Simultaneous Single-Cell
            <i>In Situ</i>
            Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

Krzywkowski, Tomasz; Ciftci, Sibel; Assadian, Farzaneh; Nilsson, Mats; Punga, Tanel

doi:10.1128/jvi.00166-17

Cited by 16 publications

(27 citation statements)

References 49 publications

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“…The PLP for HAdV-5 gDNA (NCBI reference sequence AC_000008.1) was designed to target a site adjacent to an MscI restriction site. PLP binding sites for E1A and MLTU mRNAs were positioned exactly at the exon-exon junctions to eliminate potential cross-reactivity with HAdV-5 genomic sequences (Krzywkowski et al, 2017). The PLP for cellular β-actin mRNA (ACTB, NM_001101) was reported previously (Weibrecht et al, 2013).…”

Section: Design Padlock Probementioning

confidence: 99%

“…To the best of our knowledge, simultaneous DNA and mRNA detection in individual infected cells has not been carried out before the report in Krzywkowski et al. ().…”

Section: Commentarymentioning

confidence: 99%

“…As targeted probes, design of PLPs requires prior knowledge about the DNA and/or RNA (cDNA) regions to be detected. The present protocol is based on published PLPs from Krzywkowski et al (2017). The PLP for HAdV-5 gDNA (NCBI reference sequence AC_000008.1) was designed to target a site adjacent to an MscI restriction site.…”

Section: Design Padlock Probementioning

confidence: 99%

“…Figure reproduced from Krzywkowski et al. (). ( B ) In the presented example, HeLa cells were infected with HAdV‐5 (10 FFU/cell) and analyzed at 25 hr post‐infection.…”

Section: Introductionmentioning

confidence: 99%

“…Both virus-and host-derived mRNAs are converted to cDNAs by reverse transcriptase (Figure 1A). Because the mRNA-specific PLP targets only the exon-exon junction in spliced cDNA sequences, the cross-reactivity of PLPs on genomic ssDNA is highly improbable (Krzywkowski, Ciftci, Assadian, Nilsson, & Punga, 2017).…”

Section: Introductionmentioning

confidence: 99%

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In Situ Detection of Adenovirus DNA and mRNA in Individual Cells

et al. 2018

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Infection by DNA viruses such as human adenoviruses (HAdVs) causes a high-level accumulation of viral DNA and mRNA in the cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not inevitably reflect the abundance in individual cells. As the vast majority of virus infection studies is carried out using standard experimental procedures with heterogeneous cell populations, there is a need for a method allowing simultaneous detection and quantitative analysis of viral genome accumulation and gene expression in individual infected cells within a population. This article describes a padlock probe-based rolling-circle amplification protocol that allows simultaneous detection of HAdV type 5 (HAdV-5) DNA and various virus-encoded mRNAs, as well as quantitative analysis of HAdV-5 DNA copies and mRNA species, in individual cells within a heterogeneous population. This versatile method can be used to detect the extent of pathogenic DNA virus infection in different cell types over prolonged infection times. Furthermore, simultaneous viral DNA and mRNA quantification in individual cells allows identification of cells in which persistent infections may be established. © 2018 by John Wiley & Sons, Inc.

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Section: Design Padlock Probementioning

confidence: 99%

“…To the best of our knowledge, simultaneous DNA and mRNA detection in individual infected cells has not been carried out before the report in Krzywkowski et al. ().…”

Section: Commentarymentioning

confidence: 99%

Section: Design Padlock Probementioning

confidence: 99%

“…Figure reproduced from Krzywkowski et al. (). ( B ) In the presented example, HeLa cells were infected with HAdV‐5 (10 FFU/cell) and analyzed at 25 hr post‐infection.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In Situ Detection of Adenovirus DNA and mRNA in Individual Cells

et al. 2018

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Imaging the adenovirus infection cycle

Pied

Wodrich

2019

FEBS Letters

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Edited by Urs GreberIncoming adenoviruses seize control of cytosolic transport mechanisms to relocate their genome from the cell periphery to specialized sites in the nucleoplasm. The nucleus is the site for viral gene expression, genome replication, and the production of progeny for the next round of infection. By taking control of the cell, adenoviruses also suppress cell-autonomous immunity responses. To succeed in their production cycle, adenoviruses rely on wellcoordinated steps, facilitated by interactions between viral proteins and cellular factors. Interactions between virus and host can impose remarkable morphological changes in the infected cell. Imaging adenoviruses has tremendously influenced how we delineate individual steps in the viral life cycle, because it allowed the development of specific optical markers to label these morphological changes in space and time. As technology advances, innovative imaging techniques and novel tools for specimen labeling keep uncovering previously unseen facets of adenovirus biology emphasizing why imaging adenoviruses is as attractive today as it was in the past. This review will summarize past achievements and present developments in adenovirus imaging centered on fluorescence microscopy approaches.Adenoviruses (Ads) are nonenveloped icosahedral viruses with a diameter of~90 nm with slight structural differences between genotypes. The majority of the Ad capsid shell is composed of 240 hexon trimers, and each of the 12 vertices of the icosahedron is occupied by a penton. Pentons are involved in cell attachment and receptor recognition and are composed of the pentameric penton base from which the trimeric fiber molecule extends. Minor capsid proteins IIIa, VI, VIII, and IX are embedded in the capsid and contribute to capsid stability. Protein VI is located at the inner surface of the capsid, and biochemical data suggest that it may connect the capsid to the core containing the viral genome via protein V. The genome is ã 36-kb double-stranded linear DNA molecule with the terminal protein (TP) covalently bound to each 5 0end. Adenovirus genomes are highly condensed and organized into chromatin by several hundred copies of Abbreviations ADP, adenovirus death protein

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Adenovirus in the omics era – a multipronged strategy

2020

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Human adenoviruses (HAdVs) are common pathogens associated with a wide variety of respiratory, ocular, and gastrointestinal diseases. To achieve its effective lytic mode of replication, HAdVs have to reprogram host-cell gene expression and fine-tune viral gene expression in a temporal manner. In two decades, omics revolution has advanced our knowledge about the HAdV and host-cell interplay at the RNA and protein levels. This review summarizes the current knowledge from large-scale datasets on how HAdV infections adjust coding and noncoding RNA expression, as well as how they reprogram host-cell proteome during the lytic course of infection.

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Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

Cited by 16 publications

References 49 publications

In Situ Detection of Adenovirus DNA and mRNA in Individual Cells

In Situ Detection of Adenovirus DNA and mRNA in Individual Cells

Imaging the adenovirus infection cycle

Adenovirus in the omics era – a multipronged strategy

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