tegument binding properties of pentons and hexons.
Virology, 1997
The herpes simplex virus-1 (HSV-1) capsid shell has 162 capsomers arranged on a T Å 16 icosahedral lattice. The major capsid protein, VP5 (MW Å 149,075) is the structural component of the capsomers. VP5 is an unusually large viral capsid protein and has been shown to consist of multiple domains. To study the conformation of VP5 as it is folded into capsid protomers, we identified the sequence recognized by a VP5-specific monoclonal antibody and localized the epitope on the capsid surface by cryoelectron microscopy and image reconstruction. The epitope of mAb 6F10 was mapped to residues 862-880 by immunoblotting experiments performed with (1) proteolytic fragments of VP5, (2) GST-fusion proteins containing VP5 domains, and (3) synthetic VP5 peptides. As visualized in a three-dimensional density map of 6F10-precipitated capsids, the antibody was found to bind at sites on the outer surface of the capsid just inside the openings of the trans-capsomeric channels. We conclude that these sites are occupied by peptide 862-880 in the mature HSV-1 capsid.
Extensive subunit contacts underpin herpesvirus capsid stability and interior-to-exterior allostery
Nature Structural & Molecular Biology, 2016
The herpesvirus capsid is a complex protein assembly that includes hundreds of copies of four major subunits and lesser numbers of several minor proteins, all essential for infectivity. Cryoelectron microscopy is uniquely suited for studying interactions that govern the assembly and function of such large and functional complexes. Here we report two high quality capsid structures, from human herpes simplex virus type 1 (HSV-1) and the animal pseudorabies virus (PRV), imaged inside intact virions at ~7 Å resolution. From these we developed a complete model of subunit and domainal organization and identified extensive networks of subunit contacts that underpin capsid stability and form a pathway that may signal the completion of DNA packaging from the capsid interior to outer surface for initiating nuclear egress. Differences in folding and orientation of subunit domains between herpesvirus capsids suggest that common elements have been modified for specific functions.
Proceedings of the National Academy of Sciences, 1998
Formation of herpes simplex virus-1 capsids requires the presence of intact scaffolding proteins. The C terminus of the abundant scaffolding protein associates with the major capsid shell protein VP5 through hydrophobic interactions. After cleavage by the viral encoded protease, which removes their C-terminal 25 aa, the scaffolding proteins are released from the capsid. We have used electron cryomicroscopy and computer image processing to determine, to 13 Å, the three-dimensional structures of capsids containing either cleaved or uncleaved scaffolding proteins. Detailed comparisons show that the structures of the outer icosahedral shells are almost identical in the two capsid types. Differences are apparent in the radial distribution of the density inside the capsid shell (within a radius of 460 Å) which represents the scaffolding core. However, in both capsid types, the bulk of this internal density exhibits no icosahedral symmetry. Close examination revealed localized regions of icosahedrally arranged extra density at the interface between the outer shell and the scaffold of protease-minus capsids. Rod-like densities extending inwards for Ϸ40 Å from the capsid shell are present under four of the six quasi-equivalent triplex positions. Under triplexes T b , T c , and T e , the major additional densities appear as pairs with the rods in each pair situated 37 Å apart. We propose that these rods are formed by the C-termini of the scaffolding proteins and represent the sites of interaction between the capsid shell and scaffold.
Beyond structures of highly symmetric purified viral capsids by cryo-EM
Current Opinion in Structural Biology
Cryogenic transmission electron microscopy (cryo-EM) is widely used to determine high-resolution structures of symmetric virus capsids. The method holds promise for extending studies beyond purified capsids and their symmetric protein shells. The non-symmetric genome component has been addressed in dsRNA cypoviruses and ssRNA bacteriophages Qβ and MS2. The structure of human herpes simplex virus type 1 capsids has been determined within intact virions to resolve capsid-tegument interactions. Electron tomography under cryogenic conditions (cryo-ET), has allowed resolving an early membrane fusion intermediate of Rift Valley fever virus. Antibody-affinity based sample grids allow capturing of virions directly from cell cultures or even clinical samples. These and other emerging methods will support studies to address viral entry, assembly and neutralization processes at increasingly high resolutions and native conditions. Highlights • Single-particle averaging has evolved to determine high resolution structures of large icosahedral viruses and viral genomes. • Sub-tomogram averaging continues to produce high resolution structures of viral surface proteins and nucleocapsids. • Tomography of viruses has provided insights into dynamic viral entry processes. Annotated References * [14] This paper on Kaposi's sarcoma-associated herpesvirus reports many technological advances that were required to reconstruct the capsid of this large virus to 4.2-Å resolution. This structure allowed identifying structural motifs that stabilize the capsid and that that could be targeted by antivirals.
Visualization of tegument-capsid interactions and DNA in intact herpes simplex virus type 1 virions
Journal of virology, 1999
Herpes simplex virus type 1 virions were examined by electron cryomicroscopy, allowing the three-dimensional structure of the infectious particle to be visualized for the first time. The capsid shell is identical to that of B-capsids purified from the host cell nucleus, with the exception of the penton channel, which is closed. The double-stranded DNA genome is organized as regularly spaced ( approximately 26 A) concentric layers inside the capsid. This pattern suggests a spool model for DNA packaging, similar to that for some bacteriophages. The bulk of the tegument is not icosahedrally ordered. However, a small portion appears as filamentous structures around the pentons, interacting extensively with the capsid. Their locations and interactions suggest possible roles for the tegument proteins in regulating DNA transport through the penton channel and binding to cellular transport proteins during viral infection.
Journal of General Virology, 1995
Herpes simplex virus type 1 (HSV-1) polypeptides specified by overlapping genes UL26 and UL26.5 form a scaffold around which the icosahedral capsid shell is assembled. In a series of cleavage events catalysed by the UL26-encoded protease, the full-length UL26 product is processed into capsid proteins VP24 and VP21 and the UL26.5 protein is converted into the capsid protein VP22a by the loss of 25 amino acids from its carboxy terminus. The roles of the UL26 and UL26.5 products were investigated using the baculovirus expression system, focusing on the function of the 25 residues cleaved from the UL26.5 protein. A key conclusion from electron microscopic analysis and protein expression studies is that the 25 amino acids at the carboxy terminus of the full-length UL26.5 protein are required for the interaction of the capsid shell proteins with the scaffold in the formation of intermediate capsids. When cells were multiply infected with baculoviruses expressing a truncated form of the UL26.5 product corresponding to VP22a and the essential components of the capsid shell, no capsids were detected, whereas large numbers of capsids were observed when the full-length UL26.5 product was used as a scaffold. The results are consistent with the proposal that cleavage of the UL26.5 product occurs after capsid assembly or when the UL26.5 protein is in a complex with one or more capsid shell proteins. Expression of VP22a in the absence or presence of capsid shell proteins resulted in the formation of large numbers of 60 nm scaffold-like particles. Since VP22a expressed from baculovirus was unable to participate in capsid assembly, these particles cannot be intermediates in the capsid assembly pathway but may be similar in structure to the protein cores present in HSV-1 immature (B) capsids.
Journal of Virology, 2012
Herpesviruses have an icosahedral nucleocapsid surrounded by an amorphous tegument and a lipoprotein envelope. The tegument comprises at least 20 proteins destined for delivery into the host cell. As the tegument does not have a regular structure, the question arises of how its proteins are recruited. The herpes simplex virus 1 (HSV-1) tegument is known to contact the capsid at its vertices, and two proteins, UL36 and UL37, have been identified as candidates for this interaction. We show that the interaction is mediated exclusively by UL36. HSV-1 nucleocapsids extracted from virions shed their UL37 upon incubation at 37°C. Cryo-electron microscopy (cryo-EM) analysis of capsids with and without UL37 reveals the same penton-capping density in both cases. As no other tegument proteins are retained in significant amounts, it follows that this density feature (ϳ100 kDa) represents the ordered portion of UL36 (336 kDa). It binds between neighboring UL19 protrusions and to an adjacent UL17 molecule. These observations support the hypothesis that UL36 plays a major role in the tegumentation of the virion, providing a flexible scaffold to which other tegument proteins, including UL37, bind. They also indicate how sequential conformational changes in the maturing nucleocapsid control the ordered binding, first of UL25/UL17 and then of UL36.