Browsing by Author "Kotwal, Girish J"
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- ItemOpen AccessAn investigation into the specific function of the vaccinia virus :13.8 kDa protein encoded by the N1L gene(2005) Abrahams, Melissa-Rose Hilda; Kotwal, Girish JVaccinia virus is the most extensively studied, prototype vertebrate poxvirus, which was used as a vaccine in the eradication of smallpox. The genome of this virus has characteristic variable termini encoding open reading frames that are not essential for virus replication in cell culture. One such open reading frame, N1L situated at the left terminal region of the neurovirulent Western Reserve (WR) vaccinia virus strain, encodes a protein 13.8 kDa in size. In vivo studies in mouse brains revealed that a recombinant virus, vGK5, tacking the expression of the 13.8 kDa protein was rendered replication deficient in the brain. An essential requirement of poxviruses for their replication is the energy molecule adenosine triphosphate (ATP). The supply of this molecule in the brain to support replication of a virus is limited due to the high-energy requirements and small energy reserves of this organ. The specific function of the vaccinia virus 13.8 kDa protein in relation to viral replication in the brain was investigated. The South African (SA) Lister vaccinia virus strain was confirmed to encode an identical N1L gene to that of the WR vaccinia virus by amplification, cloning and sequencing of the Lister N1L open reading frame. The Lister vaccinia virus and a 13.8 kDa deletion strain (vGK5) were cultivated and used to intracranially infect mice. Using a luciferin/luciferase bioluminescence assay system the ATP levels in Lister and vGK5 vaccinia virus-infected mouse brains were measured and found to differ significantly after a 5-day infection period. The SA vaccine Lister vaccinia virus strain was found to be a slow growing virus in the brain. Subsequently, a possible role for the vaccinia virus 13.8 kDa protein in influencing ATP levels in the brain was postulated, yet a neurovirulent wild type strain is needed for further studies to consolidate this result. The 13.8 kDa protein was successfully expressed in the P. pastoris yeast expression system and positively identified by immunodetection studies.
- ItemOpen AccessCloning and expression of a functionally active truncated N-glycosylated KSHV complement regulatory protein and immunohistochemical studies with the anti-KCP peptide antibody(2005) Gomes Pereira, Neuza Alexandra; Kotwal, Girish JKaposi sarcoma herpes virus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. The KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement-binding protein, KCP) that binds complement proteins and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Kaposi's sarcoma is an angiogenic skin lesion that has been recognized as one of the most abundant tumours found in many parts of Southern Africa and which can occasionally become highly invasive, aggressive and capable of causing death, particularly amongst AIDS patients. It is of major significance to understand how complement control proteins (CCPs) such as KCP perform their biological functions at the molecular and structural levels, because of their potentials as therapeutic agents, their implications in the pathology and importance in the etiology of many disease conditions. This study was therefore undertaken to characterise the structure-function relationship of KCP. Based on primary sequence analysis and comparison to other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by PCR, three regions of the predicted ORF 4 from human herpes virus-8 (llliV-8) DNA isolated from a primary effusion lymphoma cell line. The PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris and to produce separately, the 4 N-terminal Sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane binding domain (KCP-M, medium) and the full-length protein (KCPF, full). Expression of the viral proteins was confirmed by SDS-PAGE and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. All the KCP proteins migrated electrophoretically as higher bands compared to their expected sizes. The lower mobilities of the proteins may be due to g1ycosy1ation since there are potential N-and O-glycosylation sites in the protein's primary sequence. Also, diffused bands were obtained in all the electrophoretic gels and Western blots carried out, which is characteristic of glycoproteins. Furthermore, the antibody recognized several larger and smaller bands that may represent aggregates and/or degradation products respectively. Both partially purified KCP-S and KCP-S directly from expression media were able to inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice more efficient in inhibiting the classical pathway-mediated lysis of erythrocytes than the vaccinia virus complement control protein (VCP), which also contains 4 Sushi domains. The KCP-F and KCP-M proteins did not show any significant complement inhibitory activities. Preliminary immunohistochemical studies using the same antibody were carried out to determine the expression and distribution of KCP proteins in Kaposi's sarcoma.
- ItemOpen AccessEvaluation of the in vivo role of vaccinia virus complement control protein (VCP) following renal ischemia(2006) Ghebremariam, Yohannes T; Kotwal, Girish J; Kahn, DelIn transplantation, vascularized organs often suffer the consequences of ischemic damage as well as reperfusion injury following the reestablishment of blood flow. The induced ischemialreperfusion (I/R) damage is usually associated with the accumulation of injurious complement components. The vaccinia virus complement control protein (VCP) has the ability to simultaneously inhibit the classical and the alternative complement pathways by binding to the early components C3b and C4b. The complement component C3 is known to be the central route to all of the known complement activation pathways. As a result, it is involved in a number of complement-mediated ailments including renal ischemia/reperfusion injury. The objectives of this study were to initially evaluate the in vitro roles of the natural VCP and the humanized recombinant VCP (hrVCP), and then to establish their in vivo roles in a renal I/R injury model.
- ItemOpen AccessIn vitro characterization of the antiviral activity of Secomet V against vaccinia virus infections(2004) Rangel Lopes de Campos, Walter; Kotwal, Girish JThe anti-poxvirus agent SECOMET V was the reference name for a plant extract produced in a bioreactor from primary plant stem cells, whose antiviral activity has been widely reported in folklore medicine. It exerted its anti-vaccinia virus activity by neutralizing cell-free virus rather than interfering with the downstream events following adsorption.
- ItemOpen AccessAn investigation into the specific function of the vaccinia virus 13.8 kDa protein encoded by the N1(2005) Abrahams, Melissa-Rose Hilda; Kotwal, Girish JVaccinia virus is the most extensively studied, prototype vertebrate poxvirus, which was used as a vaccine in the eradication of smallpox. The genome of this virus has characteristic variable termini encoding open reading frames that are not essential for virus replication in cell culture. One such open reading frame, N1L situated at the left terminal region of the neurovirulent Western Reserve (WR) vaccinia virus strain, encodes a protein 13.8 kDa in size. In vivo studies in mouse brains revealed that a recombinant virus, vGK5, tacking the expression of the 13.8 kDa protein was rendered replication deficient in the brain. An essential requirement of poxviruses for their replication is the energy molecule adenosine triphosphate (ATP). The supply of this molecule in the brain to support replication of a virus is limited due to the high-energy requirements and small energy reserves of this organ. The specific function of the vaccinia virus 13.8 kDa protein in relation to viral replication in the brain was investigated. The South African (SA) Lister vaccinia virus strain was confirmed to encode an identical N1L gene to that of the WR vaccinia virus by amplification, cloning and sequencing of the Lister N1L open reading frame. The Lister vaccinia virus and a 13.8 kDa deletion strain (vGK5) were cultivated and used to intracranially infect mice. Using a luciferin/luciferase bioluminescence assay system the ATP levels in Lister and vGK5 vaccinia virus-infected mouse brains were measured and found to differ significantly after a 5-day infection period. The SA vaccine Lister vaccinia virus strain was found to be a slow growing virus in the brain. Subsequently, a possible role for the vaccinia virus 13.8 kDa protein in influencing ATP levels in the brain was postulated, yet a neurovirulent wild type strain is needed for further studies to consolidate this result. The 13.8 kDa protein was successfully expressed in the P. pastoris yeast expression system and positively identified by immunodetection studies.
- ItemOpen AccessAn investigation of the in vivo role of vaccinia virus complement control protein in head injury and Alzheimer's disease(2006) Pillay, Nirvana Shanalee; Kotwal, Girish J; Kellaway, Laurie AIncludes bibliographical references.