Adenovirus Type 40 Host Range in Tissue Culture: A Study of the E1B Region

Steinthorsdottir, Valgerdur (1991) Adenovirus Type 40 Host Range in Tissue Culture: A Study of the E1B Region. PhD thesis, University of Glasgow.

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Growth restriction in tissue culture is a distinct feature of the enteric adenoviruses and they differ in this respect from all other subgroups of human adenoviruses. Ad40 cannot be passaged on Hela cells or primary HEK cells, but will grow in cells expressing the E1 region or only the E1B region of Ad2 or Ad5. The work described here was aimed at understanding the basis for this growth restriction. In coinfection experiments Ad40 complements an Ad5 E1A mutant and is itself complemented by that mutant as well as Ad5 and Adl2 wild type and E1B 19K mutant viruses. Ad5 and Ad12 E1B 55K mutant viruses are complemented to some extent by Ad40, these mutants however do not complement Ad40 growth on Hela cells. In order to study the E1B region of Ad40 a transcription map was determined from RNA produced at late times in infected KBa+b cells, using S1 nuclease, primer extension and PCR-cDNA analysis as well as Northern blotting. E1B transcripts corresponding to Ad2 14S, 22S and 9S mRNAs were identified but no 13S mRNA equivalent was detected, a pattern similar to that seen in the Adl2 transcription map. The coding potential for E1B 19K, 55K, and 15K proteins and for ppIX is retained in the Ad40 transcripts. In addition novel E1A-E1B cotranscript counterparts of the 14S and 22S mRNAs were identified. These contain the first 40 codons of the E1A first exon linked to a site 4-5nt downstream of the E1B cap site, retaining all the coding potential of the E1B mRNAs. No new open reading frames are created by the junction, and the E1A ORF terminates with one codon added after the junction. The E1A-E1B junction is unusual in that it does not conform to splice consensus sequences and thus may not be generated by a conventional splicing mechanism. Sequences around the 5' site of the junction bear a certain resemblance to spliced leader sequences utilized in trans-splicing in trypanosomes and nematodes. A timecourse of E1 transcription and DNA replication confirmed that. E1B transcripts are not detectable at early times in infection, but appear around the onset of DNA replication. The E1A-E1B cotranscripts are first seen at the same time as transcripts from the E1B promoter. Transcription from the Ad40 E1B promoter was analysed in transient transfection assays. Chloramphenicol acetyl transferase (CAT) activity was measured in cell extracts where the CAT gene was expressed under the control of the E1B promoter in the presence or absence of E1A products of Ad5 or Ad40. Expression from the Ad40 E1B promoter was not detectable even in the presence of Ad5 E1A, suggesting that unlike its Ad5 counterpart the Ad40 E1B promoter does not respond to E1A transactivation. This would account for the lack of early E1B gene expression in tissue culture. Moreover, this suggests that in the natural host cells of the virus E1B early gene products may not be needed, or alternatively, that early expression from the Ad40 E1B promoter is dependent on cellular factors present in the natural host cell, but absent in most common tissue culture cell lines. In this study Ad40 was shown to grow in an intestinal cell line, Int407, almost as well as in KB cells expressing the Ad2 E1 region. This provides a system in which to study Ad40 growth in tissue culture in the absence of complementing viral gene products.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78243
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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