Studies on the Expression Strategy and Encoded Proteins of the Bunyavirus S RNA Segment

McGregor, Alistair (1994) Studies on the Expression Strategy and Encoded Proteins of the Bunyavirus S RNA Segment. PhD thesis, University of Glasgow.

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Abstract

A characteristic of viruses in the family Bunvaviridae is that the viral genome consists of three RNA segments of single stranded RNA, designated large (L), middle (M) and small (S). The Bunvaviridae family is sub-divided into five genera, but in all members of the family the S RNA segment encodes the RNA- binding nucleocapsid (N) protein. This study is restricted to viruses in the Bunvavirus genus and concerns the expression strategy of S segment encoded proteins and the nature of the interaction of N protein with viral RNA. For viruses of the Bunvavirus genus the S segment also encodes a non-structural protein, termed NSs, in an overlapping reading frame downstream of the start of the N open reading frame (ORF). The NSs ORF initiates from a tandem AUG in most bunyaviruses, such as Bunyamwera the prototype virus. The Maguari (MAG) virus belongs to the same serogroup as Bunyamwera virus (BUN), with which it shares high S segment homology, but the MAG S segment in addition to encoding N and NSs proteins, possess a third ORF, potentially encoding a protein of Mr 9.3K, downstream of the N and NSs ORFs. The MAG S segment is 945 bases in length and most of the MAG S segment (bases 22-937) was available on a single cDNA clone, pMAG60. In order to investigate the expression strategy of the MAG S segment fragments of the cDNA, containing either the complete S segment coding region or parts of it, were subcloned downstream of a T7 RNA polymerase promoter sequence in the vector pTZlSU. A series of recombinant pTZ plasmids were used as templates to generate message-sense RNA in T7 RNA polymerase directed run-off transcription reactions. Aliquots of the RNA were translated in a cell free translation system (rabbit reticulocyte lysate and wheat germ extract) and the synthesised proteins, which were labelled with 35S methionine, were analysed by polyacrylamide electrophoresis. Translation of RNA derived from templates containing the entire MAGS coding region produced three major products with electrophoretic mobilities equivalent to the predicted Mr of the encoded products, N, NSs and ORF3 proteins. Translation of transcripts derived from plasmids containing NSs and 9.3K ORFs or 9.3K ORF only produced two major products or one major product repectively, corresponding to NSs and/or the 9.3K protein. These experiments indicated that the proposed sites of initiation of translation for N, NSs and 9.3K proteins were correct but in addition it demonstrated that all three encoded products were capable of being translated from a single mRNA species o The NSs tandem initiation codons for NSs in the MAG S cDNA were mutated either singly or together to leave either the first or second or no potential codons at the start of the NSs ORF. Translation of transcripts derived from these templates indicated that NSs translation was initiated at the tandem AUG and that both the first and the second AUG were equally capable of initiating NSs translation. In addition NSs translation was found to be unaffected by the loss of the upstream N initiation codon or coding sequences. However, synthesis of the 9.3K protein was reduced when the upstream N initiation codon was inactivated.Although the 9.3K protein product could be translated in vitro, its presence could not be demonstrated in radiolabelled MAG virus infected cells due to a high background of labelled host proteins. In an attempt to make a specific antiserum to this protein the 9.3K protein was expressed as a beta-galactosidase (beta-gal) fusion protein in bacterial cells. A protein band corresponding to the fusion protein was isolated from preparative polyacrylamide gels and used to inoculate a rabbit at 3-4 week intervals. Test bleeds were used in immunoprecipitation assays of appropriately programmed rabbit reticulocyte lysates, but the serum failed to immunoprecipitate the 9.3k protein. However, the serum was able to recognise the beta-gal carrier protein in a seperate immunoblot assay of beta-gal protein. It was concluded that the 9.3K protein was poorly immunogenic and no further attempt at raising an antiserum to this protein was made. The N protein interacts with each RNA genome segment to form internal nucleocapids within the Bunvaviridae particle. Each nucleocapsid consists of a single species of RNA genome segment (L, M or S), multiple copies of N protein and minor amounts of L protein. In virus infected cells N protein has high specificity for full length negative-sense genomic or positive-sense antigenomic RNA, but not viral mRNAs, which have different 5' and 3' ends, and host cell RNA. The nature of this specificity is unknown and was investigated by studying in vitro the RNA binding activity of recombinant expressed N protein with various S segment derived RNA species.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Richard Elliott
Keywords: Virology, Molecular biology
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-75603
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:20
Last Modified: 19 Nov 2019 19:20
URI: http://theses.gla.ac.uk/id/eprint/75603

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