Figure 1.
GC content and observed/expected CpG ratio in parvoviral genomes.
Values are shown in percentage; viruses are listed by increasing GC content. Group I, Group II and Group III parvoviruses are indicated by red, violet and green colors respectively.
Figure 2.
CpG plot of three parvoviral genomes.
Red vertical bars represent CpG sites in the genomes, light blue shading indicates CpG islands (parameters: window 100, observed/expected CpG minimum 0.6, GC% minimum 50). A, PPV Kresse strain; B, Canine minute virus; C, AAV2 as representatives of Group I, Group II and Group III parvoviruses respectively. Figure was generated by MethPrimer program [34].
Figure 3.
CpG distribution in the coding positions of different organisms.
A, Total number of CpGs in the main coding ORFs (NS and VP) of parvoviral genomes. B, Number of CpGs in the different positions of the mRNAs of the human and drosophila proteomes. Numbers on the X axis indicate distance from the start codon of the proteins; Number of CGX (Arg codons) are indicated in 1,4,7,10… positions, numbers of XCG codons (Ser, Pro, Thr, Ala) are indicated in 2,5,8,11… positions and numbers of CGs in consecutive codons XXC GXX are indicated in 3,6,9,12… positions.
Figure 4.
Titration of the mutant PPVs with extra CpGs.
A, Schematic representation of the novel CpG sites in the mutant viruses. Red vertical bars symbolize the CpGs in the genomes. B, Titer of the mutant stocks on PT and Cos7 cells. Infected cells were detected by immunofluorescence labeling at 20 and 48 hours PI fixation, respectively. C, Titer of the mutant stocks after 10 additional passages on PT cells. Quantification was carried out by qPCR. Vertical bars indicate the standard deviation in each case.
Figure 5.
Deep sequencing of the bisulfite treated PPV genome.
Vertical bars label the position of the CpGs in the PPV genome. Height of the bars represents the frequency of unmethylated CpGs. Coverage of the CpG sites having more than 8% methylation rate was between 47 and 13694.
Figure 6.
SNP frequency of the CpG and GC sites in the PPV genome.
The frequency was calculated by dividing the rate of SNPs in the CpG and GC sites with the rate of SNPs in the total number of Gs and Cs in the genome.
Figure 7.
Initiation of the replication by differentially methylated PPV DNAs.
A, and B, PT cells on 96 well plate were transfected by 0.2 µg differentially methylated DNA and PPV infected cells were detected by immunofluorescence 24 hour post-transfection.
The fluorescent nuclei count (FNC) in one well is indicated by columns. C, Number of viral genomes in the supernatant of differentially methylated DNA transfected cells 24 and 48 hours post-transfection. Vertical bars indicate standard deviation in each case.
Figure 8.
Influence of the PPV infection on the expression of DNMTs.
A, Ratio of the mRNA levels of DNMTs in PPV infected/uninfected PT cells. Level of expressions were normalized with GADPH. B, Ratio of the alpha-tubulin normalized DNMT1 and DNMT3a/b protein levels in PPV infected/uninfected PT cells. C, Localization of DNMT1 in infected and uninfected PT cells. Nuclear staining by Hoechst 33342 is shown in blue, DNMT1 and PPV capsid proteins were visualized by indirect immunofluorescence methods and they are presented in green and red, respectively.