Adenovirus Precursor pVII Protein Stability Is Regulated By Its Propeptide Sequence

Inturi, Raviteja; Thaduri, Srinivas; Punga, Tanel

doi:10.1371/journal.pone.0080617

Cited by 19 publications

(32 citation statements)

References 52 publications

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“…Both cell lines were grown in a humidified cell incubator at 37°C in the presence of 7% CO 2 . The stable HeLa cell line expressing the HAdV-5 pVII(Δ24)Flag protein was generated by integrating a codonoptimized pVII(Δ24)Flag cDNA (14) into the HeLa-Flp-In T-Rex cell line (generously provided by Stephen Taylor [44]). pVII(Δ24)Flag protein expression with doxycycline treatment and detection in whole-cell lysates were performed as reported previously (14).…”

Section: Methodsmentioning

confidence: 99%

“…Notably, MLTU encodes an abundant histone-like protein, pVII, which is proteolytically processed into the mature protein VII during the final steps of virion maturation (13). The mature protein VII [also known as pVII(Δ24) (14)] organizes the viral genome into a nucleosome-like structure (15)(16)(17), protects viral DNA from the cellular DNA damage response (18), and also can act as a protein regulating transcription (19,20).…”

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confidence: 99%

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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

Krzywkowski

Ciftci

Assadian

et al. 2017

J Virol

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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 virusencoded 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.KEYWORDS adenovirus, persistent infection, lytic infection, rolling-circle amplification, single-cell analysis H uman adenoviruses (HAdV) are double-stranded DNA (dsDNA) viruses classified into seven distinct species, A to G, with over 60 species described so far (1). Typically, HAdV infection of epithelial cells causes an efficient cell lysis and release of

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Krzywkowski

Ciftci

Assadian

et al. 2017

J Virol

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“…The generated plasmids pcDNA3.1(Flag)PHPT1(wt) and pcDNA3.1(Flag)PHPT1(Spl) allow the expression of the PHPT1 proteins as N-terminal Flag-tagged fusion proteins in mammalian cells. Dominant negative Cullin1-5 (DN-Cul1-5) plasmids have been published before (Inturi et al, 2013).…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Indirect immunofluorescence assays were carried out as described previously (Inturi et al, 2013). Briefly, HeLa cells were grown on fibronectin coated coverslips and transfected for 24 h with the indicated plasmids.…”

Section: Indirect Immunofluorescence Assaymentioning

confidence: 99%

“…We concentrated our efforts on Cullin-RING E3 ubiquitin-ligases (CRL), as they are the most prominent class of E3 ligases in mammalian cells. An effective way to inactivate the function of Cullin-containing E3 complexes is to overexpress the dominant-negative Cullin (DNCul) proteins in the cells (Inturi et al, 2013). We took advantage of five available DN-Cul plasmids (DN-Cul 1-5), which were coexpressed along with the PHPT1(Spl) protein encoding plasmid in HeLa cells.…”

Section: The Phpt1(spl) Protein Stability Is Controlled By Cullin Promentioning

confidence: 99%

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A splice variant of the human phosphohistidine phosphatase 1 (PHPT1) is degraded by the proteasome

Inturi

Wäneskog

Vlachakis

et al. 2014

The International Journal of Biochemistry & Cell Biology

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Regulation of protein activity by phosphorylation is central in many cellular processes. Phosphorylation of serine, threonine and tyrosine residues is well documented and studied. In addition, other amino acids, like histidine can be phosphorylated, but neither the mechanism nor the function of this modification is well understood. Nevertheless, there is a 14 kDa enzyme with phosphohistidine phosphatase activity, named PHPT1, found in most animals, but not in bacteria, plant or fungi. There are a few splice variant transcripts formed from the human PHPT1 locus and some of them are predicted to form variant proteins, but studies of these proteins are lacking. In order to get insight into the possible function of the variant transcripts encoded at the PHPT1 locus, ectopic expression of PHPT1 transcript variant 6, predicted to be degraded by the non-sense mediated mRNA decay pathway, in HeLa cells was undertaken. In HeLa cells the splice variant protein was degraded by the proteasome, unlike the wild type protein. Using an in silico modeling approach the variant C-terminal end of the proteins were predicted to form different secondary structures that might explain the different properties of the two proteins. The specific degradation of the PHPT1 splice variant indicates that at least for the PHPT1 protein, the quality control and the self-guarding of the cellular system works at two levels, first at the RNA level, aberrant transcripts are degraded by the non-sense mediated mRNA decay pathway, and the small amount of proteins that are formed will be degraded by the proteasome.

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Epigenetics and the dynamics of chromatin during adenovirus infections

et al. 2019

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Edited by Urs GreberAbbreviations ADP, adenovirus death protein; Ad5 or Ad 12, human adenovirus type 5 or 12, or other type referenced; BHK, baby hamster kidney; BrdU, bromodeoxyuridine; CTCF, CCCTC binding factor; DBS, double stranded break; DDR, DNA damage response; E1A, early 1 A; E4orf3 or E4orf6, early 4 open reading frame 3 or 6; HAdV-A12, human adenovirus subgroup A type 12, or different subgroup or type referenced; HMGB, high-mobility group box; hPaf1, human RNA polymerase II-associated factor 1; ISGs, interferon-stimulated genes; PML, promyelocytic leukemia; snRNP, small nuclear ribonuclear protein; SET or TAF-1, SE translocation or template activating factor; VRCs, viral replication centers.

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Adenovirus Precursor pVII Protein Stability Is Regulated By Its Propeptide Sequence

Cited by 19 publications

References 52 publications

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

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

A splice variant of the human phosphohistidine phosphatase 1 (PHPT1) is degraded by the proteasome

Epigenetics and the dynamics of chromatin during adenovirus infections

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