Glasgow Theses Service

Characterisation of PP71 homologues encoded by mammalian cytomegaloviruses

Chaudry, Tanya N (2008) Characterisation of PP71 homologues encoded by mammalian cytomegaloviruses. PhD thesis, University of Glasgow.

Full text available as:
[img]
Preview
PDF
Download (3773Kb) | Preview

Abstract

Human cytomegalovirus (HCMV) is a human pathogen that can cause severe disease in immunocompromised or immunosupressed individuals and also in newborns infected in utero. Transcription of the viral genome occurs by a process in which three classes of HCMV genes, immediate early, early and late are expressed in a regulated temporal cascade. The HCMV protein pp71, encoded by gene UL82, is located in the tegument of the HCMV virion and is delivered to cells immediately upon infection. This protein has been identified as a transactivator of viral immediate early gene expression. It also stimulates expression from a number of heterologous promoters by a mechanism that is not promoter sequence specific. Protein pp71 has multiple properties; it can increase the infectivity of transfected viral DNA, modulate the cell cycle and interact with the retinoblastoma family of proteins. Within the cell nucleus, pp71 co-localises with the cellular proteins PML and hDaxx at sub-cellular structures named nuclear domain 10 (ND10). The interaction of pp71 with hDaxx is believed to promote the degradation of hDaxx, resulting in relief of repression at the HCMV major immediate early promoter. Protein pp71 has also been reported to have the unusual property of mediating long-term expression of reporter genes cloned into a herpes simplex virus type 1 (HSV 1) vector. This study describes a comparison of pp71 with the non-human UL82 homologues from simian CMV, baboon CMV, rhesus CMV and chimpanzee CMV, named S82, B82, RH82 and Ch82, respectively. Plasmids expressing all of the UL82 homologues as enhanced yellow fluorescent protein (EYFP) or myc-tagged proteins were constructed and analysed for expression by transfection into HFFF2 cells. The EYFP-tagged UL82 homologues all directed b-gal expression in short-term assays, while only pp71 directed both short-term and long-term gene expression. Only myc tagged pp71 was observed to direct gene expression in both the short-term and long-term assays. The EYFP-tagged proteins and myc tagged pp71 and Ch82 were cloned into a mutated HSV-1 vector to produce recombinant viruses. Functional assays in human glioblastoma (U373) cells confirmed that all of the EYFP-tagged and myc tagged non-human UL82 homologues were able to direct short-term expression but only EYFP- and myc-tagged pp71 directed long-term gene expression, confirming results obtained in transfection assays. In agreement with previous reports, pp71 was shown to promote the resumption of gene expression from quiescent HSV-1 genomes. Comparison of the pp71 and Ch82 homologues indicated that pp71 is unique in its ability to do so. No reactivation was observed in cells infected with an HSV-1 recombinant that expressed EYFP-tagged Ch82. In order to establish the region of pp71 responsible for mediating long-term gene expression six plasmids encoding EYFP-tagged hybrid proteins were constructed. The C-terminus, N-terminus and mid-regions of pp71 were substituted for the equivalent Ch82 regions using homologous restriction sites in both coding sequences. All EYFP-tagged hybrids mediated short-term gene expression, while only one protein, with the mid region of pp71 inserted between the C- and N-terminal regions of the Ch82 homologue, appeared to stimulate long-term gene expression. However, levels of expression were significantly lower than that achieved by pp71. A HSV-1 recombinant expressing the hybrid protein was used to confirm results from transfection assays, suggesting that the mid-region of pp71 may be involved in mediating its long-term properties. Given the significantly lower degree of gene expression directed by the hybrid protein in short-term assays it was concluded that alterations to pp71 may result in structural changes that prevent normal function of the protein. Immunofluorescence studies revealed further differences between the non-human UL82 homologues and pp71. In confirmation of previously published findings, in the majority of HFFF2 cells infected with a HSV-1 recombinant expressing EYFP tagged pp71, this protein localised to discrete punctate ND10 foci at all times tested. In cells infected with a HSV-1 recombinant expressing S82 a pattern distinct from that of pp71 was observed. S82 exhibited a punctate/diffuse pattern of fluorescence, which became increasingly diffuse at later times post-infection. The remaining non-human UL82 homologues, despite localising to the discrete punctate foci characteristic of pp71 at early times post infection, all showed nuclear distribution patterns akin to that of S82 at later times, in cells infected with the HSV-1 recombinants expressing EYFP-tagged Ch82, B82 and Rh82. All non-human UL82 homologues, like pp71, co-localised with the endogenous cellular proteins hDaxx and PML at the times tested. Interestingly, however, at later times post-infection, the S82 protein appeared to disperse hDaxx throughout the nucleus, a feature that was not observed with the remaining UL82 homologues. Examination of the hybrid protein observed to stimulate long-term gene expression revealed that, like pp71, it localised to discrete punctate foci, and co-localised with both PML and hDaxx at all times post-infection. In contrast to other published studies, it was not possible to demonstrate pp71-mediated hDaxx degradation, by either pp71 or the non-human UL82 homologues. The work presented in this thesis confirms the previous observation that pp71 directs long-term gene expression, reactivates quiescent genomes and co localises in the nucleus with hDaxx and PML. It also characterises the non-human UL82 homologues of pp71. This study shows that, while each non human UL82 homologue shares some characteristics with pp71, subtle functional differences exist between these proteins.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: HCMV, pp71, homologues, characterisation
Subjects: Q Science > QR Microbiology > QR355 Virology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Supervisor's Name: Preston, Dr Chris
Date of Award: 2008
Depositing User: Miss Tanya Chaudry
Unique ID: glathesis:2008-377
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 18 Sep 2008
Last Modified: 10 Dec 2012 13:18
URI: http://theses.gla.ac.uk/id/eprint/377

Actions (login required)

View Item View Item