Picobirnavirus is an unclassified dsRNA virus, which is associated with viral gastroenteritis in humans and animals. Picobirnavirus dsRNA has been detected in many cases when diagnostic PAGE screening for rotavirus dsRNA is performed. During this routine diagnosis, picobirnavirus dsRNA has been detected in the faeces of patients with and without viral gastroenteritis. Despite the common occurrence of picobirnavirus infection in humans and animals, its direct involvement in causing gastroenteritis has not been established. No molecular studies have been done on picobirnavirus except sequencing and epidemiology studies. Like all RNA viruses, picobirnavirus encodes a RNA-dependent RNA polymerase. The picobirnavirus RNA-dependent RNA polymerase has only been identified on the basis of its amino acid sequence. The catalytic activity of the polymerase has not been studied to date. In this study, a porcine picobirnavirus was studied at a molecular level to establish the activity of the protein encoded by segment 2 of its genome. To determine the identity of this putative picobirnavirus RNA-dependent RNA polymerase, its open reading frame (ORF) was successfully amplified by PCR, cloned and sequenced. Subsequently the ORF was successfully sub-cloned into baculovirus and bacterial expression vectors. The protein encoded by picobirnavirus segment 2 was successfully expressed as a recombinant protein in a soluble form in both baculovirus and bacterial expression systems. In the baculovirus system, two recombinant baculoviruses were constructed. One recombinant baculovirus expressing a histidine tagged protein and another one expressing an untagged protein. In bacterial expression systems, a recombinant protein fused to a Glutathione-S-Transferase (GST) tag at the N terminal end was expressed. The GST tag allowed easy purification of the expressed GST fusion protein by affinity chromatography on immobilized glutathione. Subsequently the GST tag could be removed from the purified recombinant protein by proteolysis with thrombin. Both tagged and untagged putative picobirnavirus RNA-dependent RNA polymerase from the bacterial expression system were shown to have an affinity for heparin. This implies that the protein might have an affinity for nucleic acids. Picobirnavirus genome segment 2 ssRNA was generated from full length picobirnavirus genome segment 2 cDNA by in vitro transcription. The recombinant E.coli expressed proteins (tagged and untagged) was tested for ssRNA binding and RNA replicase activity. No RNA binding or replicase activity was observed with either tagged or untagged recombinant protein. This study reports the first evidence other than conserved polymerase motifs, that the protein encoded by segment 2 of picobirnavirus is most likely a RNA-dependent RNA polymerase. Copyright / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/26310 |
| Date | 15 July 2009 |
| Creators | Phosiwa, Maanda Noaxe |
| Contributors | Potgieter, A.C., phosiwam@arc.agric.za |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2008, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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