Nucleotide sequence of the bacteriophage P22 genes required for DNA packaging

Eppler, Kathryn; Wyckoff, Elizabeth E.; Goates, Jeffery; Parr, Ryan; Casjens, Sherwood

doi:10.1016/0042-6822(91)90981-g

Cited by 127 publications

(138 citation statements)

References 94 publications

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“…The nucleotide sequence includes an open reading frame encoding a protein of 609 amino acids with a molecular weight of 64,366. Recently the eight aminoterminal amino acids of gp16 isolated from phage particles have been determined (Eppler et al, 1991). This amino acid sequence agrees with that deduced from the nucleotide sequence beginning at nucleotide 377 (Fig.…”

Section: Discussionsupporting

confidence: 83%

Cloning, sequencing, and overexpression of gene 16 of salmonella bacteriophage P22

Umlauf

Dreiseikelmann

1992

Virology

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It has been suggested that gene product 16 of bacteriophage P22 forms a pore for DNA transfer and/or that it functions as a pilot protein guiding the DNA across the membrane. We have cloned gene 16 and determined the nucleotide sequence. Within the sequenced region there is an open reading frame that could encode a protein of 609 amino acids having a molecular weight of 64,366. The hydropathic plot of this protein does not reveal putative membrane-spanning regions as expected for a protein forming a membrane pore. Overproduction of gene product 16 in Escherichia co/i was successful only in a mutant in which the La protease was inactivated. Gene 16 mutants of phage P22 were not able to infect recBCD mutants of Salmonella typhimurium nor was protein 16, synthesized in E. colifrom a plasmid, able to substitute for the pilot protein of phage T4. It seems that gene product 16 is not a pilot protein in the meaning of binding to the ends of linear DNA, thus protecting it from degradation by nucleases.o 1992 Academic PWS. IW.

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Section: Discussionsupporting

confidence: 83%

Cloning, sequencing, and overexpression of gene 16 of salmonella bacteriophage P22

Umlauf

Dreiseikelmann

1992

Virology

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“…As the scaffolding protein densities at the pentamers appear at a lower isosurface value, it may be possible that there is only partial occupancy of scaffolding protein at the pentamers. Regardless, this number of scaffolding proteins is likely higher than 250-300 copies as previously deduced from biochemical data (34,35).…”

Section: Resultsmentioning

confidence: 91%

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.

194

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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“…These enzymes are normally hetero-oligomeric complexes with multiple functions (17)(18)(19)(20). Although the exact mechanism of DNA translocation is not known, it is commonly accepted that this process is energy-dependent.…”

Section: Discussionmentioning

confidence: 99%

“…DNA packaging of double-stranded DNA bacteriophage requires the terminase, a complex of two proteins (14 -16). Most bacteriophage terminases are hetero-oligomers with each subunit carrying a different function (17)(18)(19)(20). The large subunit catalyzes the ATP-dependent translocation of genomic DNA into the bacteriophage procapsids and the small unit binds and cleaves concatenated DNA (21,22).…”

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confidence: 99%

ATPase Activity of the Terminase Subunit pUL56 of Human Cytomegalovirus

Hwang¹,

Bogner²

2002

Journal of Biological Chemistry

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Herpesviral DNA packaging is a complex process resulting in unit-length genomes packed into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of double-stranded DNA bacteriophages, the translocation of DNA was shown to be an energy-dependent process associated with an ATPase activity of the large terminase subunit. In the case of human cytomegalovirus it was not known which protein has the ability to hydrolyze ATP. In this study we expressed human cytomegalovirus terminase subunits, pUL89 and the carboxyl-terminal half of pUL56, as GST fusion proteins and purified these by affinity chromatography. ATPase assays demonstrated that the enzymatic activity is exclusively associated with pUL56. The characterization of the ATP hydrolysis showed that the enzymatic reaction is a fast process, whereas the spontaneous ATP decay followed slow kinetics. Interestingly, although pUL89 did not show any ATPase activity, it was capable of enhancing the UL56-associated ATP hydrolysis. Furthermore, a specific association of in vitro translated pUL89 with the carboxyl-terminal half of GST-UL56C was detected. This interaction was confirmed by co-immunoprecipitations of infected cells. Our results clearly demonstrated that (i) both terminase subunits interact with each other and (ii) the subunit pUL56 has an ATPase activity. Human cytomegalovirus (HCMV)1 DNA replication results in the formation of large head-to-tail DNA concatemers. The subsequent maturation into unit-length molecules involves site-specific cleavage at sequence motifs (pac motifs) located within the a sequence of the terminal and internal repeat segments (1-4). Unit-length genomes are then encapsidated into preassembled capsids. DNA-packaging is a complex biological process. While it is commonly accepted that ATP hydrolysis is the driving force behind it, the molecular mechanisms of DNA translocation and genome packaging remain unclear. In general, the following five steps are involved. (i) The recognition of concatemeric DNA by a specific protein (complex) able to (ii) bind and cut the DNA at specific sequence motifs (packaging signals, e.g. pac1 and pac2), (iii) translocation of the DNAprotein complex into the procapsid, (iv) packaging of one unitlength genome, and (v) completion of the packaging process by cutting off excess DNA at the portal region (5-7).Recently, we have demonstrated that the HCMV pUL56 gene product (pUL56), the homolog of the HSV-1 open reading frame UL28, is associated with sequence-specific binding of DNA containing packaging motifs leading to the suggestion that pUL56 plays a key role in DNA packaging (7-9). Comparable results were reported of the HSV-1 pUL28, demonstrating a direct interaction of pUL28 with DNA containing the pac1 motif (10). Furthermore, by the use of viral mutants it was demonstrated that the deletion of pUL28 leads to nuclear accumulation of naked nucleocapsids and uncleaved concatemeric DNA (11, 12). In addition, it has been noted that the HCMV UL...

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Nucleotide sequence of the bacteriophage P22 genes required for DNA packaging

Cited by 127 publications

References 94 publications

Cloning, sequencing, and overexpression of gene 16 of salmonella bacteriophage P22

Cloning, sequencing, and overexpression of gene 16 of salmonella bacteriophage P22

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

ATPase Activity of the Terminase Subunit pUL56 of Human Cytomegalovirus

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