CpG Distribution and Methylation Pattern in Porcine Parvovirus
Citation: Tth R, Mszros I, Stefancsik R, Bartha D, Blint , et al. (
CpG Distribution and Methylation Pattern in Porcine Parvovirus
Renta Tth 0
Istvn Mszros 0
Rajmund Stefancsik 0
Dniel Bartha 0
dm Blint 0
Zoltn Zdori 0
0 1 Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences , Budapest , Hungary , 2 Division of Genetics, FlyBase, University of Cambridge, Cambridge, United Kingdom, 3 National Food Chain Safety Office , Veterinary Diagnostic Directorate, Budapest , Hungary
Based on GC content and the observed/expected CpG ratio (oCpGr), we found three major groups among the members of subfamily Parvovirinae: Group I parvoviruses with low GC content and low oCpGr values, Group II with low GC content and high oCpGr values and Group III with high GC content and high oCpGr values. Porcine parvovirus belongs to Group I and it features an ascendant CpG distribution by position in its coding regions similarly to the majority of the parvoviruses. The entire PPV genome remains hypomethylated during the viral lifecycle independently from the tissue of origin. In vitro CpG methylation of the genome has a modest inhibitory effect on PPV replication. The in vitro hypermethylation disappears from the replicating PPV genome suggesting that beside the maintenance DNMT1 the de novo DNMT3a and DNMT3b DNA methyltransferases can't methylate replicating PPV DNA effectively either, despite that the PPV infection does not seem to influence the expression, translation or localization of the DNA methylases. SNP analysis revealed high mutability of the CpG sites in the PPV genome, while introduction of 29 extra CpG sites into the genome has no significant biological effects on PPV replication in vitro. These experiments raise the possibility that beyond natural selection mutational pressure may also significantly contribute to the low level of the CpG sites in the PPV genome.
DNA methylation is the prime form of epigenetic
modifications of the eukaryotic genome. In vertebrate cells
almost exclusively the 5th carbon atom of cytosine is
methylated within CpG dinucleotides. Methylation has a
significant impact on chromatin structure modulation, genomic
imprinting and X chromosome inactivation. It can inhibit
transcription by preventing the binding of transcription factors
or by recruiting methyl-binding proteins and histone
deacetylases leading to the formation of condensed chromatin
In mammals approximately 60-90% of CpGs comprise
methylated cytosine bases . The 5 end of the housekeeping
genes are often associated with a GC-rich stretch of DNA
containing high amounts of CpG dinucleotides, so called CpG
islands, which are free of methylation . DNA
methyltransferases (DNMTs) are responsible for the
conversion of cytosines to 5-methylcytosines. The DNMTs are
divided into two groups: maintenance (DNMT1) and de novo
(DNMT3a and DNMT3b) methyltransferases. The mechanism
of site specific CpG methylation and regulation of DNMTs to
develop specific patterns are presently not well understood .
CpGs are observed only at one-fourth to one-third of their
expected frequency [5,6] in most vertebrate genomes. Several
explanations have been proposed to account for this
discrepancy, deamination and timine conversion of methylated
cytosines, avoidance of higher stacking energy of CpG
dinucleotides during replication and prevention of autoimmune
reactions among others.
Unmethylated CpGs (UCpGs) as signature of invading
bacterial and viral organisms are immunostimulants even on
short oligonucleotides in mammals. Immune response is
triggered by the UCpGs binding to TLR9, a member of the
Tolllike receptor family on the surface of dendritic cells .
Therefore CpG methylation is not only important in the
regulation of the hosts life processes, but it also plays a key
role in the detection of microbial and viral pathogens and
inactivation of integrated foreign DNA [8,9], consequently it has
major influence on the lifecycles of DNA- and retroviruses as
well. The role of methylation in viral regulation is less
understood than in mammals. Integrated adenoviruses and
papovaviruses are generally hypermethylated, while the
actively replicating viral DNA is hypomethylated with
methylated sites in specific regions of the viral genome .
EBV is highly methylated during latency, and becomes
demethylated during active replication. It uses
methylationinduced gene silencing to evade host immunity . In
contrast, ranid herpesviruses are heavily methylated during
replication and probably code their own DNA cytosine-5
methyltransferases . CpG dinucleotides are
underrepresented in most of the small DNA viruses. This
pattern is thought to be established by evolutionary pressure to
avoid CpG-mediated immune responses and to decrease the
direct interference of methylation on the transcription of viral
RNAs and viral replication .
Parvoviruses are small single stranded DNA viruses with an
approximately 4-6 Kb linear genome. Despite their small
genome and their limited coding capacity parvoviruses are
surprisingly successful to invade a wild variety of host
organisms from insects to mammals and constitute a large,
diverse virus family . Their diversity manifests not only in
the large number of parvoviral species, but also in the
complexity of their lifecycle. Beside lytic infection some
parvoviruses are able to infect their respective host persistently
[13,14]. Adeno-associated viruses are able to insert their
genome into the host genome to establish latent infection and
subsequently are capable to parasitize the transcription
machinery of other viruses and reactivate their own replication
mechanism during helper virus infection .
Members of the Parvovirinae subfamily are among the most
dangerous and economically most harmful viruses of
domesticated and farm animals (e.g., canine parvovirus (CPV)
, goose parvovirus (GPV)  porcine parvovirus (PPV)
 and are also able to cause diseases in humans (B19) 
and human bocavirus [20,21]. PPV is responsible for syndrome
of reproductive failure in swine, included infertility, early
embryonic death, mummified fetuses and stillbirths . It
frequently causes persistent infection combined with chronic
There are some well-established connections between
persistent viral infection and CpG methylation in other virus
families [24,25] but not much is known about the role of
epigenetic modifications in parvoviruses. In this paper our aim
is to expand our knowledge about the effect of CpG
methylation in the life cycle of parvoviruses focusing on PPV
and to reveal whether methylation has any direct influence on
the evolution of the CpG poor PPV genome.
Our in silico analysis of the CpG pattern of parvoviruses
revealed that parvoviral genomes are more heterogenic in their
CpG contents than it was previously recognized and a group of
parvoviruses exists in which CpGs are not depleted despite
that their genomes are AT rich. PPV DNA was found
hypomethylated independently from its tissue origin. In vitro
methylation of PPV DNA or the introduction of additional CpG
sites into the PPV genome had no significant effect on PPV
replication in vitro. These data indicate that CpG methylation
has no regulating role in PPV life cycle and together with the
recently published findings that parvoviruses do not induce
TLR9 activated immune response  suggest that CpG
depletion in the genome of PPV and other parvoviruses is most
probably the consequence of other evolutionary forces than
Materials and Methods
Computing CpG distribution in coding positions and
Parvovirus sequences have been collected from the NCBI
nucleotide databank (Table S1a). The contiguous protein
coding sequences for Drosophila melanogaster were
downloaded in FASTA format from the FTP site of FlyBase
(release r5.24) . The core set of human coding sequences
are from The Consensus CDS (CCDS) project . The protein
coding regions of the human mRNAs were downloaded in
FASTA format from the CCDS database . Dinucleotide
frequencies as a function of coding sequence position were
calculated using custom Bash scripts (available upon request).
Single nucleotide polymorphism (SNP) was calculated from 68
PPV sequences containing the full coding regions or the
complete NS or VP genes (Table S1b) with a custom made
program. The algorithm counts the polymorphism sites
(distinguishing the transition events at C, G, CpG and GC
nucleotides) in a multiple alignment where a consensus base is
only considered with 75% confidence or above (C++ source
code available upon request).
PT (porcine testis)  and Cos 7 (African green monkey
kidney)  cell lines were used for the propagation of the
NADL-2 strain of PPV and its derivatives. Cells were grown in
Dulbeccos modified Eagles medium with high glucose (4,5 g/l)
and L-glutamine (PAA, Clbe, Germany) supplemented with
1% PenStrep (100X; penicillin: 10 000 U/ml, streptomycin: 10
mg/ml; PAA), 1% sodium-pyruvate solution (100 mM; Lonza,
Basel, Switzerland) and 10% Fetal Bovine Serum Gold (PAA).
Viral DNA extraction and determination of the
The packaged form of the viral DNA was extracted from 1 ml
tissue supernatant by the High Pure Viral Nucleic Acid Kit
(Roche, Basel, Switzerland) according to the manufacturers
recommendations. The replicative PPV DNA was purified by
using modified Hirt extraction  as it is described by Molitor
et al. . The methylation status of the viral DNAs was
determined by bisulfite PCR, cloning and sequencing. For the
bisulfite conversion the EpiTect Bisulfite Kit (Qiagen, Venlo,
Netherlands) was used according to the manufacturers
instructions. The modified CpG containing DNA fragments of
the positive and negative strands were amplified by PCR
primers (Table 1) which were designed using MethPrimer
program . PCR reactions were executed by DreamTaq
DNA Polymerase (Thermo Fisher Scientific, Waltham, MA,
USA). The DNA amplifications were carried out by initial
denaturation for 5 min at 95 C, followed by 30 cycles at 95 C
for 20 s, 52 C for 20 s, and 72 C for 20 s. The amplified DNA
fragments were cloned into pJET1.2 blunt cloning vector
MREV11F 5'-TTGTTTTAGTAAATAGGTGATTGATTAAGT-3' 262
MREV12F 5'-AATAAGAGAGTTAAAAATTAGTATTTGT-3' 185
MREV13F 5'-TGAGTATTATTTTGAAGTTAGTTGGTAGT-3' 276
MREV14F 5T'T-GAAGTAGAAAGGAATTAATTGAAGTAAG-3' 239
(Thermo Fisher Scientific) and sequenced by using BigDye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,
Foster City, CA, USA) following the instructions of the
Deep sequencing was executed on an Ion Torrent
sequenator using the IonXpress barcode set and the 316D chip
kit, after a DNA library preparation from the equimolarly pooled
bisulfite PCR fragments by the NEBNext Fast DNA
Fragmentation & Library Prep Set for Ion Torrent (New England
BioLabs, Ipswich, MA, USA). For data procession the 2.2
Torrentsuite software was used. To gain quality data, reads
under 15 average Phred score were omitted. CLC Genomics
Workbench 5.5 was used for data analysis. High confidence of
the evaluation was ensured by excluding short reads (<20
nucleotide) and setting Length fraction and Similarity fraction
parameters to 0.9.
Creation of the mutant viruses
Seven mutant viruses (3 single M1, M2, M3, and three
double M12, M23, M13 and one triple M123) were rescued
containing extra CpGs. To introduce CpG mutations into the
NcoI-SacI (3473-3935) fragments of NADL-2 strain by joining
PCR, three pairs of overlapping mutational primers (M1-3F and
M1-3R), two external (mut_exF, mut_exR) and two internal
(mut_intF, mut_intR) primers were designed (Table 2). For the
upstream fragments of the joining PCRs mut_exF and M1-3R
primers were utilized, for the downstream fragments PCR
mut_exR and M1-3F primers. For single mutants the pN2
infectious clone of the NADL-2 strain , for double mutants
the clone of the M1 and M2 mutant viruses, while for the triple
mutant the clone of M12 served as templates. The PCR
reactions included 5 l of 5X HF Buffer, 0,5 l of 10 mM dNTP,
1 l of each overlapping and external primers (15 pmol/l) of
each different mutants, 0,2 l of 2U/l Phusion Hot Start II DNA
Polymerase (Thermo Fisher Scientific), 0,05 ng template DNA
and distilled water to a final volume of 25 l. Amplifications
started by initial denaturation for 5 min at 95 C and followed by
30 cycles at 98 C for 10 s, 65 C for 15 s, and 72 C for 20 s.
Joining PCRs were carried out by mut_intF and mut_intR
primers using Hot Start II DNA Polymerase and 2 ng of the
appropriate mutant downstream and upstream fragments. DNA
amplifications started by initial denaturation for 5 min at 95 C,
followed by 30 cycles at 98 C for 10 s, 60 C for 15 s, and 72
C for 20 s. The amplified 1005 nucleotide-long products were
digested with NcoI and SacI (New England BioLabs) restriction
enzymes and cloned into the same sites of the pN2 infectious
clone. Mutant sequences were deposited into GenBank under
the accession numbers: KF913345-KF913351.
Transfection, viral stocks titration and quantification
To rescue the mutant viruses the infectious clones were
transfected into PT cells by Turbofect (Thermo Fisher
Scientific) reagent according to the suppliers
recommendations. After 48 hours 50 l culture media was
transferred from the transfected cells to a 24-well plate seeded
with PT cells and the viruses were let to multiply for 96 hours.
This was followed by the inoculation of semi confluent PT cells
with 0.5 ml of viral supernatants on 75 cm2 plates. After 96
hours the supernatants were collected and titrated by three
parallel, independent dilutions with immunofluorescence
detection technique on PT and Cos7 cells as described
previously . Shortly: virus samples were serially diluted
(10x) and cells on a 96-well plate were infected with 10 l of
the viral dilutions. PT and Cos7 cells were fixed after 20 and 48
hours respectively (to exclude the detection of progeny viruses
emerging from the cells used for titering) with 3% formaldehyde
and permeabilized with 1% Triton X-100. The 3C9 (CRL-17;
ATCC) anti-PPV antibody and Alexa Flour 488 donkey
antimouse IgG (Life Technologies Carlsbad, CA, USA) as
secondary antibody were used for visualization of the infected
nuclei (IN). Titer was calculated by multiplying the number of IN
by the dilution factors and values are given in fluorescent nuclei
For qPCR quantification of the viral production initiated by
differently methylated DNAs the transfected cells were washed
three times to ensure minimal contamination of the viral stocks
by plasmid originated viral DNA.
To monitor sequence stability viral stocks were passaged 10
times on a 24-well plate containing PT cells transferring 5 l of
the virus containing supernatant after 48 hours to freshly
seeded cells covered by 2 ml medium. After the last passages
new stocks were prepared on 75 cm2 plates as it is described
above. Following DNA extraction, the mutated regions were
amplified by mut_intF, mut_intR primers (Table 2) and
For DNA quantification qPCR were performed using the
earlier described VPS2
(5CAATACTGCACCTGTATTTCCAAATGG-3) and VPAS2
(5AAAATTTTATTGTTTTTTGGGGATAATTGG-3) primers .
The reactions included 2,5 l of 10X DreamTaq Buffer, 0,5 l
of dNTP mix, 1 l of each 15 pmol/l primers, 0,2 l of
DreamTaq DNA Polymerase, 1,25 l of 20X EvaGreenTM
(Biotium, Hayward, CA, USA), 1 l DNA template and sterile
distilled water to a final volume of 25 l. The DNA
amplifications were carried out by initial denaturation for 5 min
at 95 C, followed by 40 cycles at 95 C for 20 s, 55 C for 20 s,
and 72 C for 50 s.
A T AC CGG AAC TAC
Preparation of bacterially methylated and
nonmethylated DNA for transfection
To get unmethylated viral genome, a PCR was performed
using pN2 as template and N2F
(5GGGTTATTGTCTCATGAGCGGATACATA-3) and N2R
(5CAATTTCACACAGG AAACAGCTATGACC-3) primers. The
PCR reaction included 20 l of 5X GC Buffer, 2 l of 10 mM
dNTP, 6 l DMSO 0.6 l of each 100 pmol/l primers, 1 l of
2U/l Phusion Hot Start II DNA Polymerase (Thermo Fisher
Scientific) and distilled water to a final volume of 100 l. The
DNA amplification started by initial denaturation for 3 min at 98
C, followed by 25 cycles at 98 C for 15 s, 66 C for 20 s, and
72 C for 4 min 30 s. To obtain bacterially DAM and DCM
methylated viral genome the pN2 was digested by KpnI and
BamHI restriction enzymes (Thermo Fisher Scientific) which
cut the NADL-2 virus from the vector. In each case the viral
genomes were isolated from 0.7% agarose gel by Nucleospin
Extract II kit (Macherey-Nagel, Dueren, Germany).
In vitro CpG methylation
In a reaction 3 g viral DNA was methylated by CpG
methylase (Zymo Research, Irvine, CA, USA) according to the
manufacturers instructions. The reaction was stopped by
ethanol precipitation, washed by 70% ethanol, dried and
resolved in 20 l distilled water. The success of
hipermethylation was inspected by digestion of an aliquot with
methylation sensitive SsiI (Thermo Fisher Scientific) restriction
enzyme in 20 l final volume.
Determination of expression levels of DNA
methyltransferases in infected and non-infected tissues
The expression levels of porcine DNMT1, DNMT3a,
DNMT3b were defined by real-time PCR, and normalized with
those of the GADPH gene. RNAs were purified from PPV
infected (MOI 3) and mock infected PT cells grown on 75 cm2
flask 24 hours postinfection (HPI) by RiboZolTM RNA Extraction
Reagent (Amresco, Solon, OH, USA) according to the
manufacturers instructions. The RNA was dissolved in 15 l
DEPC-treated water. The first strand of cDNA and the
amplification were created using One-Step RT-PCR Kit
(Qiagen). The reactions included 5 l of 5X QIAGEN OneStep
RT-PCR Buffer, 1 l of dNTP mix, 1 l of each 15 pmol/l
primers (Table 3), 1 l of QIAGEN OneStep RT-PCR Enzyme
mix, 1,25 l of 20X EvaGreenTM, 1 l RNA template and
RNase-free water to a final volume of 25 l. The reaction
started with reverse transcription for 30 min at 50 C and initial
PCR activation for 15 min at 95 C, followed by 35 cycles at 95
C for 30 sec, 55 C for 30 sec, and 72 C for 40 sec, and 72
C for 5 min.
Immunofluorescence and Western blot detection of
Western blot detection of the DNMT proteins were carried
out by using DNMT1 (H-300) and DNMT3a/b (C-15) primary
antibodies (Santa Cruz Biotechnology Inc., Heidelberg,
Germany), horseradish peroxidase conjugated swine
antirabbit (DAKO, Glostrup, Denmark) and rabbit anti-goat
Primer name Sequence size (bp) number
DNMT1_F 5-TCGAACCAAAACGGCAGTAGT-3 215 NM_001032355
DNMT3a_F 5- CTGAGAAGCCCAAGGTCAAG-3 238 NM_001097437
DNMT3b_F 5-AACCCAACAAAGCAACCAG-3 275 CN_156332
GADPH_F 5'-TCGGAGTGAACGGATTTG-3' 151 AF_017079
(Southern Biotech, Birmingham, AL, USA) secondary
antibodies in a 50, 100, 500 and 8000-fold dilutions
respectively. For loading control anti-alpha tubulin antibody
(Developmental Studies Hybridoma Bank, Iowa City, IA, USA)
were used together with peroxidase conjugated rabbit
antimouse IgG (Sigma-Aldrich, St. Louis, MO, USA).
The peroxidase was revealed using the TMB cholorimetric
substrate (MIKROGEN, Neuried, Germany) according to the
suppliers recommendations. Bands were quantified with the
ImageJ programs .
For immunofluorescence detection DNMT1 (H-300) and
CF488A labeled goat anti-rabbit were used in 50 and 300-fold
dilutions respectively and the samples were examined with a
Zeiss Axio Observer D1 inverse fluorescence research
GC and CpG content of Parvoviruses
To better understand common and distinguishing features of
PPV genome organization among parvoviruses the GC content
and CpG density of 32 parvoviruses from the Parvovirinae
subfamily were calculated and compared. The GC content of
parvoviral genomes scales between 35% and 63%. In general,
it can be stated that self-replicating parvoviruses have AT-rich
genomes (GC content < 50%), while most of the
adenoassociated viruses have GC-rich genomes (GC content > 50%)
In self-replicating viruses the observed/expected CpG ratio
(oCpGr) is variable: it can take very low values (like in the case
of PPV), or high values (as it can be seen in the CaMiV
genome). Dependoviruses are more uniform regarding CpG
content: in each case the oCpGr values stay above 60% with
the exception of MDPV (which is an autonomous member of
the Dependovirus genus) and can reach more than 100%.
Considering the GC content and the oCpGr values, three
major categories can be distinguished among the members of
Parvovirinae subfamily: parvoviruses with low GC content (<
50%) and low oCpGr values (< 50%) (Group I), viruses with low
GC content (< 50%) and high oCpGr values (> 50%) (Group II)
and viruses with high GC (> 50%) content and high oCpGr (>
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
50%) values (Group III). A characteristic taxonomical
distribution can be recognized among these three groups. All
known members of Amdovirus, Erythrovirus and Parvovirus
(PPV among them) genera together with several members of
the Bocavirus genus (like the highly CpG depleted human
bocavirus and porcine bocavirus) belong to Group I. Other
members of the Bocavirus genus (e.g. the CpG-rich BPV and
CaMiV) together with the unclassified chicken and turkey
parvoviruses are members of Group II. Most viruses of the
Dependovirus genus belong to Group III.
CpG distribution in PPV and in the coding regions of
Not only the number of CpGs but their distribution in the
genome is different among parvoviruses. Very few CpG islands
can be found in members of the first group and usually they are
restricted to the terminal regions, while several potential CpG
islands can be plotted in every member of the latter two groups
scattered throughout the genome including both the coding and
non-coding regions (Figure 2).
The NADL-2 strain of PPV contains 60 CpGs 13 in the left 7
in the right non coding regions and 40 in the protein coding
sequences. The distribution of CpGs in the coding frames is
not random. Only three CpGs can be found in the first coding
position (CGX) in the Arginine codons, 13 in the second
position (XCG) (in serine, proline, threonine and alanine
codons) and 24 in the third position (XXC GXX) affecting two
amino acid codons (Figure 3a). Similar ascendant tendency of
CpG distribution can be observed by position in the majority of
the parvoviruses but also in the viral hosts, for example in
Homo sapiens (Figure 3b). Since the distribution is
independent of the absolute number of the CpGs in the viral
genomes and similar distribution can be detected in eukaryotic
organisms, which do not have CpG methylation (e.g.
Drosophila melanogaster) , it is more probable that the
particular pattern of CpG distribution is rather the effect of
coding bias and coding preference than some unknown
evolutionary effect of the methylation machinery of the different
Biological effects of additional CpGs in the PPV
The small number of CpGs in the PPV genome implies an
evolutionary pressure against this dinucleotide. To study the
biological effects of the elevated CpG ratio, seven mutants
were created in which new CpG sites were inserted into three
CpG free regions of the VP2 gene by site-specific silent
mutations of the pN2 infectious clone. The number of the new
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 .
CpG sites in the mutants ranged between 9 and 29 (Figure 4a).
The mutant clones were transfected into PT cells and the
supernatants of the transfected cells were collected 48 hour
post transfection and used to infect PT cells. The presence of
PPV was monitored by IF using 3C9 monoclonal antibodies.
The transfection of all mutant infectious clones resulted viable
virus and a stock solution was prepared from each and titrated
on permissive PT cells and semi-permissive Cos7 cells. There
was no significant difference in the infectivity of the mutant and
the wild type viral stocks indicating that the extra CpG sites
have no significant biological effects (p= 0.557 at PT cells and
p= 0.0727 at Cos7 cells by ANOVA) either on viral production
or viral growth in vitro (Figure 4b). To monitor the stability of the
mutant viruses 10 additional passages were carried out on PT
cells and new stock solutions were prepared. The titers of the
stocks were quantified by qPCR (Figure 4c) and again there
were no significant differences (p= 0.0974 by ANOVA) in the
titer among the different mutants and the wild type virus.
Sequencing of the mutant viruses from the different mutant
stocks confirmed that no new or back mutations are present on
the viruses and the introduced mutations are stable, which
indicates lack of selective disadvantage of the additional CpGs
in tissue culture.
Methylation status of PPV
To establish the methylation status of PPV genome, the
bisulfite conversion based PCR protocol was used, which
allows the independent analysis of the methylation of the
negative and the positive strands. To cover all CpG sites on the
full genome two sets of PCR primers (13 pairs for the negative
strand and 11 pairs for the positive strand) were planned. First,
the encapsidated negative strand was analyzed from virions
originated from permissive (PT) 20 HPI semi-permissive (Cos7)
cells 96 HPI and aborted pig embryos. In each case the
investigated DNAs were highly hypomethylated (Table 4, Table
S2). PPV encapsidates only negative strand, and positive
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.
strand can be found almost exclusively only in the replicative
form PPV DNA . To exclude that the hypomethylation
pattern of the viral DNA would be the result of specific
encapsidation of the unmethylated DNA, the methylation
pattern of the cellular PPV DNA purified from PPV infected PT
cells (20 HPI) was also determined (Table 4). Similarly to the
encapsidated negative strand, the positive strand proved to be
hypomethylated, suggesting that PPV DNA remains
hypomethylated during the entire life cycle of the virus including
replication and packaging.
1-2 percentage points of the CpG sites on the cloned bisulfite
treated PPV fragments remained resistant against C to T
conversion indicating a rare occurrence of methylation on the
PPV DNA. To gain more accurate data about the methylation
level, the bisulfite treated PCR fragments were deep
sequenced. Around 168000 PCR fragments were analyzed
with 0-22619 coverage of the CpG sites. Most of the CpG sites
had more than 92% conversion frequency while around 10% of
the CpG sites had less than 92% conversion rate indicating
that low level CpG methylation occurs on replicating PPV DNA
(Figure 5). Most probably there is no immediate effect of such a
low level methylation on PPV replication, however, even low
level of methylation can drive the purge of CpGs from the PPV
genome during a long period of time, since methylated
cytosines are mutational hotspots [41,42].
In fact, analysis of single nucleotide polymorphism (SNPs) of
the PPV genome based on the available sequences of the
DNA databank support the high mutability of CpG sites in the
PPV genome. On the investigated coding region, the ratio of
SNPs in the CpG sites (17 mutations, 38 CpG sites)
Table 4 (continued).
Positive strand Negative strand
CpG Clones Clones
site Position sequenced Methylation Position sequenced Methylation
1 28 6 0 29 6 0
2 38 6 0 39 6 0
3 46 6 0 47 6 0
4 48 6 0 49 6 0
5 50 6 0 51 6 0
6 55 6 0 56 6 0
7 57 6 0 58 6 0
8 59 6 0 60 6 0
9 68 6 0 69 6 0
10 79 6 0 80 6 0
11 147 6 0 148 12 0
12 168 6 0 169 12 0
13 249 4 0 250 6 0
14 299 4 0 300 6 0
15 314 4 0 315 6 0
16 455 8 0 456 6 0
17 531 4 0 532 6 0
18 547 4 0 548 6 0
19 845 3 0 846 6 0
20 1017 3 0 1018 11 0
21 1079 7 0 1080 5 0
22 1239 4 0 1240 5 0
23 1776 4 0 1777 6 0
24 2051 6 0 2052 5 0
25 2057 6 0 2058 5 0
26 2070 6 0 2071 5 1
27 2120 6 0 2121 4 0
28 2127 6 0 2128 4 0
29 2174 6 0 2175 4 0
30 2191 6 0 2192 4 0
31 2213 6 0 2214 4 0
32 2226 6 0 2227 4 0
33 2291 6 0 2292 4 0
34 2464 6 0 2465 6 0
35 2467 6 0 2468 6 0
36 2482 6 0 2483 6 0
37 2485 6 0 2486 6 0
38 2494 6 0 2495 6 0
39 2575 6 1 2576 12 0
40 2587 6 0 2588 12 0
41 2624 6 0 2625 6 0
42 2694 6 0 2695 6 0
43 2697 6 0 2698 6 0
44 2893 3 0 2894 6 0
45 2896 3 0 2897 6 0
46 2902 3 0 2903 6 0
47 3004 3 0 3005 6 0
48 3040 3 0 3041 6 0
49 3087 3 0 3088 4 0
50 3106 3 0 3107 4 1
51 3154 7 0 3155 4 0
Positive strand Negative strand
CpG Clones Clones
site Position sequenced Methylation Position sequenced Methylation
52 3180 4 0 3181 4 0
53 3334 4 0 3335 4 0
54 4940 6 0 4941 6 0
55 4950 6 0 4951 6 0
56 4962 6 0 4963 6 1
57 4971 6 1 4972 6 0
58 4983 6 0 4984 6 0
59 4987 6 0 4988 6 0
60 4990 6 0 4991 6 0
significantly exceeds the ratio of SNPs in the total number of G
and C nucleotides (113 mutations, 1619 G+C nucleotides) (p<
2.2x10-16 by Pearson's Chi-square test) or in the GC sites (29
mutations, 148 GC sites) (p= 0.0014 by Pearson's Chi-square
test). Even a higher difference (3,82) can be observed
calculating the transition rates (p= 5.413x10-13) by Pearson's
Chi-square test) (14 CT and GA in CpG sites versus 78 in
G and C nucleotides of the genome) (Figure 6).
These data, together with our observation that extra CpG
sites in PPV do not interfere with PPV replication, raise the
possibility that not only natural selection but mutational
pressure might contribute significantly to the low level balance
of the CpG sites in the PPV genome.
The effect of methylation on PPV replication
To investigate the effect of CpG methylation on the PPV
replication, PPV genomes were in vitro methylated, transfected
and their replication initiation capability was compared to that of
the bacterially cloned (DAM DCM methylated) and PCR
amplified (non-methylated) PPV genomes. In vitro methylation
was executed by M.SssI CpG methylase and in each case
almost complete CpG methylation (>95%) of the PPV genomes
could be achieved, shown by the digestion of the treated DNAs
with the SsiI methylation sensitive restriction endonuclease
(data not shown). The CpG methylated DNAs and their non
CpG methylated counterparts were transfected into PT cells
and their virus replication initiation capability was monitored by
IF assay at 24 hours post-transfection using 3C9 PPV specific
monoclonal antibody. In each case the CpG methylated DNA
induced around 62% less viral infection in PT cells than the non
CpG methylated control DNAs, indicating that CpG methylation
has a relatively modest inhibitory effect on PPV replication
(Figure 7a-b). This result was confirmed by qPCR analysis of
the supernatant of the transfected cells (Figure 7c).
Interestingly, non-methylated PPV dsDNA (PCR amplified) was
less effective to initiate viral replication than the bacterially
DAM/DCM methylated dsDNA.
The progeny viruses from CpG methylated PPV DNA
transfected cells were collected, and the methylation pattern of
their DNA was examined. The in vitro hypermethylated status
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.
of the transfected PPV genome could not be detected on the
investigated fragments of the genome of the progeny viruses,
they even proved to be hypomethylated, similarly to the
genome of the native virus (Table S2d).
To gather additional evidence on the sustainability of the
hypomethylated status of the PPV genome, the clone of the
M123 mutant was also transfected into PT cells and the
methylation pattern of the progeny virus was also examined.
The 29 newly introduced CpG sites also remained
hypomethylated similarly to the wild type CpG sites of the
native virus (Table S2e).
These experiments strongly suggest that beside the
maintenance DNMT1 the de novo DNMT3a and DNMT3b
methylases cannot methylate replicating PPV DNA effectively
either, and hypomethylation is not restricted to the existing
CpG sites: it is a generalized process which extends to the full
Influence of the PPV infection on the transcription,
translation, and cellular distribution of DNMT proteins
Hypomethylation of PPV DNA must involve a decreased
activity of the cellular DNMTs on PPV DNA and/or a weak
susceptibility of the PPV DNA to their action. Absence,
inhibition or elimination of DNMTs in infected cells, different
compartmentalization of methylases and viral DNA, or the
unability of methylases to recognize PPV DNA as substrate
singularly or synergistically can lead to hypomethylation of the
viral genome. To study the direct reasons behind the
hypomethylated status of PPV DNA we have investigated the
transcription, translation and subcellular distribution of DNMTs
in infected and non-infected host cells. Quantitative
measurement of the mRNAs of DNMT1 DNMT3a and DNMT3b
by real-time qRT-PCR reveals that the mRNAs of all the three
major form of DNMTs are present in PT cells and PPV infection
does not change their levels significantly (Figure 8a). As
demonstrated by Western-blot and immunofluorescence
experiments, the translation level of the DNMT proteins is not
affected significantly and localization of the DNMT1 is not
changed either by PPV infection in PT cells (Figure 8b-c).
The effect of overexpression of the DNMT3a on PPV
methylation was also investigated. To ensure the
coexpression of the PPV genome and the DNMT3a protein, the
pN2 and the human DNMT3a expressing
pcDNA3/MycDNMT3A [43,44] plasmid were co-transfected into PT cells.
Co-transfection of the two plasmids resulted around three times
less infectious foci (data not shown) than the cotransfection of
the pN2 and the pDSREDmonomer-N1 plasmid which
expressed the dsRED protein as a control, indicating an
inhibitory effect of DNMT3a on PPV replication.
Overexpression of DNMTs has been shown to change the
pattern of gene expression in cells and influence cell cycle
[45,46]. So it cannot be excluded that the viral inhibition of the
overexpressed DNMT3a is rather due to an indirect effect on
the host cell regulation than to direct methylase activity on the
viral DNA. Especially because bisulfite sequencing of the
rescued viruses, emerged from pcDNA3/Myc-DNMT3A and
pN2 co-transfection, revealed a hypomethylated status of the
viral DNA (Table S2f), demonstrating that DNMT3a, even
present in excess, cannot methylate effectively replicating PPV
Investigation of the GC and CpG contents of 32 parvoviral
genomes revealed three distinct groups. Dependoviruses
represent a group with high GC and high oCpGr values. As
earlier recognized, the relative CpG-rich sequence of
dependoviruses makes them an out-group among similarly
sized DNA viruses [9,47]. Just like large DNA viruses, they are
much less biased toward CpG dinucleotids than the small DNA
The majority of the known autonomously replicating
parvoviruses belong to a different group with opposing
characteristics manifesting low GC and low oCpGr values.
However, based on our findings, several recently described
autonomous parvoviruses seem to differ significantly from this
group and feature relatively high oCpGr values combined with
low GC content.
This finding somewhat extenuates the conclusion which
emerged from the earlier analysis of viral sequences that small
DNA viruses parvoviruses among them are extremely
biased against CpG dinucleotide, and only adeno-associated
viruses are exceptions from this rule [9,47].
The methylation patterns of a few DNA viruses have been
studied in details. The hypomethylated state of the replicating
PPV genome fits very well with what was described about
replicating adeno [48,49] papilloma and polyoma viruses
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.
[50,51,52,53] and reinforces the emerging view that the
genome of small DNA viruses remains hypomethylated during
replication. One possible explanation for this phenomenon is
the association of the viral DNA with host and viral proteins
during rapid viral replication and encapsidation which may
simply prevent the interactions with DNMTs. However, the lack
of (so far unknown) cis signals for de novo methylation or the
presence of chromatin insulator sequences [54,55,56] might
also contribute to the sustainment of hypomethylation in the
PPV genome. This notion is supported by the fact that while
some episomal adenoviral constructs become rapidly de novo
methylated , others, like episomal recombinant AAV and
even some bacterial plasmid constructs remain
hypomethylated for a long time after introduction into the
mammalian cells, despite being replication incompetent
[58,59]. The inverted terminal repeat (ITR) of AAV is suspected
to be an insulator  and most probably has a role in keeping
AAV recombinant constructs hypomethylated and
transcriptionally active . However, whether the ITRs of PPV
or any other parvovirus functions similarly remains to be
Interestingly, complete in vitro CpG methylation of PPV
genome has a moderate inhibitory effect (around 62%
decrease) on PPV replication initiation. The decrease was
found very reproducible in several experiments and
independent of the original methylation status of the treated
DNA. These experiments indicate a moderate sensitivity of the
PPV genome to methylation. This result is somewhat
contradictory to human parvovirus B19 studies, since B19
promoter and replication seem to be very sensitive to CpG
methylation . However, B19 has a CpG island and 42 CpGs
in its terminal 520 bp promoter/enhancer region overlapping
with Sp1, Sp3 and viral NS protein binding elements, while
PPV has no CpG island and has only 12 CpGs in its 190 bp
long P4 promoter/enhancer region and no CpGs at all in its
140-bp-long P40 promoter region. Recent investigations
revealed that the relationship between the methylation status
and transcriptional activity of a gene is more complicated than
it was originally thought [4,63,64] and different promoters react
differently to methylation [63,65]. The different sensitivity of the
two parvoviruses to methylation can be explained by the
different CpG content of their promoters since the methylation
of CpG-poor promoters frequently does not preclude
transcription  while the methylation of CpG island
promoters usually suppresses their activity [4,65].
In contrast to viruses (e.g hepatitis B virus, Mareks disease
virus, Kaposi's sarcoma-associated herpesvirus and EBV) from
other viral families [66,67,68,69], PPV infection does not
influence the mRNA or the protein level of the DNA methylases
and does not seem to change the localization of the DNMT3a.
So it looks like that replicating PPV DNA is a weak substrate of
the hosts DNA methylases and even overexpressed DNMT3a
cannot raise PPV methylation level significantly.
The genome of the majority of the autonomous parvoviruses
PPV among them similarly to their hosts are highly GC and
CpG depleted. It is widely assumed that the main reason
behind CpG depletion in small DNA viruses and RNA viruses is
natural selection coming from replicative advantage and/or
immune escape [9,47]. However, our findings that the
introduction of additional CpGs into the PPV genome has no
measurable biological effect (no disadvantage),or in vitro
hypermethylation does not significantly inhibit replication
initiation of PPV argue against the replicative advantage of
CpG depletion. A recent publication about the failure of
members of the Parvoviridae family to elicit TLR 9 activated
interferon response in plasmacytoid dendritic cells 
questions the existence of immunological pressure against
CpGs in parvoviral genomes. The ascendant distribution of
CpGs by position does not support the presence of
immunological pressure against CpGs in parvoviruses either
because it would be expected that under such pressure the
number of first position CpGs would exceed the number of
second or third position CpGs (the degenerate code makes it
easy to change the C and G of the CpGs in third and second
positions without amino acid changes while point mutations of
first position CpGs inevitably come with amino acid changes
which could be harmful to the virus).
These data together with our observation that CpG sites are
more mutable than GC or C and G sites in the PPV genome
suggest that mutational pressure could have a more significant
role in the formation of PPV genome than selective forces.
In vertebrate genomes CpG is the most mutable dinucleotide
and its occurrence is about 20 % of the expected frequency
[5,6]. The high mutability of CpGs and its frequent conversion
to TG and CA largely attributed to mutational pressure caused
by the spontaneous deamination of 5-methylcytosine. The low
level methylation of the PPV genome combined with the high
mutation rate of parvoviruses [70,71] and the single stranded
DNA genome, which is very susceptible for deamination [72,73]
may explain the higher mutability of CpGs and their loss from
the PPV genome through countless generations. Reinforcing
this argument we have to mention that not only the transition
but the transversion rates of the CpG sites are a few times
higher in mammals [74,75,76] and non-methylated CpGs still
have an approximately three times higher overall mutation rate
in the human genome than non CpG nucleotides . These
observations suggest a deamination-independent intrinsic
mutability of CpGs in the mammalian genome, and raise the
possibility that mutational pressure originating from the host
replicative machinery may have a significant influence to CpG
suppression, at least in small DNA viruses which lack their own
Similarly to other organisms, mutational pressure, genetic
drift and natural selection together shape the genome of
parvoviruses. Our in vitro investigations of different CpG
mutants of PPV were not able to reveal any significant
evolutionary advantage of CpG depletion in viral replication at
cellular level. Future in vivo experiments and monitoring our
extra CpG mutants at organism level may help us clarify how
much of the loss of CpGs from the PPV genome is the
consequence of selective or other evolutionary forces.
Table S2. Methylation level of the CpG sites in the PPV
genome. 2a, Methylation of the CpG sites in PT cells 8 hours
post infection. 2b, Methylation of the CpG sites in Cos 7 cells
96 hours post infection. 2c, Methylation of the CpG sites in
PPV originated from aborted pig embryos. 2d, Methylation of
the CpG sites in the progeny viruses of the in vitro methylated
PPV genome. 2e, Methylation of the new CpG sites in the
M123 mutant PPV. 2f, Methylation of the progeny viruses after
overexpressing of DNMT3a in PT cells.
Conceived and designed the experiments: RT ZZ AB.
Performed the experiments: IM RS DB. Analyzed the data: IM
RS DB RT ZZ AB. Contributed reagents/materials/analysis
tools: IM RS DB RT ZZ AB. Wrote the manuscript: RT ZZ AB
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