An Investigation Into the Structure and Assembly of the Herpes Simplex Virus Type 1 (HSV-1) Capsid Using the Baculovirus Expression System

Tatman, Jacqueline Dawn (1996) An Investigation Into the Structure and Assembly of the Herpes Simplex Virus Type 1 (HSV-1) Capsid Using the Baculovirus Expression System. PhD thesis, University of Glasgow.

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Intermediate (type B) capsids of HSV-1 are composed of seven proteins encoded by six genes. The proteins encoded by UL18 (VP23), UL19 (VP5), UL35 (VP26) and UL38 (VP19C) are components of the outer capsid shell whereas those specified by UL26 (VP21 and VP24) and UL26.5 (pre-VP22a) are involved in the formation of the scaffold which forms the internal core of B capsids. This scaffold plays a pivotal role in capsid assembly and is removed from the capsid concomitant with the packaging of DNA to form mature (type C) capsids. These, upon acquiring tegument and envelope proteins, exit from the cell as infectious virions. In order to gain a comprehensive understanding of the protein interactions that are important for the capsid assembly process, it was necessary to devise a system in which to examine the structures formed by, and the interactions taking place between, the capsid proteins in isolation from other herpesvirus proteins. The initial aim of this work was to clone and express the four outer shell capsid proteins in baculovirus so that in conjunction with baculoviruses recombinant for the genes UL26 and UL26.5 (which had been made previously by V. Preston) a complete panel of recombinant baculoviruses would exist. Each recombinant produced a protein profile with a unique band of the correct size, as judged by its co-migration with the respective protein in preparations of purified HSV-1 capsids. In addition, the autoprocessing by the protease into VP21 and VP24 and the processing of the scaffolding protein, into VP22a; an event which is an essential requirement for DNA packaging, occurred as in wild type HSV-1. Co-expression of the six genes in insect cells resulted in the formation of capsids that were indistinguishable in appearance, as viewed in the electron microscope, and protein composition from those made during HSV-1 infection of mammalian cells. This demonstrated that the proteins encoded by the known capsid genes contain all the structural information necessary for capsid assembly and that other virus-encoded proteins are not required for this process. The requirements for capsid assembly were then analysed in further depth. Omission of single recombinant baculoviruses from this system allowed the role of individual HSV-1 proteins in capsid assembly to be determined. Capsid assembly did not take place in the absence of VP5, VP19C or VP23, whereas lack of VP26 had no discernible effect on capsid formation. These results agreed with findings from the analysis of HSV-1 mutants with lesions in the UL19, UL38 and UL18 genes which had demonstrated that these genes were essential for capsid assembly. Lack of a UL35 mutant virus meant that it was previously not known whether this protein was required for capsid assembly. Capsids which assembled in the absence of the UL26 gene products had a large-cored phenotype resembling that previously described for the HSV-1 mutant ts1201 which has a lesion in this gene. Some apparently intact capsid shells were also made in the absence of the major scaffolding protein, pre-VP22a thereby demonstrating that the outer shell proteins could polymerise into the correct conformation in the absence of this protein. However, in these experiments, VP21 which is identical to VP22a except for a short N-terminal extension, was present and could have compensated for the lack of the scaffolding protein. Indeed when both UL26 and UL26.5-expressing baculoviruses were omitted from the reaction, this resulted in the appearance of large numbers of partial and deformed capsid shells which suggested that the products of one or other of these genes is required to direct correct capsid shell assembly. Capsids made in the baculovirus system were purified and analysed by collaborators W. Chiu and H. Zhou who had previously performed high resolution (2.6nm) three-dimensional cryo-electron microscopic analysis on wild type HSV-1 capsids. Comparison of the three-dimensional structures of wild type HSV-1 B capsids and of baculovirus generated capsids lacking VP26 revealed the presence of a horn shape mass of density present on all the hexons, but not the pentons, in the wild type capsid which was absent from the hexons in the VP26 negative capsid. Analysis of the difference map of the two capsids revealed a star shape mass of density on the distal tip of the hexons composed of six copies of VP26. In addition to confirming the location of VP26 this also provided information as to the structure of this protein; which appears to consist of a large and small domain which interact with each other and with the underlying VP5 molecules to form bridges between the hexon subunits. The baculovirus system was also used to examine whether any of the capsid proteins could interact to form discrete sub-capsid structures. Co-infection with the viruses expressing the products of the UL26 and UL26.5 genes resulted in the formation of 40- 60nm diameter spheres which resembled the internal scaffold-cores of HSV-1 B capsids.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Frazer Rixon
Keywords: Virology
Date of Award: 1996
Depositing User: Enlighten Team
Unique ID: glathesis:1996-75600
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:20
Last Modified: 19 Nov 2019 19:20

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