Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Show others and affiliations
Number of Authors: 52017 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 91, no 11, article id UNSP e00166-17Article in journal (Refereed) Published
Abstract [en]

An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells. IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.

Place, publisher, year, edition, pages
2017. Vol. 91, no 11, article id UNSP e00166-17
Keywords [en]
adenovirus, persistent infection, lytic infection, rolling-circle amplification, single-cell analysis
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-144695DOI: 10.1128/JVI.00166-17ISI: 000402166500007OAI: oai:DiVA.org:su-144695DiVA, id: diva2:1128061
Available from: 2017-07-21 Created: 2017-07-21 Last updated: 2017-10-11Bibliographically approved
In thesis
1. iLocks: a novel tool for RNA assays with improved specificity
Open this publication in new window or tab >>iLocks: a novel tool for RNA assays with improved specificity
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Central Dogma of molecular biology describes a framework for how genetic information is transferred in cells, placing RNA as a messenger between DNA and translated proteins. During the last years, interest in RNA research has grown tremendously due to the increasing understanding and recognition of the importance of RNA in regulation of gene expression, biochemical catalysis, and genome integrity surveillance. Most importantly, RNA content, unlike DNA, changes constantly, fine-tuning the cellular response to match the environmental conditions. There is a clear potential for RNA biomarkers, reflecting both the natural and pathological conditions in vivo.

Various methods have been developed to study RNA, of which the most common tools and techniques are described in this thesis. Since many of these gold standard methods are based on detecting RNA derivative (cDNA), there is a wide scope for efficient alternative tools directly targeting RNA. In Paper I, the spatiotemporal expression of human adenovirus-5 mRNA in epithelial and blood cells infected with the virus has been studied. For this, padlock probes and rolling circle amplification (RCA) were used to visualize, quantify and analyse both viral and host cell cDNAs in different infection scenarios, at single cell level. In Paper II, direct RNA detection fidelity has been evaluated using padlock probes. A novel type of probe (iLock) that is activated on RNA via invasive cleavage mechanism, prior to RCA was developed in this approach. Using iLocks, a substantial improvement of direct RNA sensing fidelity has been observed. In Paper III, RNA modifications were introduced in otherwise DNA iLock probes to enhance the probes’ efficiency on miRNAs. Using chimeric iLock probes, multiplexed differentiation of conserved miRNA family members were performed with next- generation sequencing-by-ligation readout. Efficient replication of chimeric probes used in Paper III implies that the Phi29 DNA polymerase readily accepts RNA-containing circles as amplification substrates. In Paper IV, real-time RCA monitoring for measurement of amplification rates and analysis of amplification patterns of various RNA-containing circles was achieved. Moreover, the RCA products were sequenced as a proof for the reverse-transcriptase activity of the Phi29 DNA polymerase.

This thesis effectively contributes to a better understanding of mechanisms influencing RNA detection with, but not limited to, padlock probes. It expands the available RNA analyses toolkit with novel strategies and solutions, which can be potentially adapted for RNA-focused research, in general and molecular diagnostics, in particular.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2017. p. 59
Keywords
RNA, miRNA, non-coding RNA, padlock probes, rolling circle amplification, invader, single cell, in situ, adenovirus, virology, diagnostics
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-147734 (URN)978-91-7797-043-9 (ISBN)978-91-7797-044-6 (ISBN)
Public defence
2017-11-24, Högbomsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2017-10-31 Created: 2017-10-11 Last updated: 2017-11-01Bibliographically approved

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full text

Search in DiVA

By author/editor
Krzywkowski, TomaszCiftci, SibelNilsson, Mats
By organisation
Department of Biochemistry and BiophysicsScience for Life Laboratory (SciLifeLab)
In the same journal
Journal of Virology
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 11 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf