Capsid fine structure of T-even bacteriophages binding and localization of two dispensable capsid proteins into the P23* surface lattice

Aebi, Ueli; Driel, Roel van; Bijlenga, R.K.L.; Heggeler, B. ten; Broek, R. van den; Steven, Alasdair C.; Smith, Paul

doi:10.1016/s0022-2836(77)80084-3

Cited by 63 publications

(36 citation statements)

References 20 publications

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“…The T4 phage soc and hoc proteins are examples of such phage-encoded proteins. These bind to the capsid surface after DNA has entered and are thought to reinforce the capsid structure (1,10). The location of UL25 at multiple distinct sites in the capsid suggests a similarity to soc, hoc, and other similar proteins, such as the phage lambda D protein (9) and L phage Dec protein (7), that are also located at multiple sites.…”

Section: Discussionmentioning

confidence: 99%

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

Newcomb

Homa

Brown

2006

J Virol

105

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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.Like replication of double-stranded DNA bacteriophage, herpesvirus replication involves a step in which progeny DNA molecules are introduced into a preformed capsid (4, 29). DNA encapsidation takes place in the infected-cell nucleus, where the virus DNA is synthesized and where progeny capsids are assembled. Once a capsid is filled with DNA, it transits to the cytoplasm, where it acquires tegument and membrane layers prior to exiting the host cell.Studies of herpes simplex virus 1 (HSV-1) replication have led to the identification of seven genes involved specifically in DNA encapsidation (UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes) (2,4,5,13,15,27,28,30,38). When cells are infected with a mutant unable to produce any one of the seven gene products, capsids are assembled normally and the virus DNA is replicated but capsids are not filled. Concatemeric DNA and closed capsids accumulate in the infected-cell nucleus, indicating that the missing gene is involved specifically in DNA encapsidation and not, for instance, in capsid formation or DNA replication. Homologs for six of the seven HSV-1 DNA processing/packaging proteins are encoded by all herpesviruses (6).Analysis of the DNA encapsidation process has defined the functions for three of the processing/packaging proteins, UL6, UL15, and UL28. UL6 is the structural subunit of the portal, a funnel-shaped 12-mer found at one of the 12 capsid vertices (22). DN...

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Section: Discussionmentioning

confidence: 99%

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

Newcomb

Homa

Brown

2006

J Virol

105

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“…Another is that the capsid-binding sites for BPP may become available only after the protein(s) involved has been incorporated into the capsid and has possibly also undergone maturational rearrangements. Such conformational changes occur in both bacteriophage (1,4,5,39) and eukaryotic viruses, including HSV (42), and may help drive the assembly process forward. If BPP or other proteins do associate with nascent particles through sites formed (Fig.…”

Section: Discussionmentioning

confidence: 99%

Cytomegalovirus Basic Phosphoprotein (pUL32) Binds to Capsids In Vitro through Its Amino One-Third

Baxter

Gibson

2001

J Virol

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The cytomegalovirus (CMV) basic phosphoprotein (BPP) is a component of the tegument. It remains with the nucleocapsid fraction under conditions that remove most other tegument proteins from the virion, suggesting a direct and perhaps tight interaction with the capsid. As a step toward localizing this protein within the molecular structure of the virion and understanding its function during infection, we have investigated the BPP-capsid interaction. In this report we present evidence that the BPP interacts selectively, through its amino one-third, with CMV capsids. Radiolabeled simian CMV (SCMV) BPP, synthesized in vitro, bound to SCMV B-capsids, and C-capsids to a lesser extent, following incubation with either isolated capsids or lysates of infected cells. Human CMV (HCMV) BPP (pUL32) also bound to SCMV capsids, and SCMV BPP likewise bound to HCMV capsids, indicating that the sequence(s) involved is conserved between the two proteins. Analysis of SCMV BPP truncation mutants localized the capsid-binding region to the amino one-third of the molecule-the portion of BPP showing the greatest sequence conservation between the SCMV and HCMV homologs. This general approach may have utility in studying the interactions of other proteins with conformation-dependent binding sites.The basic phosphoprotein (BPP) is an abundant constituent of the cytomegalovirus (CMV) virion. On the basis of its presence in preparations of virions, noninfectious enveloped particles, and cytoplasmic nucleocapsids, but not immature nuclear capsids or dense bodies, BPP was classified as a tegument protein (13,16,21). More recent evidence from cryoelectron microscopy suggests that it contacts the capsid through the distal end of the capsomers or through the triplex subunits that interlink them (8,43). BPP is phosphorylated in vivo (12,24,35), like many other herpesvirus tegument proteins (e.g., references 17, 29, and 31), and is a predominant phosphate acceptor in vitro for the virion-associated protein kinase(s) (35).BPP homologs are recognized in other betaherpesviruses (e.g, see Fig. 2) but not in alpha-or gammaherpesviruses. The human CMV (HCMV) BPP homolog is encoded by open reading frame (ORF) UL32 (7, 23) and is predicted to have a mass of 113 kDa. However, its relative electrophoretic mobility is closer to that of the 150-kDa major capsid protein (MCP; pUL86) and is influenced by the specific conditions of electrophoresis (21, 24), potentially complicating its identification on the basis of size or relative electrophoretic mobility alone. BPP is expressed late (6, 36) and has been detected in the nuclei of infected cells (20,33), although at later times it accumulates in the perinuclear region of the cytoplasm (20,36,38). Unlike its simian CMV (SCMV) counterpart, HCMV BPP is modified by the attachment of O-linked N-acetylglucosamine to two serines near its carboxyl end (2, 19). It elicits a strong humoral immune response (16,24,27), and there is evidence from the effects of antisense sense RNAs (30) and a spontaneous mutation in the gene...

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“…Two proteins, highly antigenic outer capsid protein (hoc, 39.1 kDa) and small outer capsid protein (soc, 9.7 kDa), are attached to the capsid surface (8)(9)(10). Whereas the soc protein helps to stabilize the capsid against extremes of pH and temperature, hoc only has a marginal effect on head stability (11,12).…”

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confidence: 99%

Molecular architecture of the prolate head of bacteriophage T4

Fokine

Chipman

Leiman

et al. 2004

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

266

322

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The head of bacteriophage T4 is a prolate icosahedron with one unique portal vertex to which the phage tail is attached. The three-dimensional structure of mature bacteriophage T4 head has been determined to 22-Å resolution by using cryo-electron microscopy. The T4 capsid has a hexagonal surface lattice characterized by the triangulation numbers Tend ‫؍‬ 13 laevo for the icosahedral caps and Tmid ‫؍‬ 20 for the midsection. Hexamers of the major capsid protein gene product (gp)23* and pentamers of the vertex protein gp24*, as well as the outer surface proteins highly antigenic outer capsid protein (hoc) and small outer capsid protein (soc), are clearly evident in the reconstruction. The size and shape of the gp23* hexamers are similar to the major capsid protein organization of bacteriophage HK97. The binding sites and shape of the hoc and soc proteins have been established by analysis of the soc ؊ and hoc ؊ soc ؊ T4 structures. Bacteriophage T4 is a large, tailed, double-stranded DNA (dsDNA) virus (family Myoviridae) that uses Escherichia coli as a host. The mature T4 virion, which contains Ϸ50 different proteins, consists of a prolate capsid, 172-kbp genomic DNA, and a tail with a contractile sheath terminating in a base plate to which are attached six long tail fibers. The architecture and the molecular composition of the T4 head, tail, and fibers have been characterized extensively by using a variety of techniques (1-3) leading to a structural model (4). Recent studies of the T4 components by cryo-electron microscopy (cryo-EM) and x-ray crystallography extended the structural knowledge to higher resolution (5).T4 has one of the most complex structures of any virus that has been studied. There are Ͼ2,000 protein molecules of at least five different gene products (gps) in the head alone. The molecular mass of the DNA-filled head is 194 MDa and of the capsid alone is 82 MDa (6). The T4 head assembly proceeds via a number of intermediate stages. First, a DNA-free precursor, or prohead, is assembled that is processed proteolytically. Next, the genomic DNA is packaged into the prohead in a process that requires ATP energy (1). The prohead assembly is initiated by the portal protein gp20. The prohead contains an internal core made up of the major core protein, gp22, the minor core proteins, gpalt, a serine-type protease, gp21, and other internal proteins (1). The major capsid protein, gp23, is assembled around the scaffolding core together with the minor capsid protein, gp24. After completion of prohead assembly, the inactive gp21 enzyme is converted to the active protease, which cleaves the scaffold proteins into small peptides. A 65-residue-long amino-terminal ''⌬-piece'' is also cleaved from the 56-kDa gp23 molecule, thus yielding 48.7-kDa gp23* (1) ʈ . In addition, a 2.2-kDa amino-terminal piece of the 48.4-kDa gp24 is cleaved, giving rise to gp24* during head maturation. Most of the small peptides produced by the gp21 protease are expelled from the prohead, thus providing space necessary to accommodate the geno...

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Capsid fine structure of T-even bacteriophages binding and localization of two dispensable capsid proteins into the P23* surface lattice

Cited by 63 publications

References 20 publications

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

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

Cytomegalovirus Basic Phosphoprotein (pUL32) Binds to Capsids In Vitro through Its Amino One-Third

Molecular architecture of the prolate head of bacteriophage T4

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