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Adenovirus L4-22K stimulates major late transcription by a mechanism requiring the intragenic late-specific transcription factor-binding site
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Göran Akusjärvi)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Göran Akusjärvi)
2010 (English)In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 151, no 2, 220-228 p.Article in journal (Refereed) Published
Abstract [en]

The adenovirus major late promoter (MLP) generates a primary transcript that undergoes a complex pattern of regulated alternative RNA splicing and polyadenylation events. The late-specific activation of the MLP requires binding of two infected-cell specific transcription factor complexes, DEF-A and DEF-B, to the so-called DE sequence located downstream of the MLP start site. Previous studies have shown that DEF-B is a homodimer of the viral IVa2 protein and suggested that DEF-A is a heterodimer of IVa2 and an unknown protein. Here we have searched for a possible DEF-A candidate protein. The adenovirus L4-33K protein functions as a virus-encoded alternative RNA splicing factor, stimulating cytoplasmic accumulation of most late viral mRNAs. Interestingly, the L4 region also encodes for a second related protein, L4-22K, which share the 105 amino-terminal amino acids with L4-33K. Here we show that L4-22K both in vivo and in vitro stimulates transcription from the MLP in a DE sequence dependent manner. We also show that the viral pIX promoter is a natural target, activated by L4-22K. Interestingly, the position of the L4-22K DNA binding site in a promoter does not appear to be critical for function. Thus, tethering L4-22K, as a BPV E2 DNA binding domain fusion protein either to a position upstream or downstream of the MLP start site, or upstream of a minimal E1B promoter, resulted in an activation of transcription. Collectively, our results are compatible with the hypothesis that L4-22K may be the elusive component of DEF-A that partakes in activation of the MLP.

Place, publisher, year, edition, pages
2010. Vol. 151, no 2, 220-228 p.
Keyword [en]
Adenovirus, L4-22K, MLP, transcription, DE elements
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-101310DOI: 10.1016/j.virusres.2010.05.013ISI: 000280210000015PubMedID: 20621673OAI: oai:DiVA.org:uu-101310DiVA: diva2:212570
Available from: 2009-04-22 Created: 2009-04-22 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Regulation of Adenoviral Gene Expression by the L4-33K and L4-22K Proteins
Open this publication in new window or tab >>Regulation of Adenoviral Gene Expression by the L4-33K and L4-22K Proteins
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The splicing pattern during an adenovirus infection is shifted at the late phase towards using weaker splice sites, splicing out larger introns. Splicing of weak 3´ splice sites usually requires recognition of the 3´AG dinucleotide before the first catalytic step of splicing. Such splicing events are said to be AG-dependent and requires an interaction of both subunits of the cellular splicing factor U2AF with the 3´ splice site. We show that splicing of transcripts that are AG-dependent in uninfected nuclear extracts (NE) becomes AG-independent in nuclear extracts prepared form adenovirus late-infected HeLa cells (Ad-NE). Further we demonstrate that the first step in splicing of a model transcript, IgM, becomes completely U2AF-independent in Ad-NE. This finding supports our working model that 3´ splice site recognition in Ad-NE is altered, and in fact might be U2AF-independent.

We further show that the adenovirus late protein L4-33K acts as a virus encoded alternative splicing factor. L4-33K activates splicing of both cellular and viral transcripts containing weak 3´ splice sites. This supports the hypothesis that adenovirus alter splicing during the infection to favour usage of weak, suboptimal 3´ splice sites. However, we were unable to find an alternative U2AF-related factor that could stimulate L4-33K splicing enhancer activity. Furthermore, we demonstrate that the serine residues in the C-terminal part of L4-33K are important for the splicing enhancer activity but also for its nuclear localisation.

The adenovirus major late promoter is highly activated after the onset of viral genome replication. Protein complexes binding to downstream elements of the promoter are required for full enhancement of this promoter. We show that an L4-33K-related protein, L4-22K, stimulates transcription from the major late promoter. This stimulation is mainly via the downstream elements and does not require the viral IVa2 protein, which is a transcription factor of the major late promoter.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 45 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 458
National Category
Microbiology in the medical area
Research subject
Medical Virology
Identifiers
urn:nbn:se:uu:diva-101324 (URN)978-91-554-7529-1 (ISBN)
Public defence
2009-06-04, C10:305, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-05-13 Created: 2009-04-22 Last updated: 2011-02-24Bibliographically approved
2. The Multifunctional Nature of the Adenovirus L4-22K Protein
Open this publication in new window or tab >>The Multifunctional Nature of the Adenovirus L4-22K Protein
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The adenovirus major late transcription unit (MLTU) encodes for most of the mRNAs that are translated into the structural proteins of the virus capsid. Transcription from the MLTU is directed by the major late promoter (MLP), which is highly activated during the late phase of infection. This thesis discusses how the adenovirus-encoded L4-22K protein regulates the MLP at both the level of transcription and pre-mRNA splicing. The study shed new light on the complex regulation of the early to late shift of adenoviral gene expression.

Here we show that the L4-22K protein has opposing effects on MLP transcription, functioning both as an activator and a repressor protein. The stimulatory effect mainly depends on the direct interaction of the L4-22K protein with the downstream element (DE element) located approximately 100 nucleotides downstream of the transcription initiation site. In addition to the DE element we also show that the promoter-proximal upstream element (UPE) acts as an L4-22K responsive enhancer element in the MLP. Preliminary data suggests that the activation of MLP transcription via DE and UPE differs mechanistically. The transactivation domain of the L4-22K protein is localized to the conserved carboxy-terminus of the protein.

Our results also defined a novel low affinity L4-22K binding site, the R1 region, which functions as a repressor element in MLP transcription. At high concentrations L4-22K binds to R1 and recruits the cellular transcription factor Sp1 to a DNA segment covering the major late first leader 5´ splice site that is embedded in the R1 region. Sp1 binding to R1 results in a suppression of L4-22K-mediated activation of MLP transcription. This self-limiting effect on MLP transcription might have a function to fine-tune the MLTU gene expression.

Interestingly, the L4-22K protein binds with the same sequence specificity to both the R1 double-stranded DNA and R1 single-stranded RNA (ssRNA). L4-22K binds to the R1 ssRNA with the same polarity as the MLTU nascent RNA. This binding results in the recruitment of U1 snRNA to the major late first leader 5´ splice site. This enhanced U1 snRNA recruitment leads to a suppression of MLP transcription and simultaneously an increase of major late first intron splicing.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 53 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1185
Keyword
L4-22K, MLP, RNA, transcription, splicing, adenovirus
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-278135 (URN)978-91-554-9486-5 (ISBN)
Public defence
2016-04-15, A1:111a, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2016-03-22 Created: 2016-02-23 Last updated: 2016-04-04

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