Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796688
Title: Analysis of four capsid protein genes of HSV-1
Author: Nicholson, Peter
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1992
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Abstract:
The initial goal of this work was the cloning and expression of the genes UL18, UL19 and UL38 of HSV-1, These genes encode the three capsid proteins VP23, VP5 (the major capsid protein) and VP19C respectively, which are all present in cored and in coreless intranuclear capsids. Cloning having been achieved, each gene was expressed in a recombinant vaccinia virus; each recombinant producing a protein profile with a unique band of the correct size, as judged by its comigration with the respective protein in preparations of purified capsids. A further recombinant vaccinia virus was constructed, which expresses the HSV-1 assembly protein VP22a, the product of the gene UL26.5. Expression of VP22a by this recombinant was confirmed using a monoclonal antibody (5010) against VP22a. These viruses were used in a study of HSV-1 capsid structure and assembly. One aim of this research was to be able to assemble HSV-1 capsids using capsid genes cloned and expressed in a heterologous system. However, electron microscopy demonstrated that the products of the four capsid genes UL18, UL19, UL26.5 and UL38 are insufficient for the assembly of capsids or capsid-like particles in the vaccinia expression system. Possible reasons for this, and implications for the capsid assembly process, are discussed. VP19C has previously been reported to be a DNA-binding protein. Attempts were made to confirm this using the VP19C expressed from a recombinant vaccinia virus, but VP19C did not exhibit significant DNA-binding activity in this system. The vaccinia virus expressing VP23 was used to identify VP23 as the target antigen for a previously unassigned monoclonal antibody (1060). This antibody was then used to study the properties of the UL18 gene product in HSV-1-infected cells. UL18 mRNA has been reported to be regulated with early-late kinetics in lytic infections. In an attempt to confirm this, the kinetics of synthesis of VP23 were examined. VP23 was detectable by immunoprecipitation from 2 to 3 hours post-infection (personal communication, Dr A.M.Cross). Production of VP23 was not affected by the presence of the DNA synthesis inhibitor phosphonoacetic acid. Thus VP23 is produced early in infection and is not dependent on viral DNA synthesis, in agreement with the designation of this gene as an early-late. To investigate the subcellular distribution of VP5, VP19C, VP23 and VP22a when expressed from the vaccinia vectors, cells infected with the recombinant viruses were separated into cytoplasmic and nuclear fractions. A control experiment using wild-type HSV-1 showed these proteins to separate predominantly with the nuclear fraction. By contrast, in recombinant-vaccinia-infected cells expressing VP5 or VP23, these proteins were almost exclusively cytoplasmic. However, when expressed by a recombinant vaccinia virus, VP19C and VP22a showed intrinsic ability to locate to the nucleus, the site of capsid assembly. The availability of antibodies against VP23 and VP22a allowed further investigation of the subcellular distributions of these proteins by immunofluorescence experiments on infected cells. These experiments again showed that when expressed by the recombinant vaccinia virus, VP23 remains localised in the cytoplasm in contrast to the situation obtaining during infections with wild-type HSV-1, when VP23 localised to the nucleus. It was thought unlikely that this failure of the herpes protein to accumulate in the nucleus was a consequence of the vaccinia vector interfering with the normal transport mechanisms since other herpesvirus proteins expressed from the same vector are known to enter and to accumulate in the nucleus. However, to eliminate this possibility the distribution of VP23 expressed from a plasmid vector was examined. The UL18 coding sequences were placed under the control of the HSV-1 glycoprotein D promoter, and this construct was cotransfected into cells along with plasmids expressing the two HSV-1 immediate early transactivating proteins VmwllO and Vmwl75. When the distribution of VP23 expressed in these cells was examined by fluorescent staining with 1060, it was identical to that in the recombinant vaccinia-infected cells, ie predominantly cytoplasmic. Use of 5010, directed against VP22a in immunofluorescence experiments showed that when expressed from the vaccinia vector, VP22a localises to the nucleus as in wild-type infections, in direct contrast to the cytoplasmic location of VP23. This observation provides further confirmation that the vaccinia vector does not interfere with the processes of transport of the foreign protein. This result has been confirmed by immunoelectron microscopy. A significant finding was that immunofluorescence studies suggest that VP22a is able to affect the subcellular location of VP23. Use of 1060 showed that while VP23 adopted a cytoplasmic location when expressed by a recombinant vaccinia virus, in a dual infection with a VP22a-expressing recombinant VP23 localised predominantly in the nucleus. VP22a is thought to function as a scaffolding protein during HSV capsid assembly, and this result suggests that it may also function in intracellular transportation of another capsid protein.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796688  DOI: Not available
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