Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796693
Title: Expression of the Bunyamwera virus M genome segment gene products
Author: Nakitare, George W.
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1992
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Abstract:
Bunyamwera virus is the prototype of both the Bunyavirus genus and the Bunyaviridae family, which comprises more than 300 viruses. The virus possesses a negative stranded RNA genome of three segments designated Large (L), Middle (M) and Small (S). The M segment codes for the two virion glycoproteins, G1 and G2, and a non-structural polyprotein, termed NSm. The mature gene products are derived from a polyprotein precursor which is probably cotranslationally cleaved as it has not been detected in virus-infected cells. Genetic studies have indicated that the M segment gene products are involved in many of important biological attributes of the virus, including determinants of virulence, neutralisation and haemagglutination. The M segment RNA of bunyaviruses has a different gene organisation from those of Phiebovirus genus in having NSm in the middle of the precursor, whereas some members of the Phlebovirus genus have NSm at the beginning of the precursor. The Uukuniemi group of phieboviruses and members of the Hantavirus genus have no NSm. A common feature among members of the Bunyaviridae is maturation at the smooth surfaced membranes of the Golgi complex. The glycoproteins encoded by the M segment RNA are thought to be the main determinants of this feature. It was therefore of interest to express the Bunyamwera virus M RNA segment in order to study the biogenesis of the glycoproteins and NSm, and with the long-term view of studying and targetting of these proteins to the Golgi. The overall aims of this work were to express the M RNA segment of Bunyamwera virus from a cloned cDNA; to determine the cellular residence of NSm; and to investigate the transport of the proteins from the endoplasmic reticulum to the Golgi complex. No immunological reagents specific for NSm were available, and two approaches were utilized in the production of specific antisera to NSm. One involved synthesis of fusion proteins with beta-galactosidase using the pUEX3 vector system, and the second synthesis of branching peptides to selected sequences of NSm. Rabbits were immunized separately with these immunogens and the antisera characterised by enzyme-linked immunosorbent assays and radioimmunoprecipitations. Antisera to fusion proteins failed to recognise NSm in immunoprecipitations. However, immune sera raised against the branching peptides recognised NSm specifically in Bunyamwera virus-infected cells by radioimmunoprecipi tation. The antipeptide sera were used in indirect immunofluorescence experiments to determine that NSm localized to the Golgi complex in virus-infected cells; this was confirmed by dual staining with rhodamine conjugated wheat germ agglutinin which predominantly stains the Golgi complex. For in vitro translation of mRNA synthesized from Bunyamwera virus cDNA, the full length cDNA was cloned in the pT7T3 transcription vector under the control of the T3 RNA promoter. Full length and various truncated transcripts were made using T3 RNA polymerase. Attempts to express the transcripts in vitro were unsuccessful in both rabbit reticulocyte lysate and in wheat germ cell free translation systems. However, a transcript representing nucleotides 1352 to 4001 of the M segment mRNA, with an internal in-frame AUG at nucleotides 1430-1432, was translated in vitro in the rabbit reticulocyte lysate system to yield a 90K protein which was immunoprecipitated by antivirus serum. These experiments suggested that sequences at the 5' end of the M segment mRNA inhibited translation in vitro. Since translation in vitro appeared to be unsuitable in the study of the biogenesis of the M RNA gene products, attempts were made to express the proteins using recombinant vaccinia viruses. The cDNA was cloned in the vaccinia virus transfer vector vTF7-5 under control of the T7 promoter. Recombinant viruses were obtained, as demonstrated by dot blot and Southern blot analyses, and one designated recBUN M. In cells dually infected with recBUN M and VTF7-3, a recombinant vaccinia virus, which expresses T7 RNA polymerase, G1, G2 and NSm were synthesised. These proteins were demonstrated to be antigenically authentic by immunoprecipitation reactions with anti-BUN serum, which recognises both G1 and G2, and the antipeptide serum to NSm.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796693  DOI: Not available
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