An Investigation Into Factors That Influence the Incorporation of Proteins Into the HSV-1 Tegument

Leslie, Jenny (1996) An Investigation Into Factors That Influence the Incorporation of Proteins Into the HSV-1 Tegument. PhD thesis, University of Glasgow.

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The tegument of herpesvirus particles is an amorphous region between the capsid and envelope and consists of a complex array of virus-encoded polypeptides. It has no apparent regular structure but, along with the envelope components, the tegument proteins of herpes simplex virus type 1 (HSV-1) can assemble to give non-infectious particles termed light particles that lack nucleocapsids. Although most of the HSV-1 genes that specify tegument proteins have been identified, little is known about the mode of entry of the proteins into virus particles. To examine the processes controlling incorporation into the tegument, manipulatable experimental systems that would enable the characterisation of sequences that direct proteins to the tegument were developed. Initial studies involved the construction of a fusion gene composed of the sequences from a tegument gene (UL41, vhs) linked to a non-viral, non-structural gene (chloramphenicol acetyltransferase (CAT)). The resultant fusion product, vhs-CAT, had an apparent molecular weight of 84KDa and retained CAT activity. By analysis of fractions across Ficoll gradients which were used to purify virions and light particles, the fusion protein was shown to be present in both types of particle. The protein could be detected by both Western blotting and enzymatic assays but was not incorporated in quantities that could be measured by staining methods. Subsequently, the commercial antibody which was used to detect the CAT component of the fusion protein was found to have poor avidity for the CAT protein and gave unreliable data. This necessitated the production of a new CAT monoclonal antibody which delayed progress and prevented further use of CAT as a marker polypeptide. To examine the processes controlling incorporation into the tegument, studies were initiated on VP22, a major tegument protein encoded by UL49. Using a virus vector called 1802, a HSV-1 recombinant, vUL49ep, was constructed that expresses two copies of UL49; one copy specified the unmodified form of VP22 under the control of the native promoter while the second was placed under the control of the human cytomegalovirus (HCMV) immediate early (IE) promoter. To distinguish between the two versions of VP22, the inserted copy was tagged at the C-terminus with an epitope from the HCMV UL83 gene product. In cells infected with the recombinant virus, the overall levels of VP22 synthesised were about 5-fold higher than for wild type virus and this increase was due to the high levels of expression of the tagged protein. Comparison of the polypeptide compositions of particles revealed that the amount of VP22 in the tegument was approximately 2- to 3-fold higher in recombinant virions and light particles than in particles produced by wild-type virus. To demonstrate that the tag sequence did not influence the incorporation of VP22 into the tegument, a HSV-1 recombinant virus containing the epitope linked to CAT was constructed. In this case, the distribution of epitope-tagged CAT across Ficoll gradients did not correspond with that of virions and light particles made by the virus and therefore epitope-tagged CAT could not be classified as a structural component. The high abundance of VP22 in vUL49ep virus particles correlated with a reduction in the quantities of VP 13/14 present in virions and light particles and there was a decrease in the amount of untagged VP22 that was incorporated. In addition, the recombinant virions and light particles had noticeably increased migration on Ficoll gradients. These results provided the first evidence that, for certain proteins, the level of polypeptide synthesis could act as a positive controlling factor for the amount of protein incorporated into the tegument. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John McLauchlan
Keywords: Virology
Date of Award: 1996
Depositing User: Enlighten Team
Unique ID: glathesis:1996-75470
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 20:01
Last Modified: 19 Nov 2019 20:01

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