Parvovirus b19 DNA CpG dinucleotide methylation and epigenetic regulation of viral expression

Abstract

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.The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections.The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19.

Figure 1. Identification of CpG islands within B19V genome.

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 http://www.ebi.ac.uk/Tools/emboss/cpgplot/).

Figure 2. Presence and distribution of CpG dinucleotides in B19V genome.

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 . Sp1/3, Sp factors 1/3 recognition sequences.

Figure 3. CpG methylation and EGFP reporter gene expression in UT7/EpoS1 cells.

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×.

Figure 4. Detection of CpG methylation in B19V DNA from bioptic samples.

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).

Figure 5. Distribution of CpG methylation in B19V DNA from bioptic samples.

Representative diagrams derived from sequencing chromatograms of the amplification products obtained by means of BSP primers on viral DNA from bioptic samples. Top line, schematic diagram of the left terminal region and proximal part of the internal region of B19V genome, as in Figure 2. BSP and MSP primer positions are indicated. Bottom lines, diagrams indicating the methylation status of CpG dinucleotides as determined by sequencing of BSP amplification products, for the selected samples. The relative abundance of methylated or unmethylated cytosines within CpG dinucleotides is indicated by the gray scale code shown at the bottom of the figure.