A P22 Scaffold Protein Mutation Increases the Robustness of Head Assembly in the Presence of Excess Portal Protein

Moore, Sean D.; Prevelige, Peter E.

doi:10.1128/jvi.76.20.10245-10255.2002

Cited by 41 publications

(34 citation statements)

References 33 publications

(64 reference statements)

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“…Morphologically normal (T ϭ 7; T, triangulation number) procapsids are formed both in vivo and in vitro in the absence of the portal protein, indicating that the portal is not obligatorily involved in the initiation process (8,9). The portal was found to have little effect on the rate of procapsid formation, and results with mutants in which portal incorporation was affected suggested that the portal could be incorporated at any stage of assembly (1,8). It is possible, therefore, that viruses differ in the manner in which the portal becomes incorporated into the nascent procapsid.…”

Section: Discussionmentioning

confidence: 99%

“…The mechanism of portal assembly into a polyhedral capsid has been studied for many years in dsDNA bacteriophages, such as T4, 29, P22, and SPP1 (2,7,8,9,25,33). As in the case of HSV-1, bacteriophage portals are (i) cone-shaped structures formed from 12 or 13 copies of a single polypeptide, the portal protein; (ii) located at a unique vertex of the capsid polyhedron, where they serve as a channel for DNA to enter and exit; and (iii) a Capsids were assembled in vitro as described in Materials and Methods in the presence or absence of added portals.…”

Section: Discussionmentioning

confidence: 99%

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Involvement of the Portal at an Early Step in Herpes Simplex Virus Capsid Assembly

Newcomb

Homa

Brown

2005

J Virol

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DNA enters the herpes simplex virus capsid by way of a ring-shaped structure called the portal. Each capsid contains a single portal, located at a unique capsid vertex, that is composed of 12 UL6 protein molecules. The position of the portal requires that capsid formation take place in such a way that a portal is incorporated into one of the 12 capsid vertices and excluded from all other locations, including the remaining 11 vertices. Since initiation or nucleation of capsid formation is a unique step in the overall assembly process, involvement of the portal in initiation has the potential to cause its incorporation into a unique vertex. In such a mode of assembly, the portal would need to be involved in initiation but not able to be inserted in subsequent assembly steps. We have used an in vitro capsid assembly system to test whether the portal is involved selectively in initiation. Portal incorporation was compared in capsids assembled from reactions in which (i) portals were present at the beginning of the assembly process and (ii) portals were added after assembly was under way. The results showed that portal-containing capsids were formed only if portals were present at the outset of assembly. A delay caused formation of capsids lacking portals. The findings indicate that if portals are present in reaction mixtures, a portal is incorporated during initiation or another early step in assembly. If no portals are present, assembly is initiated in another, possibly related, way that does not involve a portal.Assembly of herpes simplex virus type 1 (HSV-1) can be considered to begin with formation of the capsid. Capsids are produced in the infected cell nucleus, where they are also packaged with the double-stranded DNA (dsDNA) genome. Promptly after DNA is introduced, capsids transit to the cytoplasm, where they are enveloped to form mature virions (5, 21, 23).The HSV-1 capsid is closely similar in structure to the capsids of other herpesviruses. It is icosahedral in shape, and its major structural features are 162 capsomers. The capsomers are of three types: (i) hexons that form the capsid faces and edges, (ii) pentons that are located at 11 of the 12 capsid vertices, and (iii) the portal found at one of the 12 vertices. The 150 hexons are hexamers of the major capsid protein (UL19), while the 11 pentons are UL19 pentamers. The portal is a 12-mer of UL6, the portal protein. The portal is about the size of a hexon or penton and cylindrical, with an axial channel through which HSV-1 DNA passes as it is introduced into the capsid (32). The capsomers are connected in groups of three by the triplexes, trigonal structures that lie on the outer surface of the capsid floor (15,27,32,36).The mechanism of HSV-1 capsid formation has been examined in infected cells, in insect cells infected with recombinant baculoviruses (rBV) encoding HSV-1 capsid proteins, and in an in vitro assembly system (14,28,31,34). Such studies have demonstrated that assembly of closed icosahedral capsids requires the major capsid protein, the tr...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Involvement of the Portal at an Early Step in Herpes Simplex Virus Capsid Assembly

Newcomb

Homa

Brown

2005

J Virol

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“…An expanding list of putative helicases may not be involved in duplex unwinding, but rather in RNA or DNA translocation, modulating RNA-protein interactions, or other functions (13,14,16,39,53,56). Moreover, a wide variety of ds-and ssDNA viruses and dsRNA viruses use hexameric NTPases or NTPase complexes to package their nucleic acid into preformed empty capsids (26,29,43). In at least some of these cases, the NTPase activity is related to helicase functions.…”

Section: Discussionmentioning

confidence: 99%

Brome Mosaic Virus 1a Nucleoside Triphosphatase/Helicase Domain Plays Crucial Roles in Recruiting RNA Replication Templates

Wang

Lee

Watanabe

et al. 2005

J Virol

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Positive-strand RNA virus RNA replication is invariably membrane associated and frequently involves viral proteins with nucleoside triphosphatase (NTPase)/helicase motifs or activities. Brome mosaic virus (BMV) encodes two RNA replication factors: 1a has a C-terminal NTPase/helicase-like domain, and 2a pol has a central polymerase domain. 1a accumulates on endoplasmic reticulum membranes, recruits 2a pol , and induces 50-to 70-nm membrane invaginations (spherules) serving as RNA replication compartments. 1a also recruits BMV replication templates such as genomic RNA3. In the absence of 2a pol , 1a dramatically stabilizes RNA3 by transferring RNA3 to a membrane-associated, nuclease-resistant state that appears to correspond to the interior of the 1a-induced spherules. Prior results show that the 1a NTPase/helicase-like domain contributes to RNA recruitment. Here, we tested mutations in the conserved helicase motifs of 1a to further define the roles of this domain in RNA template recruitment. All 1a helicase mutations tested showed normal 1a accumulation, localization to perinuclear endoplasmic reticulum membranes, and recruitment of 2a pol . Most 1a helicase mutants also supported normal spherule formation. Nevertheless, these mutations severely inhibited RNA replication and 1a-induced stabilization of RNA3 in vivo. For such 1a mutants, the membrane-associated RNA3 pool was both reduced and highly susceptible to added nuclease. Thus, 1a recruitment of viral RNA templates to a membrane-associated, nuclease-resistant state requires additional functions beyond forming spherules and recruiting RNA to membranes, and these functions depend on the 1a helicase motifs. The possibility that, similar to some double-stranded RNA viruses, the 1a NTPase/helicase-like domain may be involved in importing viral RNAs into a preformed replication compartment is discussed.Positive-strand RNA viruses are a large class of viral pathogens causing numerous clinically and economically important diseases of humans, animals, and plants. Although such viruses encompass substantial variation in morphology, genetic organization, host range, and other properties, they all share fundamental similarities in their basic replication mechanisms. For example, genome replication by positive-strand RNA viruses is universally associated with intracellular membranes, which usually are induced by viral replication proteins to form invaginations or vesicles (51). In the early steps of positive-strand RNA virus replication, the viral genomic RNAs first serve as templates for translating these replication proteins and often other viral proteins. Once such proteins induce formation of the membraneassociated replication complexes, the incoming viral genomic RNA must be recruited away from translation to serve as a template for RNA replication. This genomic RNA transition from translation to RNA replication is a crucial step in early infection and must be tightly regulated to effectively balance translation and replication (44). Nevertheless, the mechanisms of...

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“…2D and Movie S2). The presence of the scaffolding proteins here explains why the portal is not incorporated into procapsids in the absence of scaffolding proteins (40) and the mutation (G287E) in the loop, proximal to the portal, of the helix-loop-helix motif in the C-terminal portion of scaffolding protein influences the portal incorporation into procapsids (41).…”

Section: Interactions Between Scaffolding Proteins and The Unique Pormentioning

confidence: 99%

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.

197

319

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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...

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A P22 Scaffold Protein Mutation Increases the Robustness of Head Assembly in the Presence of Excess Portal Protein

Cited by 41 publications

References 33 publications

Involvement of the Portal at an Early Step in Herpes Simplex Virus Capsid Assembly

Involvement of the Portal at an Early Step in Herpes Simplex Virus Capsid Assembly

Brome Mosaic Virus 1a Nucleoside Triphosphatase/Helicase Domain Plays Crucial Roles in Recruiting RNA Replication Templates

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

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