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Regulation of HIV-1 mRNA Processing by Cellular Splicing Factors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

According to UNAIDS there were 34 million people living with human immunodeficiency virus (HIV) infection at the end of 2010. HIV is the causative agent of acquired immunodeficiency syndrome (AIDS) and the number of people dying of AIDS-related causes at the end of 2010 was 1.8 million. Due to the high mutability of the virus, there is a constant need for new approaches to attack the virus.

Splicing of HIV-1 pre-mRNA is a highly regulated process. In order to produce all mRNAs needed to be infectious HIV-1 utilizes alternative splicing ­- from one single transcript more than 35 differently spliced mRNAs can be produced. A new approach to fight HIV-1 could be to interfere with the essential splicing. In this thesis, I describe the regulation of HIV-1 pre-mRNA splicing.

SR proteins are involved in the regulation of splicing, both in an early and a late stage. We find that the intracellular concentration of SR proteins is of great importance for HIV-1 to be able to produce the correct amounts of mRNAs. Variations in concentrations of SR proteins lead to big changes in the HIV-1 pre-mRNA splicing pattern.

The functions of HIV-1 protein Vpr are diverse and it is essential in vivo. HIV-1 vpr mRNA 13a7 is partially spliced, containing an intron, and the regulation of it is not fully understood. We find that SRp55 and SRp75 induce the production of HIV-1 vpr mRNA 13a7 by inhibiting splice donor 3. We also conclude that this inhibition at least for SRp55 is due to an interaction with the viral RNA element GAR. In the presence of SRp55 we also see an increase in cytoplasmic amounts of intron containing vpr mRNA due to increased nuclear export. Our results show that SRp55 can have several functions in the regulation of HIV-1 splicing: by inhibiting splice donors and by facilitating the export of incompletely spliced mRNAs to the cytoplasm.

In conclusion, this thesis describes SRp55 as a regulator of HIV-1 vpr mRNA, both in splicing as well as in nuclear export. These discoveries provide an insight into the regulation of HIV-1 mRNA processing.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 57 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 749
Keyword [en]
HIV-1, SR protein, SRp55, SFRS6, splicing, indole derivative
National Category
Microbiology in the medical area
Research subject
Medical Virology
Identifiers
URN: urn:nbn:se:uu:diva-169256ISBN: 978-91-554-8300-5 (print)OAI: oai:DiVA.org:uu-169256DiVA: diva2:509352
Public defence
2012-04-26, C10:301, BMC, Husargatan 3, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2012-04-04 Created: 2012-02-25 Last updated: 2012-04-19Bibliographically approved
List of papers
1. Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5 '-Splice Site of Exon 3
Open this publication in new window or tab >>Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5 '-Splice Site of Exon 3
2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, ISSN 20685659, Vol. 285, no 41, 31537-31547 p.Article in journal (Refereed) Published
Abstract [en]

HIV-1 non-coding exon 3 can either be spliced to exons 4, 4a, 4b, 4c, and 5 to generate tat, rev, and nef mRNAs or remain unspliced to produce the 13a7 vpr mRNA. Here we show that serine-and arginine-rich proteins 55 and 75 (SRp55 and SRp75) inhibit splicing from the 5'-splice site of exon 3 thereby causing an accumulation of the partially unspliced 13a7 vpr mRNA. In contrast, serine- and arginine-rich protein 40 (SRp40) induces splicing from exon 3 to exon 4, thereby promoting the production of the 1347 tat mRNA. We demonstrate that SRp55 stimulates vpr mRNA production by interacting with the previously identified HIV-1 splicing enhancer named GAR and inhibiting its function. This inhibition requires both serine arginine-rich and RNA-binding domains of SRp55, indicating that production of HIV-1 vpr mRNA depends on the interaction of SRp55 with an unknown factor.

Keyword
RNA/Splicing, Viruses/HIV, Gene Regulation, Human Immunodeficiency Virus, RNA Processing, SR Protein, SRp55, Alternative Splicing, vpr
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-135095 (URN)10.1074/jbc.M109.077453 (DOI)000282764600048 ()
Available from: 2010-12-06 Created: 2010-12-03 Last updated: 2017-12-11Bibliographically approved
2. Inhibition of splicing by serine-arginine rich protein 55 (SRp55) causes the appearance of partially spliced HIV-1 mRNAs in the cytoplasm
Open this publication in new window or tab >>Inhibition of splicing by serine-arginine rich protein 55 (SRp55) causes the appearance of partially spliced HIV-1 mRNAs in the cytoplasm
2011 (English)In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 157, no 1, 82-91 p.Article in journal (Refereed) Published
Abstract [en]

We have previously shown that SRp55 inhibits splicing from HIV-1 exon 3, thereby generating partially spliced mRNAs encoding HIV-1 vpr. Here we show that SRp55 also inhibits splicing from HIV-1 exon 5 to generate HIV-1 vpu/env mRNA, albeit with lower efficiency. We also show that inhibition of HIV-1 splicing by SRp55 causes the appearance of partially spliced vpu, env and vpr mRNAs in the cytoplasm. SRp55 could also induce production of extracellular p24gag from a rev-defective HIV-1 provirus. These results indicate that SRp55 aids in the generation of partially spliced and unspliced HIV-1 mRNAs.

Keyword
HIV-1, vpr, SRp55, Splicing
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-153549 (URN)10.1016/j.virusres.2011.02.010 (DOI)000289864600011 ()21345357 (PubMedID)
Available from: 2011-05-16 Created: 2011-05-16 Last updated: 2017-12-11Bibliographically approved
3. Indole-related compounds from the National Cancer Institute (USA) repository do not inhibit HIV-1 replication in primary blood mononuclear cells
Open this publication in new window or tab >>Indole-related compounds from the National Cancer Institute (USA) repository do not inhibit HIV-1 replication in primary blood mononuclear cells
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Indole derived substances have been shown previously to inhibit HIV-1 replication, presumably by interfering with cellular splicing factors of the SR protein family. Here we have tested the effect of indole-related substances obtained from the NCI repository on HIV-1 gene expression in transient transfections and in primary cell cultures infected by X4 and R5 tropic HIV-1 strains. Although these substances displayed a significant inhibitory effect on HIV-1 gene expression in transiently transfected cells, they did not inhibit replication of HIV-1 in primary peripheral blood mononuclear cells. These results demonstrate important differences between transfection assays performed in cell lines and infection assays performed in primary cells and indicate that only a subset of indole-related compounds can inhibit HIV-1 replication.

National Category
Basic Medicine
Research subject
Medical Virology
Identifiers
urn:nbn:se:uu:diva-170300 (URN)
Available from: 2012-03-11 Created: 2012-03-11 Last updated: 2012-04-19

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