Bacteriophage P22 portal vertex formation in vivo

Moore, Sean D.; Prevelige, Peter E.

doi:10.1006/jmbi.2001.5275

Cited by 40 publications

(57 citation statements)

References 87 publications

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“…A wealth of structural and functional data has been obtained for portal complexes of dsDNA 1 bacteriophages (e.g. 29, , SPP1, P22, T4, and T-odd phages (1)(2)(3)(4)(5)(6), but the molecular basis of the mechanochemical coupling is not understood. This is partly because of the complexity of portal complexes, in which the packaging ATPase is a multifunctional enzyme and associates transiently with the packaging machinery (i.e.…”

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Enzymatic Mechanism of RNA Translocation in Double-stranded RNA Bacteriophages

Lísal

Kainov

Bamford

et al. 2004

Journal of Biological Chemistry

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Many complex viruses acquire their genome by active packaging into a viral precursor particle called a procapsid. Packaging is performed by a viral portal complex, which couples ATP hydrolysis to translocation of nucleic acid into the procapsid. The packaging process has been studied for a variety of viruses, but the mechanism of the associated ATPase remains elusive. In this study, the mechanism of RNA translocation in doublestranded RNA bacteriophages is characterized using rapid kinetic analyses. The portal complex of bacteriophage 8 is a hexamer of protein P4, which exhibits nucleotide triphosphatase activity. The kinetics of ATP binding reveals a two-step process: an initial, fast, second-order association, followed by a slower, first-order phase. The slower phase exhibits a high activation energy and has been assigned to a conformational change. ATP binding becomes cooperative in the presence of RNA. Steady-state kinetics of ATP hydrolysis, which proceeds only in the presence of RNA, also exhibits cooperativity. On the other hand, ADP release is fast and RNA-independent. The steady-state rate of hydrolysis increases with the length of the RNA substrate indicating processive translocation. Raman spectroscopy reveals that RNA binds to P4 via the phosphate backbone. The ATP-induced conformational change affects the backbone of the bound RNA but leaves the protein secondary structure unchanged. This is consistent with a model in which cooperativity is induced by an RNA link between subunits of the hexamers and translocation is effected by an axial movement of the subunits relative to one another upon ATP binding.Genome encapsidation in many viruses is realized through energy-dependent packaging into a preformed capsid (procapsid). This requires molecular motors (portal complexes) converting chemical energy (ATP hydrolysis) into mechanical work (translocation). A wealth of structural and functional data has been obtained for portal complexes of dsDNA 1 bacteriophages (e.g. 29, , SPP1, P22, T4, and T-odd phages (1-6), but the molecular basis of the mechanochemical coupling is not understood. This is partly because of the complexity of portal complexes, in which the packaging ATPase is a multifunctional enzyme and associates transiently with the packaging machinery (i.e. the ATPase is a non-structural protein) (4). In particular, very little is known about the enzymatic action of these ATPases during packaging (7-9).The packaging system of dsRNA bacteriophages from the Cystoviridae family (phages 6-14 (10, 11)) is considerably simpler. The ssRNA translocation is fueled by a single structural protein P4. P4 forms stable hexamers (12, 13), which possess nonspecific nucleotide triphosphatase activity and resides at the 5-fold vertices of the procapsid (14, 15). The dodecahedral procapsid results from co-assembly of the major structural protein P1 with P4 hexamers and minor proteins P2 (RNA polymerase) and P7 (assembly and functional cofactor) (16,17).Cystoviruses have a tripartite dsRNA genome. The procapsid-associa...

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confidence: 99%

Enzymatic Mechanism of RNA Translocation in Double-stranded RNA Bacteriophages

Lísal

Kainov

Bamford

et al. 2004

Journal of Biological Chemistry

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“…The aberrant procapsids that form in the presence of excess P22 portal protein apparently contain extra portal vertices (23). This finding provided a unique opportunity to investigate the process of portal vertex formation in vivo.…”

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confidence: 95%

“…P22 is rather unique among the dsDNA bacteriophages in that the fidelity of head assembly is not affected by the absence of portal protein (23). In most other instances, when portal proteins are absent during assembly, coat and scaffold proteins assemble into aberrant spiral structures or long open-ended tubes (17,31).…”

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confidence: 99%

“…Therefore, portal proteins appear to play some role in head form determination. Interestingly, when wild-type P22 portal protein is overexpressed in trans during a P22 infection, the fidelity of procapsid assembly is greatly reduced, resulting in the formation of heads with the wrong triangulation (T) number and spiral shells (23). This finding suggests that the P22 portal protein can also play a role in form determination under certain circumstances.…”

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confidence: 99%

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

Moore

Prevelige

2002

J Virol

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Bacteriophage with linear, double-stranded DNA genomes package DNA into preassembled protein shells called procapsids. Located at one vertex in the procapsid is a portal complex composed of a ring of 12 subunits of portal protein. The portal complex serves as a docking site for the DNA packaging enzymes, a conduit for the passage of DNA, and a binding site for the phage tail. An excess of the P22 portal protein alters the assembly pathway of the procapsid, giving rise to defective procapsid-like particles and aberrant heads. In the present study, we report the isolation of escape mutant phage that are able to replicate more efficiently than wild-type phage in the presence of excess portal protein. The escape mutations all mapped to the same phage genome segment spanning the portal, scaffold, coat, and open reading frame 69 genes. The mutations present in five of the escape mutants were determined by DNA sequencing. Interestingly, each mutant contained the same mutation in the scaffold gene, which changes the glycine at position 287 to glutamate. This mutation alone conferred an escape phenotype, and the heads assembled by phage harboring only this mutation had reduced levels of portal protein and exhibited increased head assembly fidelity in the presence of excess portal protein.Because this mutation resides in a region of scaffold protein necessary for coat protein binding, these findings suggest that the P22 scaffold protein may define the portal vertices in an indirect manner, possibly by regulating the fidelity of coat protein polymerization.

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“…To successfully package a full-length genome, incorporation of one and only one connector vertex is essential (4). In vivo, nearly every assembled procapsid has one and only one connector vertex and is able to package DNA and mature into an infectious phage (5).…”

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A Docking Model Based on Mass Spectrometric and Biochemical Data Describes Phage Packaging Motor Incorporation

Uetrecht

Kang

et al. 2010

Molecular & Cellular Proteomics

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The molecular mechanism of scaffolding protein-mediated incorporation of one and only one DNA packaging motor/connector dodecamer at a unique vertex during lambdoid phage assembly has remained elusive because of the lack of structural information on how the connector and scaffolding proteins interact. We assembled and characterized a 29 connector-scaffolding complex, which can be incorporated into procapsids during in vitro assembly. Native mass spectrometry revealed that the connector binds at most 12 scaffolding molecules, likely organized as six dimers. A data-driven docking model, using input from chemical cross-linking and mutagenesis data, suggested an interaction between the scaffolding protein and the exterior of the wide domain of the connector dodecamer. The connector binding region of the scaffolding protein lies upstream of the capsid binding region located at the C terminus. This arrangement allows the C terminus of scaffolding protein within the complex to both recruit capsid subunits and mediate the incorporation of the single connector vertex. Molecular & Cellular Proteomics 9:1764 -1773, 2010.The DNA packaging motor of double-stranded DNA bacteriophages translocates genomic DNA into a preformed procapsid to near crystalline density and is the strongest motor characterized to date. The packaging motor of the Bacillus subtilis phage 29 can work against 57 piconewtons of internal force and translocate 2 bp of DNA per ATP hydrolyzed at a maximum velocity of 103 bp/s (1, 2). The motor complex is assembled on a dodecamer of the connector protein, which replaces a pentameric vertex in the procapsid and serves both as a portal for DNA passage and the docking site for the other packaging components (3).To successfully package a full-length genome, incorporation of one and only one connector vertex is essential (4). In vivo, nearly every assembled procapsid has one and only one connector vertex and is able to package DNA and mature into an infectious phage (5). This narrow distribution in which 95% of particles have a single connector vertex cannot be explained by random statistical incorporation. The control mechanism is coupled to the procapsid assembly process. Procapsid assembly requires the copolymerization of hundreds of copies each of the capsid and scaffolding proteins as well as a dodecamer of the portal or connector protein. The scaffolding protein acts to both activate the coat protein for assembly and ensure proper form determination. In the absence of scaffolding protein, uncontrolled polymerization results in the assembly of aberrant structures. In a properly assembled procapsid, the portal protein is located at one vertex, whereas scaffolding protein occupies the bulk of the interior space and is subsequently removed during DNA packaging by either proteolysis or simple release. Mutational studies have indicated that scaffolding protein is involved either directly or indirectly in the incorporation of the connector vertex during procapsid assembly in a variety of phages (6 -8).In 29, the con...

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Bacteriophage P22 portal vertex formation in vivo

Cited by 40 publications

References 87 publications

Enzymatic Mechanism of RNA Translocation in Double-stranded RNA Bacteriophages

Enzymatic Mechanism of RNA Translocation in Double-stranded RNA Bacteriophages

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

A Docking Model Based on Mass Spectrometric and Biochemical Data Describes Phage Packaging Motor Incorporation

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