Cryo-EM structure of a bacteriophage T4 gp24 bypass mutant: The evolution of pentameric vertex proteins in icosahedral viruses

Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054, USA.
Journal of Structural Biology (Impact Factor: 3.23). 07/2006; 154(3):255-9. DOI: 10.1016/j.jsb.2006.01.008
Source: PubMed


Many large viral capsids require special pentameric proteins at their fivefold vertices. Nevertheless, deletion of the special vertex protein gene product 24 (gp24) in bacteriophage T4 can be compensated by mutations in the homologous major capsid protein gp23. The structure of such a mutant virus, determined by cryo-electron microscopy to 26 angstroms, shows that the gp24 pentamers are replaced by mutant major capsid protein (gp23) pentamers at the vertices, thus re-creating a viral capsid prior to the evolution of specialized major capsid proteins and vertex proteins. The mutant gp23* pentamer is structurally similar to the wild-type gp24* pentamer but the insertion domain is slightly more distant from the gp23* pentamer center. There are additional SOC molecules around the gp23* pentamers in the mutant virus that were not present around the gp24* pentamers in the wild-type virus.

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    • "It is likely that the special gp24 vertex protein of phage T4 is a relatively recent evolutionary addition as judged by the ease with which it can be bypassed. Cryo-electron microscopy showed that in the bypass mutants that substitute pentamers of the major capsid protein at the vertex, additional Soc decoration protein subunits surround these gp23* molecules, which does not occur in the gp23*-gp24* interfaces of the wild-type capsid [7]. Nevertheless, despite the rationalization of major capsid protein affecting head size mutations, it should be noted that these divert only a relatively small fraction of the capsids to altered and variable sizes. "
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    ABSTRACT: The bacteriophage T4 capsid is an elongated icosahedron, 120 nm long and 86 nm wide, and is built with three essential proteins; gp23*, which forms the hexagonal capsid lattice, gp24*, which forms pentamers at eleven of the twelve vertices, and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. The past twenty years of research has greatly elevated the understanding of phage T4 head assembly and DNA packaging. The atomic structure of gp24 has been determined. A structural model built for gp23 using its similarity to gp24 showed that the phage T4 major capsid protein has the same fold as that found in phage HK97 and several other icosahedral bacteriophages. Folding of gp23 requires the assistance of two chaperones, the E. coli chaperone GroEL and the phage coded gp23-specific chaperone, gp31. The capsid also contains two non-essential outer capsid proteins, Hoc and Soc, which decorate the capsid surface. The structure of Soc shows two capsid binding sites which, through binding to adjacent gp23 subunits, reinforce the capsid structure. Hoc and Soc have been extensively used in bipartite peptide display libraries and to display pathogen antigens including those from HIV, Neisseria meningitides, Bacillus anthracis, and FMDV. The structure of Ip1*, one of the components of the core, has been determined, which provided insights on how IPs protect T4 genome against the E. coli nucleases that degrade hydroxymethylated and glycosylated T4 DNA. Extensive mutagenesis combined with the atomic structures of the DNA packaging/terminase proteins gp16 and gp17 elucidated the ATPase and nuclease functional motifs involved in DNA translocation and headful DNA cutting. Cryo-EM structure of the T4 packaging machine showed a pentameric motor assembled with gp17 subunits on the portal vertex. Single molecule optical tweezers and fluorescence studies showed that the T4 motor packages DNA at a rate of up to 2000 bp/sec, the fastest reported to date of any packaging motor. FRET-FCS studies indicate that the DNA gets compressed during the translocation process. The current evidence suggests a mechanism in which electrostatic forces generated by ATP hydrolysis drive the DNA translocation by alternating the motor between tensed and relaxed states.
    Full-text · Article · Dec 2010 · Virology Journal
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    • "The T4 genome is completely sequenced and the functions of the majority of its genes are known (Miller, Kutter, et al. 2003). The structure and function of many of T4's essential components have been dissected in genetic, biochemical, enzymological (Karam and Konigsberg 2000; Fokine et al. 2004, 2006; Letarov et al. 2005), crystallographic (Shamoo et al. 1995; Karam and Konigsberg 2000; Thomassen et al. 2003; Fokine et al. 2005), and electron micrographic studies (Leiman et al. 2003; Fokine et al. 2004, 2006; Rossmann et al. 2004). Apart from T4 itself, a number of other distant T4-like phages have been studied in recent years, including Aeromonas spp. "
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    ABSTRACT: The Escherichia coli bacteriophage T4 has served as a classic system in phage biology for more than 60 years. Only recently have phylogenetic analyses and genomic comparisons demonstrated the existence of a large, diverse, and widespread superfamily of T4-like phages in the environment. We report here on the T4-like major capsid protein (MCP) sequences that were obtained by targeted polymerase chain reaction (PCR) of marine environmental samples. This analysis was then expanded to include 1,000 s of new sequences of T4-like capsid genes from the metagenomic data obtained during the Sorcerer II Global Ocean Sampling (GOS) expedition. This data compilation reveals that the diversity of the major and minor capsid proteins from the GOS metagenome follows the same general patterns as the sequences from cultured phage genomes. Interestingly, the new MCP sequences obtained by PCR targeted to MCP sequences in environmental samples are more divergent (deeper branching) than the vast majority of the MCP sequences coming from the other sources. The marine T4-like phage population appears to be largely dominated by the T4-like cyanophages. Using approximately 1,400 T4-like MCP sequences from various sources, we mapped the degree of sequence conservation on a structural model of the T4-like MCP. The results indicate that within the T4 superfamily there are some clear phylogenetic groups with regard to the more conserved and more variable domains of the MCP. Such differences can be correlated with variations in capsid morphology, the arrangement of the MCP lattice, and the presence of different capsid accessory proteins between the subgroups of the T4 superfamily.
    Full-text · Article · Aug 2008 · Molecular Biology and Evolution
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    ABSTRACT: Viral genomes are encapsidated within protective protein shells. This encapsidation can be achieved either by a co-condensation reaction of the nucleic acid and coat proteins, or by first forming empty viral particles which are subsequently packaged with nucleic acid, the latter mechanism being typical for many dsDNA bacteriophages. Bacteriophage PRD1 is an icosahedral, non-tailed dsDNA virus that has an internal lipid membrane, the hallmark of the Tectiviridae family. Although PRD1 has been known to assemble empty particles into which the genome is subsequently packaged, the mechanism for this has been unknown, and there has been no evidence for a separate packaging vertex, similar to the portal structures used for packaging in the tailed bacteriophages and herpesviruses. In this study, a unique DNA packaging vertex was identified for PRD1, containing the packaging ATPase P9, packaging factor P6 and two small membrane proteins, P20 and P22, extending the packaging vertex to the internal membrane. Lack of small membrane protein P20 was shown to totally abolish packaging, making it an essential part of the PRD1 packaging mechanism. The minor capsid proteins P6 was shown to be an important packaging factor, its absence leading to greatly reduced packaging efficiency. An in vitro DNA packaging mechanism consisting of recombinant packaging ATPase P9, empty procapsids and mutant PRD1 DNA with a LacZ-insert was developed for the analysis of PRD1 packaging, the first such system ever for a virus containing an internal membrane. A new tectiviral sequence, a linear plasmid called pBClin15, was identified in Bacillus cereus, providing material for sequence analysis of the tectiviruses. Analysis of PRD1 P9 and other putative tectiviral ATPase sequences revealed several conserved sequence motifs, among them a new tectiviral packaging ATPase motif. Mutagenesis studies on PRD1 P9 were used to confirm the significance of the motifs. P9-type putative ATPase sequences carrying a similar sequence motif were identified in several other membrane containing dsDNA viruses of bacterial, archaeal and eukaryotic hosts, suggesting that these viruses may have similar packaging mechanisms. Interestingly, almost the same set of viruses that were found to have similar putative packaging ATPases had earlier been found to share similar coat protein folds and capsid structures, and a common origin for these viruses had been suggested. The finding in this study of similar packaging proteins further supports the idea that these viruses are descendants of a common ancestor. Virukset muodostuvat perimäaineksesta, DNA:sta tai RNA:sta, ja sitä ympäröivästä proteiinikuoresta. Viruksen perimän pakkaaminen proteiinikuoren sisään on tärkeä osa viruksen elinkiertoa solussa, ja kuori varjelee perimäainesta ympäristön rasituksilta viruksen ollessa solun ulkopuolella. Virus voi rakentua joko siten, että kuoriproteiinimolekyylit sitoutuvat perimään, ja perimä ja kuoriproteiinit tiivistyvät valmiiksi virukseksi, tai rakentamalla pallomainen, tyhjä proteiinikuori ensin, minkä jälkeen perimä pakataan kuoren sisään. Bakteriofagi eli bakteerivirus PRD1 on muodoltaan ikosaedri (12-kulmio), mutta poikkeuksellisen PRD1:stä tekee sen proteiinikuoren alla oleva rasvakalvo. Vaikka PRD1:stä on tiedetty, että virus rakentuu tyhjien kuorien eli prokapsidien kautta, tarkkaa mekanismia PRD1:n tai minkään sisäkalvollisen viruksen DNA:n pakkaukselle ei ole aikaisemmin tunnettu. Tässä väitöskirjatyössä on tunnistettu useita PRD1:n pakkauksessa toimivia virusproteiineja sekä havaittu niiden sijaitsevan yhdessä kulmassa, erillään kuoren muissa kulmissa sijaitsevista isäntäsolun tunnistukseen tarvittavista proteiineista. Työssä on tutkittu PRD1:n eri pakkausproteiinien toimintaa, ja mm. kehitetty PRD1:lle ns. in vitro -pakkausmenetelmä, jossa DNA:ta voidaan pakata tyhjiin prokapsideihin koeputkessa, ilman eläviä soluja. Virukset voivat lysogenisoitua, eli vaipua lepotilaan, jossa uusia viruksia ei tuoteta eikä isäntäsolu tuhoudu. Joskus tässä tilassa olevan viruksen perimään voi osua haitallinen mutaatio, joka estää virusta aktivoitumasta. Työssä tunnistettiin bioinformatiikan keinoin PRD1:n ja sen sukulaisen Bam35-viruksen kaltaiseksi eräs Bacillus cereus -bakteerin perimästä löytynyt osa, pBCLin15. pBClin15:n arvellaan olevan joko lepotilassa oleva virus tai mutaatioita kerännyt viruksen jäänne. Työssä havaittiin myös Bam35:n voivan lysogenisoitua. PRD1:n kuoriproteiinin rakenteen on aiemmin todettu olevan hyvin samankaltainen mm. ihmisen adenoviruksen kuoriproteiinin kanssa, ja onkin ehdotettu, että nämä virukset olisivat sukua toisilleen. Sama kuorityyppi on sittemmin löydetty monista eläinten tai muiden aitotumallisten eliöiden viruksista. Tässä tutkimuksessa verrattiin bioinformatiikan keinoin eri virusten DNA-pakkausproteiineja, ja PRD1-tyyppisiä pakkausproteiineja todettiin löytyvän bakteerien, eläinten, levien ja jopa arkkien viruksista. Kyseessä olivat samat virukset, joilla on todettu olevan samankaltainen kuoriproteiini, ja joista monilla on PRD1:n kaltainen sisäkalvo. Tämä havainto tukee siten aiemmin esitettyjä ajatuksia näiden virusten periytymisestä samasta kantaviruksesta. Vaikka PRD1 onkin bakteerien virus, voi samankaltaisten pakkausproteiinien löytyminen muista viruksista viitata siihen, että näiden virusten perimän pakkaus toimii samalla tavoin. Siten PRD1:stä saatu tieto voi auttaa ymmärtämään muidenkin virusten pakkausta, ja jopa tarjota kohteen uusille viruslääkkeille. Ensisijaisesti tämä työ on kuitenkin perustutkimusta ja lisää ymmärtämystämme virusten toiminnasta ja niiden evoluutiosta.
    Full-text · Article · Jan 2006
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