Expression of the Bunyamwera Virus Polymerase Gene

Jin, Hong (1991) Expression of the Bunyamwera Virus Polymerase Gene. PhD thesis, University of Glasgow.

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Bunyamwera virus is the prototype of the family Bunyaviridae and has a tripartite negative-sense RNA genome. The largest RNA segment (L) contains 6875 nucleotides and encodes a high molecular weight protein (259K) also termed L. The L protein is presumed to be the virion-associated transcriptase or RNA polymerase, although direct proof of this is lacking. The large size of the L protein suggests it is multifunctional and most probably catalyzes initiation, elongation, and termination of RNA synthesis and perhaps also plays a role in events which generate the host-derived primers for transcription, such as cap-binding and endonuclease activities. With the long-term aim of defining the functional domains within the bunyavirus L protein, the available cloned cDNAs were exploited to establish systems in which the L protein can be expressed. As a prelude to characterizing the expressed L protein, monospecific antisera to portions of the L protein fused to B-galactosidase were prepared. Five fusion proteins were expressed using the bacterial expression vector pUEX, and two antisera raised against the C and N termini of the L protein specifically recognized the L protein. A full-length cDNA to the L gene of Bunyamwera virus was constructed from the existing cDNA subclones and synthetic oligonucleotides. The assembled L gene cDNA was subsequently cloned into various expression vectors and several expression systems were explored in order to obtain a biologically active L protein for domain mapping. The full-length cDNA to the L gene was cloned into two vaccinia virus expression systems. In the first, the L gene is under control of vaccinia virus p7.5 promoter by using the plasmid transfer vector pSC11; in the second, the L gene is under control of the bacteriophage T7 &phis;10 promoter by using the plasmid transfer vector pTF7-5, and expression of the L gene requires coinfection with a second recombinant vaccinia virus vTF7-3 which synthesizes T7 RNA polymerase. Both systems expressed a protein which is the same size as the Bunyamwera virus L protein and was recognized by the monospecific L anti sera. The L protein was also transiently expressed by transfection of vTF7-3 infected cells with recombinant plasmid DNAs which contained the cDNA for the L gene under control of the T7 promoter. Expression of the L protein in a cell free system (rabbit reticulocyte lysate) by translation of an in vitro transcribed L gene was performed. Minor amounts of an L sized protein were synthesized, but many prematurely terminated polypeptides were also produced. The L gene was also cloned under the T7 promoter in a bacterial expression vector (pET8c) to express the L protein in E. coli. However, no L protein was expressed by either IPTG induction or infection of the culture with a bacteriophage (CE6) to provide the T7 RNA polymerase. To test the functionality of the L protein expressed by the recombinant vaccinia viruses, three assay systems were explored: complementation of bunyavirus ts L gene mutants; in vitro transcription assay; and the ability of the expressed L protein to replicate an RNA template in infected cultured cells. The transcriptase activity of the expressed L protein present in cytoplasmic extracts from the recombinant vaccinia virus infected cells was assayed in vitro using Bunyamwera virus intracellular nucleocapsids as templates. However, the activity of the authentic L protein which was associated with the RNP was stimulated by mock infected cell extracts. Thus, the intracellular nucleocapsid was not an appropriate template for this assay. The attempts to complement bunyavirus ts mutants led to noninterpretable results. Efforts t reconstitute a synthetic RNA template containing the 5' and 3' end sequences of the Bunyamwera virus S RNA were also made. Linearized recombinant plasmid pUC-BUNS292 containing bases 1-99 and 768-961 nucleotides of the S RNA under control of the T7 promoter, was transfected into vTF7-3 infected cells, and positive-sense RNA should have been transcribed. The cells also expressed N protein, either transiently or from Bumyamwera virus infection, to encapsidate any short S-like RNA. However, it was observed that the transfected linearized shorter S cDNA was not transcribed by the T7 RNA polymerase in vTF7-3 infected cells. The L protein, expressed from either of the two vaccinia virus systems or transiently expressed, was demonstrated to be functional by its capability of replicating viral S RNA in a nucleocapsid (RNP) transfection assay. Recombinant vaccinia virus-infected cells were transfected with purified Bunyamwera virus nucleocapsids, and subsequently total cellular RNA was analyzed by Northern (RNA) blotting or the cells were labelled with 35S-methionine to analyze viral protein synthesis. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78346
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 28 Feb 2020 12:09
Last Modified: 28 Feb 2020 12:09

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