Identification and Characterisation of a Novel RNA-Binding Partner for the US11 Protein of HSV-1

Attrill, Helen Louise (2001) Identification and Characterisation of a Novel RNA-Binding Partner for the US11 Protein of HSV-1. PhD thesis, University of Glasgow.

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The US11 protein herpes simplex virus type-1 is a small, highly basic phosphoprotein, which localises to the nucleoli of infected cells. Although this non-essential protein has a number of activities attributed to it, its actual role during infection is unknown. The two major functions that have been associated with US 11 are the antagonism of host-mediated translational shut-off and the ability to bind RNA, the latter of which is the focus of this thesis. To date, US11 has been shown to bind five RNAs; the antisense transcript of the US11 5' UTR, a truncated transcript of the UL34 gene, termed A34, the Rev-response element of human immunodeficiency virus type-1, the Rex-response element of human T-cell lymphotrophic virus type-1 and rRNA derived from the 60S ribosome subunit. The function of the RNA-binding activity of US1 1 during infection is as yet unclear. The RNAs bound by USl 1 appear, at least superficially to possess little in common, both in terms of their sequence and the biological influence of US11 on them. US11 may potentially interact with other RNAs in infected cells and the aim of this work was to attempt to test this hypothesis. Firstly, methods in which US11-RNA complexes could be isolated from infected cell lysates were examined. Using a GST-US11 fusion protein to pull out interacting RNAs from lysates it proved possible to isolate the known binder, ?34 RNA. Secondly, a reverse transcription-polymerase chain reaction (RT-PCR) method was developed to allow the amplification of sequences pulled out with GST-US11. This lead to the identification of a 585nt sequence that was present in three HSV-13' co-terminal genes, UL12, UL13 and UL14, which encode alkahne nuclease, a protein kinase and a nuclear protein of unknown function, respectively. The interaction between this RNA, termed 12/14 RNA, and US 11 was examined in vitro. The binding was found to be sequence-specific and mediated by the C-terminal domain of US11. US11 bound 12/14 RNA in a multimeric fashion, for which the N-terminal domain was not required. The affinity of the C-terminal domain of US11 for 12/14 RNA is less that that for A34 RNA, indicating that these RNAs may be bound in a slightly different fashion. The binding site for US11 was mapped to a 232nt region which encompasses 108nt of the UL12 5' UTR, 225nt of the 3' ORP and 7nt of the 3' UTR of UL13 and lies within the 3' UTR of UL14. The interaction between this shorter RNA and US11 was examined and the binding found to be dependent on secondary structure. The influence of US 11 on the expression of UL12, UL13 and UL14 was examined in infected cells. In the absence of US11 the levels of UL12 and UL14 remain unchanged, but the expression of UL13 is elevated at early times post-infection. It therefore appears that US11 can down-regulate the expression of the UL13 protein kinase at early times during infection.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Richard Elliott
Keywords: Virology
Date of Award: 2001
Depositing User: Enlighten Team
Unique ID: glathesis:2001-75746
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15

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