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Functional Characterization of the Evolutionarily Conserved Adenoviral Proteins L4-22K and L4-33K
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Göran Akusjärvi)ORCID iD: 0000-0002-0797-8007
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Regulation of adenoviral gene expression is a complex process directed by viral proteins controlling a multitude of different activities at distinct phases of the virus life cycle. This thesis discusses adenoviral regulation of transcription and splicing by two proteins expressed at the late phase: L4-22K and L4-33K. These are closely related with a common N-terminus but unique C-terminal domains. The L4-33K protein is an alternative RNA splicing factor inducing L1-IIIa mRNA splicing, while L4-22K is stimulating transcription from the major late promoter (MLP). The L4-33K protein contains a tiny RS-repeat in its unique C-terminal end that is essential for the splicing enhancer function of the protein. Here we demonstrate that the tiny RS-repeat is required for localization of the protein to the nucleus and viral replication centers. Further, we describe an auto-regulatory loop where L4-33K enhances splicing of its own intron. The preliminary characterization of the responsive RNA-element suggests that it differs from the previously defined L4-33K-responsive element activating L1-IIIa mRNA splicing.

L4-22K lacks the ability to enhance L1-IIIa splicing in vivo, and here we show that the protein is defective in L1-IIIa or other late pre-mRNA splicing reactions in vitro. Interestingly, we found a novel function for the L4-22K and L4-33K proteins as regulators of E1A alternative splicing. Both proteins selectively upregulated E1A-10S mRNA accumulation in transfection experiments, by a mechanism independent of the tiny RS-repeat.

Although L4-22K is reported to be an MLP transcriptional enhancer protein, here we show that L4-22K also functions as a repressor of MLP transcription. This novel activity depends on the integrity of the major late first leader 5’ splice site. The model suggests that at low concentrations L4-22K activates MLP transcription while at high concentrations L4-22K represses transcription.

So far, characterizations of the L4-22K and L4-33K proteins have been limited to human adenoviruses 2 or 5 (HAdV-2/5). We expanded our experiments to include HAdV-3, HAdV-4, HAdV-9, HAdV-11 and HAdV-41. The results demonstrated that the transcription- or splicing-enhancing properties of L4-22K and L4-33K, respectively, are evolutionarily conserved and non-overlapping. Thus, the sequence-based conservation is mirrored by the functions, as expected for functionally important proteins.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 74 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1062
Keyword [en]
L4-22K, L4-33K, RNA, splicing, adenovirus, nuclear localization, replication, transcription, evolution, SR protein, MLP, promoter, E1A, serotypes
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:uu:diva-238487ISBN: 978-91-554-9132-1 (print)OAI: oai:DiVA.org:uu-238487DiVA: diva2:771955
Public defence
2015-02-13, C8:301, BMC, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-01-23 Created: 2014-12-12 Last updated: 2015-03-09
List of papers
1. Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers
Open this publication in new window or tab >>Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers
2012 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 433, no 2, 273-281 p.Article in journal (Refereed) Published
Abstract [en]

The adenovirus L4-33K protein is a key regulator involved in the temporal shift from early to late pattern of mRNA expression from the adenovirus major late transcription unit. L4-33K is a virus-encoded alternative splicing factor, which enhances processing of 3’ splice sites with a weak sequence context. Here we show that L4-33K expressed from a transfected plasmid has a diffuse nuclear localization with strong enrichment in the nuclear membrane. We further show that the highly conserved part of the carboxy-terminal end of L4-33K, which functions as the splicing enhancer domain, is sufficient for nuclear localization of the protein. Interestingly, infection of the transfected cells caused a redistribution of L4-33K from the nuclear membrane into discrete ring-like structures corresponding to the viral transcription sites. We also show that serine 192 in the tiny RS repeat, which is critical for the splicing enhancer function of L4-33K, is necessary for the nuclear localization and redistribution of L4-33K protein into viral transcription sites. Collectively, our results show a good correlation between the activity of L4-33K as a splicing enhancer protein and its localization to viral transcription sites.

Keyword
L4-33K, L4-22K, splicing, localization, adenovirus, replication centers, transcription sites, peripheral replicative zone
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-159614 (URN)10.1016/j.virol.2012.08.021 (DOI)000310095700001 ()22944109 (PubMedID)
Available from: 2011-10-05 Created: 2011-10-05 Last updated: 2017-12-08Bibliographically approved
2. A suppressive effect of the first leader 5’ splice site on L4-­22K-­mediated activation of major late transcription
Open this publication in new window or tab >>A suppressive effect of the first leader 5’ splice site on L4-­22K-­mediated activation of major late transcription
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-238486 (URN)
Available from: 2014-12-12 Created: 2014-12-12 Last updated: 2015-03-09
3. RNA elements involved in adenovirus L4-33K regulation of alternative splicing
Open this publication in new window or tab >>RNA elements involved in adenovirus L4-33K regulation of alternative splicing
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-237965 (URN)
Available from: 2014-12-12 Created: 2014-12-08 Last updated: 2015-03-09
4. Conservation of the transcriptional and post-­transcriptional activities of serotype-­specific adenovirus L4 proteins
Open this publication in new window or tab >>Conservation of the transcriptional and post-­transcriptional activities of serotype-­specific adenovirus L4 proteins
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-238482 (URN)
Available from: 2014-12-12 Created: 2014-12-12 Last updated: 2015-03-09

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Östberg, Sara

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