Purification and organization of the gene 1 portal protein required for phage P22 DNA packaging

Bazinet, Christopher; Benbasat, Julyet Aksiyote; King, Jonathan; Carazo, José Marı́a; Carrascosa, José L.

doi:10.1021/bi00406a009

Cited by 78 publications

(52 citation statements)

References 60 publications

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“…Brown, unpublished observations). U L 6 contrasts in this respect with the P22 portal protein (gp1), in which monomers and other oligomers are observed (1,19,20). The presence of intact portals in scaffold-portal particles is most consistent with the view that U L 6 is incorporated into the nascent capsid as a preformed, intact dodecamer.…”

Section: Discussionmentioning

confidence: 60%

Assembly of the Herpes Simplex Virus Capsid: Identification of Soluble Scaffold-Portal Complexes and Their Role in Formation of Portal-Containing Capsids

Newcomb

Thomsen²,

Homa³

et al. 2003

J Virol

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The herpes simplex virus type 1 (HSV-1) portal complex is a ring-shaped structure located at a single vertex in the viral capsid. Composed of 12 U L 6 protein molecules, the portal functions as a channel through which DNA passes as it enters the capsid. The studies described here were undertaken to clarify how the portal becomes incorporated as the capsid is assembled. We tested the idea that an intact portal may be donated to the growing capsid by way of a complex with the major scaffolding protein, U L 26.5. Soluble U L 26.5-portal complexes were found to assemble when purified portals were mixed in vitro with U L 26.5. The complexes, called scaffold-portal particles, were stable during purification by agarose gel electrophoresis or sucrose density gradient ultracentrifugation. Examination of the scaffold-portal particles by electron microscopy showed that they resemble the 50-to 60-nm-diameter "scaffold particles" formed from purified U L 26.5. They differed, however, in that intact portals were observed on the surface. Analysis of the protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that portals and U L 26.5 combine in various proportions, with the highest observed U L 6 content corresponding to two or three portals per scaffold particle. Association between the portal and U L 26.5 was antagonized by WAY-150138, a small-molecule inhibitor of HSV-1 replication. Soluble scaffold-portal particles were found to function in an in vitro capsid assembly system that also contained the major capsid (VP5) and triplex (VP19C and VP23) proteins. Capsids that formed in this system had the structure and protein composition expected of mature HSV-1 capsids, including U L 6, at a level corresponding to ϳ1 portal complex per capsid. The results support the view that U L 6 becomes incorporated into nascent HSV-1 capsids by way of a complex with U L 26.5 and suggest further that U L 6 may be introduced into the growing capsid as an intact portal.All herpesviruses have the same basic structure, a rigid icosahedral capsid surrounded by a membrane envelope. The capsid contains the virus DNA in a highly condensed form in which it is protected from mechanical and other damage. The capsid also functions to release the viral DNA into the host cell nucleus shortly after a new cycle of infection is initiated (33,34).The structure of the herpes simplex virus type 1 (HSV-1) capsid has been studied extensively and was found to be similar to that of other herpesviruses (38,44,49). Composed entirely of protein, the HSV-1 capsid is an icosahedral shell ϳ125 nm in diameter and 15 nm thick. Its major structural features are 162 capsomers (150 hexons and 12 pentons) that lie on a T ϭ 16 icosahedral lattice. The hexons are found on the capsid edges and faces, while the pentons are located at the vertices. The capsomers are connected in groups of three by the triplexes, trivalent structures composed of U L 18 and U L 38 that lie above the capsid floor.The 150 hexons resemble each other in that all have...

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Section: Discussionmentioning

confidence: 60%

Assembly of the Herpes Simplex Virus Capsid: Identification of Soluble Scaffold-Portal Complexes and Their Role in Formation of Portal-Containing Capsids

Newcomb

Thomsen²,

Homa³

et al. 2003

J Virol

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“…[148][149][150][151][152][153][154][155] A DNA-translocating motor, powered by ATP hydrolysis, accomplishes this energetically unfavorable DNA transportation process. A RNA molecule called pRNA has been shown to have a novel and indispensable role in phi29 DNA packaging motor.…”

Section: Motor Prna Of Bacteriophages Phi29mentioning

confidence: 99%

RNA Nanotechnology: Engineering, Assembly and Applications in Detection, Gene Delivery and Therapy

Guo

2005

j nanosci nanotechnol

159

135

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Biological macromolecules including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. RNA is unique in nanoscale fabrication due to its amazing diversity of function and structure. RNA molecules can be designed and manipulated with a level of simplicity characteristic of DNA while possessing versatility in structure and function similar to that of proteins. RNA molecules typically contain a large variety of single stranded loops suitable for inter-and intra-molecular interaction. These loops can serve as mounting dovetails obviating the need for external linking dowels in fabrication and assembly.The self-assembly of nanoparticles from RNA involves cooperative interaction of individual RNA molecules that spontaneously assemble in a predefined manner to form a larger two-or threedimensional structure. Within the realm of self-assembly there are two main categories, namely template and non-template. Template assembly involves interaction of RNA molecules under the influence of specific external sequence, forces, or spatial constraints such as RNA transcription, hybridization, replication, annealing, molding, or replicas. In contrast, non-template assembly involves formation of a larger structure by individual components without the influence of external forces. Examples of non-template assembly are ligation, chemical conjugation, covalent linkage, and loop/loop interaction of RNA, especially the formation of RNA multimeric complexes. The best characterized RNA multiplier and the first to be described in RNA nanotechnological application is the motor pRNA of bacteriophage phi29 which form dimers, trimers, and hexamers, via hand-in-hand interaction. phi29 pRNA can be redesigned to form a variety of structures and shapes including twins, tetramers, rods, triangles, and 3D arrays several microns in size via interaction of programmed helical regions and loops. 3D RNA array formation requires a defined nucleotide number for twisting and a palindromic sequence. Such arrays are unusually stable and resistant to a wide range of temperatures, salt concentrations, and pH. Both the therapeutic siRNA or ribozyme and a receptorbinding RNA aptamer or other ligands have been engineered into individual pRNAs. Individual chimeric RNA building blocks harboring siRNA or other therapeutic molecules have been fabricated subsequently into a trimer through hand-in-hand interaction of the engineered right and left interlocking RNA loops. The incubation of these particles containing the receptor-binding aptamer or other ligands results in the binding and co-entry of trivalent therapeutic particles into cells. Such particles were subsequently shown to modulate the apoptosis of cancer cells in both cell cultures and animal trials. The use of such antigen-free 20-40 nm particles holds promise for the repeated long-term treatment of chronic diseases. Other potentially useful RNA molecules that form multimers include HIV RNA that contain kissing loop to form di...

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“…Enzymes involved in the viral DNA packaging process, so-called terminases, are responsible for site-specific duplex nicking and insertion of the DNA into the procapsids, (4,5). Terminases were first described for double-stranded DNA bacteriophages and are highly conserved throughout many double-stranded DNA (dsDNA) viruses, e.g., herpesviruses and adenoviruses (6)(7)(8)(9)(10)(11)(12)(13).…”

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confidence: 99%

Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93

Köppen-Rung

Dittmer

Bogner

2016

J Virol

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DNA packaging into procapsids is a common multistep process during viral maturation in herpesviruses. In human cytomegalovirus (HCMV), the proteins involved in this process are terminase subunits pUL56 and pUL89, which are responsible for sitespecific cleavage and insertion of the DNA into the procapsid via portal protein pUL104. However, additional viral proteins are required for the DNA packaging process. We have shown previously that the plasmid that encodes capsid-associated pUL77 encodes another potential player during capsid maturation. Pulse-chase experiments revealed that pUL77 is stably expressed during HCMV infection. Time course analysis demonstrated that pUL77 is expressed in the early late part of the infectious cycle. The sequence of pUL77 was analyzed to find nuclear localization sequences (NLSs), revealing monopartite NLSm at the N terminus and bipartite NLSb in the middle of pUL77. The potential NLSs were inserted into plasmid pHM829, which encodes a chimeric protein with ␤-galactosidase and green fluorescent protein. In contrast to pUL56, neither NLSm nor NLSb was sufficient for nuclear import. Furthermore, we investigated by coimmunoprecipitation whether packaging proteins, as well as pUL93, the homologue protein of herpes simplex virus 1 pUL17, are interaction partners of pUL77. The interactions between pUL77 and packaging proteins, as well as pUL93, were verified. IMPORTANCEWe showed that the capsid-associated pUL77 is another potential player during capsid maturation of HCMV. Protein UL77 (pUL77) is a conserved core protein of HCMV. This study demonstrates for the first time that pUL77 has early-late expression kinetics during the infectious cycle and an intrinsic potential for nuclear translocation. According to its proposed functions in stabilization of the capsid and anchoring of the encapsidated DNA during packaging, interaction with further DNA packaging proteins is required. We identified physical interactions with terminase subunits pUL56 and pUL89 and another postulated packaging protein, pUL93, in infected, as well as transfected, cells. Human cytomegalovirus (HCMV) belongs to the betaherpesviruses and is characterized by its narrow host range and prolonged replication cycle (1-3). Viral DNA packaging is a key step in HCMV replication. Enzymes involved in the viral DNA packaging process, so-called terminases, are responsible for site-specific duplex nicking and insertion of the DNA into the procapsids, (4, 5). Terminases were first described for double-stranded DNA bacteriophages and are highly conserved throughout many double-stranded DNA (dsDNA) viruses, e.g., herpesviruses and adenoviruses (6-13).The HCMV enzymes required for this process, terminase subunits pUL56 and pUL89 (14-18), have already been identified. Together with the portal protein pUL104, they form the most powerful biological nanomotor (19,20). Terminase subunit pUL56 provides the ATP-hydrolyzing activity required for DNA translocation (21,22). The toroidal structure of pUL56 is in accordance with its func...

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Purification and organization of the gene 1 portal protein required for phage P22 DNA packaging

Cited by 78 publications

References 60 publications

Assembly of the Herpes Simplex Virus Capsid: Identification of Soluble Scaffold-Portal Complexes and Their Role in Formation of Portal-Containing Capsids

Assembly of the Herpes Simplex Virus Capsid: Identification of Soluble Scaffold-Portal Complexes and Their Role in Formation of Portal-Containing Capsids

RNA Nanotechnology: Engineering, Assembly and Applications in Detection, Gene Delivery and Therapy

Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93

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