Parvovirus B19 DNA CpG Dinucleotide Methylation and Epigenetic Regulation of Viral Expression

CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression

Mechanisms involved in Parvovirus B19 replication and regulation of gene expression

Il Parvovirus B19, virus patogeno umano della famiglia Parvoviridae, mostra uno specifico tropismo per i precursori eritroidi e una limitata replicazione in alcune linee cellulari megacarioblastoidi. Allo scopo di sviluppare sistemi utili allo studio delle caratteristiche biologiche del virus, diversi laboratori si sono occupati della costruzione di cloni genomici di B19 dotati di competenza funzionale e capaci di generare virus infettante. Parte del presente lavoro ha riguardato l’analisi funzionale di diversi cloni genomici di B19 e ha permesso di caratterizzare le regioni terminali del virus e di identificare requisiti essenziali per la loro funzionalità. Nel contesto intracellulare, esistono differenti livelli di restrizione in relazione alla capacità della cellula di supportare la replicazione virale, non ancora del tutto caratterizzati. Inoltre si sono accumulate evidenze circa la capacità del B19 di instaurare persistenza in numerosi tessuti. Non sono ancora note le caratteristiche funzionali del genoma virale in questo stato, è possibile che il virus persista in forma silente e meccanismi epigenetici possano regolare tale silenziamento. In questo studio è stato analizzato lo stato di metilazione del genoma di B19 e il suo possibile effetto sul ciclo replicativo virale ed è stata investigata la possibile associazione del DNA virale agli istoni cellulari nel corso di infezione in vitro. I risultati ottenuti confermano la presenza di questi meccanismi epigenetici, potendo ipotizzare che giochino un importante ruolo nella regolazione della funzionalità virale e nell’interazione B19-cellula e siano un elemento critico per l’adattamento del virus nell’ambiente in cui si trova. Inoltre l’ipotesi che anche i microRNA possano assumere un importante significato nell’interazione B19-cellula è stata proposta da diversi lavori e nel presente studio è stata valutata la produzione di queste piccole molecole durante l'infezione in vitro, ricercando microRNA (cellulari e/o virali) con omologia di sequenza per il genoma di B19 e quindi specifici per il virus.Parvovirus B19, human pathogenic virus of the Parvoviridae family, shows a specific tropism for erythroid precursors and a limited replication in some megakaryoblastic cell lines. In order to develop an in vitro system and study the biological features of this virus, several groups have constructed genomic clones of B19 with functional competence and able to generate infectious particles. Part of this work has focused on the functional analysis of several genomic clones of B19 to characterize the terminal regions of the virus and to identify essential requirements for their functionality. Once the virus is inside the cell, there are different levels of restriction depending on the cell's ability to support viral replication, which are not yet fully characterized. Furthermore there is increasing evidence that B19 is able to establish persistence in several tissues. The functional characteristics of the persistent viral genome are not yet known; the virus probably persists in a silent state and epigenetic mechanisms may regulate such silencing. In this study we analyzed the methylation status of the genome of B19 and its possible effect on the viral replication cycle and we investigated the potential association of viral DNA with histones during in vitro infection. The results confirm that these epigenetic events take place, supporting the hypothesis that they play an important role in the regulation of viral functionality and B19-cell interaction and are critical for the adaptation of the virus to the cellular environment. Moreover several works have proposed the hypothesis that microRNAs may also have an important value in B19-cell interaction, so in this study we evaluated the production of these small molecules during in vitro infection, searching for microRNAs (cellular and/or viral) showing sequence homology with the B19 genome and being thus specific for the virus

Glycemic Index Values of Pasta Products: An Overview

Durum wheat pasta is considered a low-glycemic index (GI) food. In recent years, the interest in developing enriched pasta has increased. Since both the formulation and processing technologies may affect the GI, this study aimed to investigate the GI values of pasta products (pp) reported in the literature until 2020. GI values of pp analyzed following the ISO guidelines were included in this survey. A total of 95 pp were identified and, according to their formulation, classified into 10 categories (n, mean GI): category n 1: 100% refined wheat (35, 55); category n 2: 100% whole wheat (6, 52); category n 3: other cereal-based products (8, 52); category n 4: containing egg (5, 52); category n 5: gluten free (11, 60); category n 6: containing legumes (9, 46); category n 7: noodles and vermicelli (9, 56); category n 8: containing vegetable or algae (6, 51); category n 9: containing other ingredients (5, 37); category n 10: stuffed (1, 58). Overall, pasta is confirmed to be a medium–low-GI food, even if a high variability among or within each category emerged. The formulation of enriched pp able to elicit a controlled glycemic response could represent a strategy to improve the nutritional value of pasta.</jats:p

Glycemic Index Values of Pasta Products: An Overview

Durum wheat pasta is considered a low-glycemic index (GI) food. In recent years, the interest in developing enriched pasta has increased. Since both the formulation and processing technologies may affect the GI, this study aimed to investigate the GI values of pasta products (pp) reported in the literature until 2020. GI values of pp analyzed following the ISO guidelines were included in this survey. A total of 95 pp were identified and, according to their formulation, classified into 10 categories (n, mean GI): category n 1: 100% refined wheat (35, 55); category n 2: 100% whole wheat (6, 52); category n 3: other cereal-based products (8, 52); category n 4: containing egg (5, 52); category n 5: gluten free (11, 60); category n 6: containing legumes (9, 46); category n 7: noodles and vermicelli (9, 56); category n 8: containing vegetable or algae (6, 51); category n 9: containing other ingredients (5, 37); category n 10: stuffed (1, 58). Overall, pasta is confirmed to be a medium–low-GI food, even if a high variability among or within each category emerged. The formulation of enriched pp able to elicit a controlled glycemic response could represent a strategy to improve the nutritional value of pasta

Detection of CpG methylation in B19V DNA from bioptic samples.

<p>A. Distribution of methylation indexes of viral DNA present in bioptic samples, as calculated by means of MSP assay [DNA_met/(DNA_met+DNA_unmet]. B. Melting profiles of the amplification products obtained by either MSP primer pair, specific for methylated or unmethylated target sequences (shown for standards and samples with methylation index >0.40).</p

Presence and distribution of CpG dinucleotides in B19V genome.

<p>The left terminal region and the proximal part of the internal region of B19V genome are shown in the diagram (nt.1–586, scale at the top line). Upper line: thick segment, left terminal region and axis of dyad symmetry; thin segment, internal region. Middle line: distribution of CpG dinucleotides, TATA box and start of transcription. Lower line: distribution of cis- recognition sequence elements. NSBE 1–4, NS protein binding elements 1–4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033316#pone.0033316-Guan1" target="_blank">[19]</a>. Sp1/3, Sp factors 1/3 recognition sequences.</p

Analytical characteristics of bisulfite and methylation specific real-time, quantitative PCR.

a)<p>bisulfite-treated B19V insert excised from pB19-M20.</p>b)<p>amplification of residual, unmethylated target following in vitro M.SssI treatment.</p><p>Unmethylated or in vitro methylated, bisulfite-treated B19V inserts from pB19-M20 plasmid have been quantified spectrophotometrically and 10-fold diluted to obtain quantitative standards for the definition of calibration curves. Analytical characteristics have been determined from triplicate amplification of standards in the range 10²–10⁸ target copies/reaction. For calibration curves, R² is >0.99 and CV is 0.16–0.24.</p

DNA methylation and quantitative analysis of B19V nucleic acids in a time course of infection.

<p>UT7/EpoS1 and U937 cells were infected with B19V at a multiplicity of infection of 10³ geq/cell, and samples collected from 0 to 27 days post infection (dpi). Quantitative analysis was carried out for the determination of the amounts of viral DNA and total viral RNA from cell fractions (copies/10⁴ cells). Methylation index was calculated by parallel amplification with both MSP primer pairs on bisulfite-modified DNA and is expressed as ratio of methylated over total viral DNA. ND: not detected.</p

CpG methylation and EGFP reporter gene expression in UT7/EpoS1 cells.

<p>Activity of viral P6 promoter, as determined by direct detection of EGFP fluorescence following transfection in UT7/EpoS1 cells of: unmethylated (A) or in vitro methylated (B) pEGFP-P6 plasmid DNA; linear products derived from ligation of the viral promoter (unmethylated, C and E, or methylated, D and F), upstream of the reporter gene in pEGFP-1 vector backbone (unmethylated, C and D, or methylated, E and F). Transfected UT7/EpoS1 cells were harvested at 72 hpt, about 50000 cells were spotted on glass slides, fixed with 4% paraformaldehyde in PBS, and counterstained with Evans Blue. FITC filter set, original magnification 40×.</p

Identification of CpG islands within B19V genome.

<p>Analysis of Parvovirus B19 genome sequence for the presence and distribution of CpG dinucleotides was performed by means of EMBOSS CpGPlot. Submission consisted of a consensus sequence of B19 genome, genotype 1, using standard parameters (Window length 100, Obs/Exp CpG ratio 0.6, Min C+G 50%, Min Length 200). Results are shown for positive sense (A) and complementary (B) strands. Upper graphs, distribution of observed/expected ratios of CpG dinucleotides; middle graphs, distribution of C+G; lower graphs, identification of putative CpG islands according to set parameters. (EMBOSS CpGPlot accessed at <a href="http://www.ebi.ac.uk/Tools/emboss/cpgplot/" target="_blank">http://www.ebi.ac.uk/Tools/emboss/cpgplot/</a>).</p