INHIBITING HEPATITIS B VIRUS GENE EXPRESSION WITH HAMMERHEAD RIBOZYMES THAT TARGET THE HBx OPEN READING FRAME

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2002Hepatitis B virus (HBV) infection is endemic to several populous regions and is often complicated by cirrhosis and hepatocellular carcinoma (HCC). Present treatment of chronic HBV infection is usually ineffective and novel therapeutic approaches are an important medical objective. The X open reading frame (ORF) of HBV, HBx, is a conserved sequence that overlaps with the polymerase ORF and viral c/'s-elements, and is present within all viral transcripts. In addition, the HBx ORF encodes a 17 kDa transactivator protein, HBx, which is required for the establishment of viral infection and has been implicated in HBV-associated hepatocarcinogenesis. The HBx sequence thus represents a compelling target for applying nucleic acid hybridisation-based therapeutic agents for the inhibition of HBV gene expression and replication.IT201

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

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

Recent CMV Research

I am very pleased with this Viruses Special Issue. Of particular interest to families and caregivers affected by CMV diseases are several papers: addressing prevention of CMV infection of trophoblast cells (Zydek et al., 2014), CMV latency (Sinclair and Reeves, 2013), as well as of CMV lung infections in non-HIV infected children (Restrepo-Gualteros et al., 2014). Our ability to enhance immune responses for controlling CMV infection (Hanley and Bollard, 2014) and new strategies for CMV vaccine development guided by non-human primate studies (Deere and Barry, 2014) are discussed in two excellent reviews. Several articles address the CMV manipulation of the immune system, both innate and adaptive immune responses (Stevenson et al., 2014, Fink et al, 2013, 2014, Raghavan et al., 2014) and of DNA damage responses (E and Kowalik, 2014; Kulkarni and Fortunato, 2014). [...

Tailing cDNAs with terminal deoxynucleotidyl transferase in RT-PCR assays to identify ribozyme cleavage products.

Polytailing a cDNA with terminal deoxynucleotidyltransferase (TdT) results in the addition of a homopolymeric sequence at its 3'-end. Here we describe the use of tailing in competitive RT-PCR assays to evaluate cleavage efficiency of ribozymes. Using a system that perfectly mimics intracellular cleavage, we were able to detect as few as 1% of cleaved moieties. Furthermore, employing primers overlapping the junction between tails and the cleaved RNA moiety in non-competitive assays, the sensitivity of the method could be improved to <10 fg. Using the latter protocol and reactions employing a trans -acting hairpin ribozyme targeting the nucleocapsid mRNA of the mumps virus, we were able to demonstrate ribozyme-induced cleavage