RNA transcription and DNA replication of this still poorly characterized ssDNA virus, we cloned the full-length genome of TTV genotype 6 and transfected it into cells of several types..
Trang 1molecular clone of human Torque teno virus (TTV)
genotype 6
Laura Kakkola1, Johanna Tommiska1, Linda C L Boele2, Simo Miettinen1, Tea Blom1,
Tuija Kekarainen2, Jianming Qiu3, David Pintel3, Rob C Hoeben2, Klaus Hedman1
and Maria So¨derlund-Venermo1
1 Department of Virology, Haartman Institute and Helsinki University Central Hospital, University of Helsinki, Finland
2 Department of Molecular Cell Biology, Leiden University Medical Center, the Netherlands
3 Department of Molecular Microbiology and Immunology, University of Missouri–Columbia, Life Sciences Center, Columbia, MO, USA
TT-virus (TTV) recently named Torque teno virus [1]
was found in 1997 in Japan from a patient with
post-transfusion hepatitis of unknown etiology [2] The
virus is non-enveloped and contains a single-stranded
circular DNA genome of approximately 3.8 kb [3,4]
To date, five major phylogenetic groups have been
defined [1] Due to its genome organization and
struc-ture, TTV resembles the chicken anemia virus (CAV)
of the Circoviridae family This family of veterinary
viruses comprises the genus Gyrovirus, including CAV,
and the genus Circovirus, including the porcine
circo-virus (PCV) and the beak and feather disease circo-virus of birds The human TT-virus is currently classified as a member of a new, floating genus, Anellovirus [1] The TTV genome consists of an approximately 2.6 kb coding and an approximately 1.2 kb noncoding region The latter contains a GC-rich area, a promoter and transcriptional enhancer elements [3,5–8] The transcriptional capacity of the minute viral genome is greatly expanded by splicing [9,10], resulting in six dis-tinct yet partially overlapping viral proteins [11] Little
is known of their functions However, the longest gene,
Keywords
Anellovirus; replication; Torque teno virus;
transcription
Correspondence
L Kakkola, Department of Virology,
Haartman Institute, Haartmaninkatu 3,
PO Box 21, University of Helsinki,
FIN-00014, Finland
Fax: +358 9 19126491
Tel: +358 9 19126676
E-mail: laura.kakkola@helsinki.fi
Website: http://www.hi.helsinki.fi/english
Note
Nucleotide sequence data are available in
the DDBJ ⁄ EMBL ⁄ GenBank databases under
the accession number AY666122
(Received 11 April 2007, revised 10 July
2007, accepted 11 July 2007)
doi:10.1111/j.1742-4658.2007.06020.x
Torque teno virus (TTV) is a non-enveloped human virus with a circular negative-sense (approximately 3800 nucleotides) ssDNA genome TTV resembles in genome organization the chicken anemia virus, the animal pathogen of the Circoviridae family, and is currently classified as a member
of a new, floating genus, Anellovirus Molecular and cell biological research
on TTV has been restricted by the lack of permissive cell lines and func-tional, replication-competent plasmid clones In order to examine the key biological activities (i.e RNA transcription and DNA replication) of this still poorly characterized ssDNA virus, we cloned the full-length genome of TTV genotype 6 and transfected it into cells of several types TTV mRNA transcription was detected by RT-PCR in all the cell types: KU812Ep6, Cos-1, 293, 293T, Chang liver, Huh7 and UT7⁄ Epo-S1 Replicating TTV DNA was detected in the latter five cell types by a DpnI-based restric-tion enzyme method coupled with Southern analysis, a novel approach to assess TTV DNA replication The replicating full-length clone, the cell lines found to support TTV replication, and the methods presented here will facilitate the elucidation of the molecular biology and the life cycle of this recently identified human virus
Abbreviations
CAV, chicken anemia virus; DIG, digoxigenin; PBMC, peripheral blood mononuclear cells; PCV, porcine circovirus; TTV, Torque teno virus.
Trang 2ORF1, is assumed to encode a capsid protein that may
also participate in DNA replication [3,12] as is the case
with CAV [13]
The infection mechanisms and pathogenicity of TTV
are unknown Putative replicative forms of TTV DNA
have been found in peripheral blood mononuclear cells
(PBMC), bone marrow and liver [14–16], suggesting
replication at these sites Low-level infectivity of TTV
has been shown in activated PBMC and in a few
human cell lines [17–19] The TTV promoter has been
shown to be active in both human [5,8,11] and
non-human cells [8,9] TTV mRNAs have been detected in
human PBMC [19], bone marrow [10] and several
other organs [20] However, the main host cells and
the target organs of this virus are still undefined
The study of the biological functions of TTV is
par-ticularly challenging Replication does not appear to
be very efficient in primary cells [17–19], nor in the few
cell lines supporting virus growth [17] On the other
hand, the veterinary circoviruses CAV and PCV have
been studied successfully with infectious plasmid clones
[21–24] With a full-length TTV plasmid clone of
geno-type 1, RNA transcription and splicing was studied in
Cos-1 cells However, neither DNA replication, nor
cell permissiveness was demonstrated [9]
The present study aimed to construct a full-length
TTV clone that can be used as a tool for exploring the
viral determinants important in virus–cell interactions,
and to find permissive cell lines for further molecular
and cell biological studies of this peculiar human virus
For this purpose, we have cloned and sequenced the
full-length genome of TTV genotype 6, and used it to
detect the key biological functions (i.e RNA
transcrip-tion and DNA replicatranscrip-tion) in a number of different
cell lines
Results
Genotype 6 cloning and sequence analysis
A full length molecular clone, pTTV, in plasmid
pST-Blue-1 was constructed of TTV genotype 6 (Fig 1A)
The cloned genome was sequenced (GenBank
acces-sion number AY666122; nucleotide numbering
accord-ingly), and found to be 3748 nucleotides in length A
TATA-box (TATAA) was located at nucleotides 83–87
and a poly(A) sequence ATTAAA at nucleotides
2978–2983 A GC-rich area was 107 nucleotides in
length In the present study, two forms of the
full-length clone were used for transfection experiments;
the excised linear genome (linTTV) and the intact
plas-mid pTTV (Fig 1B,C) The full-length plasplas-mid clone
contains at its left end the NG136 primer sequence [25]
in which two nucleotides differ from the in vivo geno-type 6 sequence However, the BspEI-excised linear construct excludes these primer-derived nucleotides
Production of TTV RNA Prior to the studies, all the cells were tested and found
to be TTV DNA negative by generic UTR-PCR and
by genotype 6 specific PCR
All seven cell lines were analyzed with RT-PCR and were found to produce identical TTV RNA upon transfection with either pTTV or linTTV In RT-PCR analysis of the TTV clone-transfected cells, two ampli-cons were observed (Fig 2): one from the spliced TTV mRNA (454 bp) and the other from TTV DNA (555 bp) RNase treatment prior to RT-PCR abolished the 454 bp amplicon; and DNase treatment abolished the 555 bp but not the 454 bp amplicon (Fig 2D) In addition, RT-PCR without the RT step, and RT-PCR
of the input DNA constructs, yielded only the 555 bp amplicon (Fig 2D) Furthermore, the sequence data of both amplicons showed that in the 454 bp amplicon the intron had, indeed, been spliced out These experi-ments substantiated that the 454 bp amplicon origi-nated from the transcribed viral RNA and not from DNA The TTV RNA was shown by RT-PCR to per-sist in subcultured cells for at least 11 days Nontrans-fected cells and cells transNontrans-fected with the backbone plasmid pSTBlue-1, remained negative for TTV RNA (Fig 2B,C) confirming the absence of endogenous, transcriptionally active TTV RT-PCR of retinoblas-toma mRNA yielded the expected amplicons (data not shown) demonstrating mRNA integrity The results were identical for all the cell lines
Replication of TTV DNA All seven cell lines were transfected with linTTV and with the intact pTTV For detection of TTV replication, total DNA from the transfected cells was treated with the restriction enzymes BamHI and DpnI, and subjected
to Southern analysis Two different probes were used (Fig 1): the one labelled with32P differentiates by size the replicating TTV DNA from the input; and the other labelled with digoxigenin (DIG) additionally documents the susceptibility of the input DNA to DpnI
In cells transfected with linTTV, the input DNA (after BamHI digestion) was seen with the DIG-probe as a 3004-bp fragment (Figs 1B and 3A,B, marked with filled circles), which was further digested by DpnI into a fragment of 2162 bp (Figs 1B and 3A,B, marked with filled squares) On day 3 post transfection, a full-length TTV DNA of 3748 bp (after BamHI digestion,
Trang 3detected with either probe) emerged in 293T, Huh7 and
UT7⁄ Epo-S1 cells, and less pronounced also in 293 and
Chang liver cells (Fig 3A,C, marked with an arrow)
However, no such bands appeared in KU812Ep6 and
Cos-1 cells That BamHI digestion yielded a full-length
fragment indicates that circularization of the input
lin-ear construct had occurred Furthermore, this 3748 bp
fragment was resistant to DpnI (Fig 3A,C, marked with
an arrow), indicating TTV DNA replication The
linear-ized backbone plasmid, not separated from the excised
linear TTV construct, did not replicate in these cells As
an additional specificity control for the DpnI assay,
three (HindIII, EcoNI or ScaI) restriction enzymes,
other than BamHI, were used in Southern analysis,
resulting in identical DpnI-resistant bands on day 3 The
DpnI-resistant DNA progressively accumulated in the
transfected cells from day 0 to day 3, as shown for 293T
cells in Fig 4 However, on days 3, 5 and 8–10 post
transfection upon cell passage, the amount of
DpnI-resistant (replicating) TTV DNA declined, and was
detectable in Southern analyses for up to day 5
Interest-ingly, in those cells that permitted replication of the
excised linear construct, high molecular weight double bands (sensitive to DpnI) were visible (Fig 3) Single-stranded DNA (ssDNA) could, however, not be visual-ized by Southern analysis, suggesting that its production remained below the detection limit Of note, when com-paring the 293 cells, with and without the SV40 large
T antigen, the amount of DpnI-resistant replicating TTV DNA detected on day 3 post transfection (relative
to a standard amount of total cellular DNA) was invari-ably much lower in 293 than in 293T cells (Fig 3A, and more pronounced in Fig 3C), suggesting a possible helper function for the SV40 large T antigen
In cells transfected with the intact pTTV, the input DNA (after BamHI digestion) was seen with the DIG-probe as a 3165 bp fragment (Figs 1C and 3A,B, marked with filled circles), which was further digested by DpnI into a fragment of 2162 bp (Figs 1C and 3A,B, marked with filled squares) On day 3 post transfection, the same 3165 bp fragment (Fig 3A, marked with an asterisk) was DpnI resis-tant, indicating replication of the complete pTTV The results with the 32P-probe verified this: the input
TTV genome
3748 bp
3004 bp
2162 bp
DIG
32 P
pSTBlue-1
3851 bp
32 P
2162 bp
3165 bp
DIG
32 P
32 P
pTTV total length
7775 bp
3748/1
TTV genotype6
•
NsiI
*
BspEI
pSTBlue-1
BspEI tscr tlat BspEI
•
TTVGCF NG136
A
Fig 1 The full-length clone and the
con-structs for transfection (A) The cloning
strategy of TTV into the pSTBlue-1 plasmid.
The GC-rich area is shown as a striped box
and the overlapping area in the clone as
spotted boxes The three products used in
the construction of the TTV clone are
indi-cated with black lines The key restriction
enzymes (see text for details), primers
(arrows: forward TTVGCF, reverse NG136),
the TATA-box (*), the poly(A) (d), the
tran-scription initiation (tscr) and the translation
initiation (tlat) sites are shown Schematic
representations of (B) linear BspEI-excised
construct (linTTV), and (C) pTTV construct
used in transfection experiments The viral
genome is represented by an empty bar and
the backbone plasmid by a thin black line.
The DIG- and the32P-labelled probes are
indicated BamHI (vertical bars) and DpnI
(r) restriction enzymes were used for the
analysis of DNA replication The predicted
TTV DNA products in replication analyses:
linTTV-derived 3004 bp fragment and
pTTV-derived 3165 bp fragment after BamHI
digestion are marked with d; linTTV and
pTTV derived 2162 bp fragments after
BamHI ⁄ DpnI-digestion are marked with j.
Trang 4pTTV was seen (after BamHI digestion) as two
DpnI-sensitive restriction fragments of 3165 bp and
4610 bp (Fig 3C) and, on day 3 post transfection,
these fragments had become DpnI resistant (Fig 3C)
In all pTTV-transfected cells, as detected with either
probe, a full-length DpnI-resistant 3748 bp fragment
(which would indicate rescue and replication of the
TTV genome from the backbone plasmid) remained absent
As opposed to the five other cell lines, in KU812Ep6 and Cos-1 cells, no replicating DNA (or, in some experiments with the latter cells, barely exceeding detection threshold) were detected upon transfection with either construct (Fig 3A) No apparent cyto-pathological changes were microscopically detected in the cells supporting TTV DNA replication
The results were the same regardless of the DNA iso-lation method (total cellular; Hirt extraction) and of the detection probes (DIG- and 32P-labelled probe) (data shown for 293 and 293T cells in Fig 3A,C) The non-transfected cells and the backbone plasmid-non-transfected cells were always negative for TTV DNA In Southern analysis, the input DNA served as an internal control to verify restriction enzyme activity: the input DNA was sensitive to DpnI (Fig 3A,B, marked with filled squares) whereas the newly synthesized DNA was resistant (Fig 3A, marked with an arrow) but remained digest-ible with other restriction enzymes (data not shown)
Circularization of the linear construct
In the linTTV-transfected cells on day 3, the emer-gence of the 3748 bp TTV DNA after BamHI diges-tion (Fig 3A) indicated that the input linear TTV DNA had circularized To confirm this, two other restriction enzymes (HindIII and ScaI, Fig 5A) that, like BamHI, cut the TTV genome only once, were used with identical results (i.e a single product of approximately 3.7 kb was detected; data not shown) The circularization was further confirmed by digestion
of the DNA samples with pairs of restriction enzymes that cut on both sides of the linearization breakpoint (Fig 5) BamHI⁄ SalI or XhoI ⁄ SalI double-digestions
of the input linear construct yielded three restriction fragments of approximately 2100, 740 and 890 bp with the first enzyme pair, and of 2200, 630 and 890 bp with the latter However, the same double-digestions
of replicating TTV DNA from 293T cells on day 3 post transfection yielded additional fragments of approximately 1630 bp and 1520 bp, respectively (Fig 5B), indicating fusion of the linearization break-point ends Taken together, these results show that, in the linTTV-transfected 293T cells, circular forms of the TTV genome had been formed
Effect of aphidicolin on TTV DNA replication
To reconfirm that TTV DNA replication had occurred and to investigate whether it utilizes the cellular replica-tion machinery, aphidicolin (an inhibitor of eukaryotic
C
B
D
DNA
nt 112 nt 182 nt 284
RT1F
nt 129-147
RT1R
nt 683-660
RNA
poly-A
D
non-transf.
cells Mw Mw
linTTV
500
400
300
bp
day3
Mw
pSTBlue-1 pTTV
Mw
500
400
600
day3
linTTV day3
400
500
600
400
500
600
input
linTTV
input pTTV +RT +RT
R
Fig 2 RT-PCR of TTV RNA (A) A schematic drawing of TTV
RT-PCR Transcription initiates at nucleotides 112, and splicing
removes nucleotides 182–284 [11] RT-PCR primers are shown
with arrows Amplicon sizes are 555 bp for DNA and 454 bp for
spliced mRNA RT-PCR results of representative 293T cells
trans-fected with (B) the linear excised TTV (linTTV) and (C) intact pTTV.
Nontransfected cells and cells transfected with the backbone
plas-mid (pSTBlue-1) were included as controls (D) RT-PCR controls
from 293T cells transfected with linTTV (day 3) and from the input
constructs +RT, normal RT-PCR; –RT, without the RT-step; R,
RNase-treated; D, DNase-treated.
Trang 5C
A
B
U bp 4899 3639 2799
Mw
input linTTV
U bp 3639 2799
Mw
input pTTV
4899
4899
3639
2799
293T
B B/D B B/D B B/D B B/D B B/D
bp
pSTBlue-1 Mw
293
B B/D B B/D B B/D B B/D B B/D
bp
pSTBlue-1
4899 3639 2799 Mw
4899 2799
Mw
Cos-1
KU812Ep6
B B/D B B/D B B/D B B/D B
cells
4899
3639
2799
Mw
3639
UT7/Epo-S1
cells
4899 2799 Mw
B B/D B B/D B B/D B B/D B B/D
bp
4899
3639
2799
Mw
Huh7
Chang liver
bp
4899
3639
2799
Mw
linTTV
input
bp 4625 3165 Fig 3 Southern analysis of TTV DNA replication (A) 293T, 293, KU812Ep6, UT ⁄ Epo-S1, Huh7, Cos-1 and Chang liver cells transfected with the excised linear (linTTV) or the intact pTTV construct The key products of the replication assay are marked in the 293T-cell figure: the input linTTV yielding a 3004 bp fragment and the input pTTV yielding a 3165 bp fragment after BamHI digestion are marked with d; the input linTTV and pTTV yielding 2162 bp fragments after BamHI⁄ DpnI digestion are marked with j; the DpnI-resistant circularized full-length TTV DNA is marked with an arrow; DpnI-resistant pTTV is marked with * (B) Southern analysis of the input constructs The products of the restriction enzyme digestions are marked as those in the 293T-cell figure (Note the absence of a 3748 bp product.) U, undigested (C) South-ern analysis of Hirt-extracted (BamHI ⁄ DpnI-digested) DNA from the 293T and 293 cells (with and without T antigen, respectively) transfected with pTTV or linTTV Input pTTV digested with BamHI as a control Arrows indicate DpnI-resistant full-length TTV DNA For detection of TTV DNA, either a DIG- (A,B) or a 32 P- (C) labelled probe was used.
Trang 6nuclear DNA replication) [26,27] was used The 293T
cells were transfected either with linTTV or with
pTTV, and were grown in the presence (versus
absence) of aphidicolin Upon aphidicolin treatment,
TTV DNA replication was blocked (Fig 6), indicating
nuclear DNA replication
Discussion Most of the data published on TTV are from PCR studies of clinical patient materials However, to apprehend the full impact of this human virus, more information on the molecular biology and host–cell interactions of this virus is needed To this end, we have cloned and sequenced the full-length genome of TTV genotype 6, and studied the key biological activi-ties of this virus The cloned viral genome was
3748 nucleotides in length; 105 nucleotides shorter than the TTV prototype TA278 (genotype 1, accession number AB017610) In genomic organization, geno-type 6 turned out to resemble the other known TTV types: a TATA-box and a poly(A) sequence flanking the coding region, and a GC-rich area of 107 bp located in the noncoding region
The biological activity of our full-length plasmid clone was analyzed in seven cell lines In a previous study, the TTV expression profile (i.e mRNAs tran-scribed and proteins translated) in 293 cells was deter-mined by using this full-length clone [11] In the present study, cells of all seven types, transfected with either pTTV or linTTV, produced TTV RNA The RNA transcripts were unequivocally documented with RT-PCR designed to flank a common splice site in the TTV genome In a previous study, TTV RNAs were
bp
4899
3639
2799
Mw
Fig 4 Accumulation of DpnI-resistant full-length TTV DNA (marked
with an arrow) in 293T cells from day 0 to day 3 post transfection.
293T cells were transfected with linTTV, and DIG-labelled probe
was used to detect the TTV DNA.
A
B
lin day3 Mw
BamHI/SalI
lin day3 Mw
XhoI/SalI
1953 1882 1515 1482
992
718
bp
2799 3639
H a B
I
la S
I
1631 bp
la S
I
1521 bp
744 bp
TTV
genome
3748 bp
SalI
H a B
I
o X
I
aI i d H
I
2227 bp
2117 bp
Fig 5 Circularization of the linear construct in 293T cells (A)
Sche-matic representation of the restriction enzyme cutting sites and the
corresponding restriction-fragment sizes (B) The input linear
con-struct (lin) and the total cellular DNA (from the linTTV-transfected
293T cells on day 3 post transfection) were digested with two
enzyme pairs (BamHI ⁄ SalI or XhoI ⁄ SalI), each pair cutting at both
sides of the linearization breakpoint Southern detection was
per-formed with a probe prepared by nick translation (the band
corre-sponding to the cloning vector pSTBlue-1 is indicated) The
fragments from the circularized TTV DNA are marked with arrows.
A
B
bp 4899 3639 2799
Mw
Fig 6 The effect of aphidicolin (APC) on TTV DNA replication 293T cells were transfected with pTTV or the linear (linTTV) con-struct in the absence (–APC) or presence (+APC) of aphidicolin (A) Southern analysis with the 32 P-probe of Hirt-extracted
(Bam-HI ⁄ DpnI-digested) DNA (B) Southern analysis with the DIG-labelled probe of BamHI- (B) and BamHI ⁄ DpnI-digested (B ⁄ D) DNA on day 3 post transfection Arrows are pointing to the sites of replicat-ing, DpnI-resistant TTV DNA.
Trang 7detected in Cos-1 cells transfected with a plasmid clone
of TTV genotype 1 [9] The cloning strategy used by
this group was similar to ours: the plasmid clone
con-tained overlapping genomic regions and the cloning
breakpoint was in the noncoding region Their
geno-type 1 clone and our genogeno-type 6 clone differed in the
lengths of the overlaps and in the locations of the
cloning breakpoints However, upon transfection, both
constructs produced RNA, indicating that cloning did
not impair the function of the promoter The promoter
area of TTV genotype 1 has been shown to be active
in many cell types, including Huh7 and cells of
ery-throid origin [5,8] Our results with genotype 6 were
similar, displaying TTV RNA expression (i.e promoter
activity) in diverse cell types
For the detection of replicating TTV DNA, we used
restriction enzyme analysis combined with Southern
hybridization This straightforward approach was
dem-onstrated to be useful for the study of TTV DNA
rep-lication TTV DNA replication was detected in the
majority of cell types When the excised linear
geno-type 6 DNA was transfected into 293T, 293, UT7⁄
Epo-S1, Chang liver and Huh7 cells, circularized and
replicating (DpnI resistant) forms of TTV DNA
accu-mulated up to days 3–4 It is possible that the DpnI
resistance might arise from replication-independent
demethylation of the input DNA, or from DNA
repli-cation not related to TTV However, both the input
linTTV and the cotransfected (together with linTTV)
backbone plasmid always remained sensitive to DpnI
This indicates that the DpnI-resistance of TTV DNA
was not due to replication-independent demethylation
of the input DNA, or due to general DNA replication,
but instead was a result of TTV DNA-specific
replica-tion That the production of the DpnI-resistant forms
could be abolished with a polymerase inhibitor further
verifies that replication had occurred within the cells
The high-molecular weight double bands that appeared
restrictively in the cells that supported TTV DNA
rep-lication (after transfection with linTTV), could
theoret-ically originate from concatamers formed from the
transfected DNA The bands were DpnI sensitive and
visible also in aphidicolin treated cells, suggesting that
they are formed without the cellular replication
machinery Whether these intracellular DNA forms are
required for initiation of replication remains to be
studied Of our seven cell lines, 293T showed the
high-est yield of replicating viral DNA, even exceeding that
of the Chang cells, which have been recently reported
to sustain TTV infection [17]
Upon transfection of five cell types with the intact
pTTV, DpnI-resistant (i.e replicating) DNA was
detected However, rescued forms of the TTV genome
were not seen, which could indicate inefficient excision from the backbone plasmid With other ssDNA-virus clones, the extent of rescue and replication seems to vary For example, a rodent parvovirus, HaPV, repli-cates and produces infectious virions when transfected
as an intact plasmid clone containing one copy of the viral genome [28] whereas, among the circoviruses, PCV gives a higher virus titer when cloned as tandem repeats [29] and CAV requires either tandem-repeat cloning or excision of the viral genome from the plas-mid before transfection [30] The rescue and replication
of the viral genomes could involve cellular and⁄ or viral proteins, with mechanisms possibly dependent on the particular virus (e.g nicking of DNA strands, recombi-nation, and rolling circle replication) [31,32] In com-parison with other ssDNA virus molecular clones, ours appear to resemble that of CAV; in order to produce genome-size replicating DNA, the viral genome needs
to be excised from the backbone plasmid before trans-fection
With PCV, it has been shown that the mere origin
of replication, cloned into the plasmid, can lead to replication of the entire plasmid, if transfected into PCV-infected cells [33] This raises the question whether replication of our pTTV (and maybe also linTTV) might have been assisted by coinfection with
a homologous virus However, all our cell lines were shown to be TTV DNA negative by generic PCR This suggests that the replicating TTV DNA in the present study was not produced with the help of endogenous TTV Interestingly, the amount of replicating TTV DNA was much lower in 293 than in 293T cells, suggesting that the SV40 large T antigen might provide some helper functions CAV and PCV have been shown to replicate efficiently in heterologously infected cells: CAV in MDCC-MSB-1 cells transformed with Marek’s virus [34] and PCV in pk-15 cells infected with swine papova virus [35–37] It is therefore possible that TTV indeed might benefit from some helper functions of other viruses for efficient produc-tive infection
By contrast to the other cell lines in the present study, the TTV clone showed little or no replication in KU812Ep6 and Cos-1 cells The former are of ery-throid origin, as are UT7⁄ Epo-S1, and could thus be thought to support TTV replication However, accord-ing to our unpublished data, the viability of KU812Ep6 cells declines during electroporation TTV,
a minute virus apparently lacking DNA polymerase, most likely depends on the cellular S-phase for replica-tion Indeed, our results with aphidicolin, which inhib-its eukaryotic nuclear DNA replication and blocks the cell cycle at early S-phase, strongly suggest that TTV
Trang 8utilizes the cellular DNA polymerase and S-phase for
replication The requirement for rapid cell division,
together with the diminished viability of the
electropo-rated KU812Ep6 cells, could explain the lack of
repli-cation of our full-length clone in those cells The
deficiency of TTV replication in monkey
kidney-derived Cos-1 cells could represent species specificity
However, because mRNAs were produced, the
poten-tial species barrier does not appear to affect
transcrip-tion In previous studies, the sites of TTV replication
in humans have been suggested to be the
hematologi-cal compartment and the liver [15,16] Our results,
demonstrating replication of cloned TTV genotype 6
DNA in erythroid UT7⁄ Epo-S1, and hepatic Huh7
and Chang liver cells, further support this concept In
any case, the species and cell-type specificity of TTV
replication needs to be examined further The genetic
variation among TT viruses is extremely high To what
extent the various genotypes share biological functions
and possibly influence each others in coinfections
remains to be investigated At least the RNA
tran-scription of genotypes 1 and 6, which belong to the
same genogroup, appears to be similar [9,11]
The production of infectious virions from the
trans-fected cells could not be verified unequivocally with
the methods in use, and thus remains to be
investi-gated In our experiments, upon cell subculture, even
though the more sensitive RT-PCR continued to be
positive, the levels of replicating TTV DNA in
South-ern analysis declined below detection limit These
results suggest that, if infectious virions were
pro-duced, the infection did not spread efficiently to the
neighbouring cells, and⁄ or that the amounts of
prog-eny virions were relatively low It is possible that the
cells used in the present study did not express the
unknown TTV receptor, or that TTV simply does not
grow well in ordinary tissue culture, thereby
resem-bling many other viruses such as human
parvovi-rus B19, hepatitis C viparvovi-rus and human papillomaviparvovi-rus
A low infectivity of TTV is concordant with previous
studies showing that infection of Chang liver cells gave
rise to only low amounts of progeny virus [17] and
that, even in ex vivo PBMC cultures, the production of
TTV is scanty and requires cell activation [18,19]
Because TTV in vivo infects healthy individuals
chroni-cally, strict regulation of virus multiplicity and of cell
damage is mutually beneficial for the virus and its
host
The research on TTV-like animal circoviruses has
been greatly assisted by the availability of full-length
plasmid clones producing infectious virions in vitro
and clinical disease in vivo [21–24,29] Indeed, the
genetic map (defining viral mRNAs and proteins) of
TTV has been recently elucidated with this full-length TTV clone [11] In the present study, we demonstrated TTV-promoter activity in all cell types studied and, by a novel approach, identified five cell lines that supported TTV replication In the absence of knowledge on TTV proteins, replication and cell bio-logy, the full-length plasmid clone and replication assays presented here will be valuable tools for the examination of the mechanisms pertaining to the molecular biology, the cell tropism and the clinical sig-nificance of this recently discovered human virus
Experimental procedures
Cell lines and transfection methods
Seven cell lines, Cos-1, Huh7, Chang liver, KU812Ep6,
cells (African green monkey kidney; transformed with SV40) were maintained in DMEM containing 10% fetal
Invitro-gen, Carlsbad, CA, USA) and antibiotics The cells were transfected 1 day after subculture, at approximately 95% confluency, with 30 lL Lipofectamine2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) and 5 lg DNA per
main-tained in MEM, with 10% fetal bovine serum and antibiot-ics, and were transfected as the Cos-1 cells Human liver cells, Chang liver (ECACC 88021102), were maintained as Huh7 cells, and transfected as Cos-1 cells (except that the amount of Lipofectamine2000 was 16 lL) Human kidney-derived 293T cells (expressing the SV40 T antigen,
were maintained in DMEM, 10% fetal bovine serum and antibiotics The 293T and 293 cells were transfected as the Cos-1 (except that the amount of Lipofectamine2000 was
25 lL); or by the calcium phosphate technique [40] The human erythroid leukemia cell line KU812Ep6 [41], kindly provided by Dr Miyagawa (Fujirebio Inc., Tokyo, Japan), was maintained in suspension in RPMI1640, 10% fetal
Janssen-Cilag, Berchem, Belgium) and antibiotics For transfection, the cells were harvested on days 3–4 For one reaction,
DNA was added The cells were electroporated immediately
at 300 or 350 V, and 960 lF (‘Gene Pulser’; Bio-Rad, Her-cules, CA, USA) A human erythroblastoid cell line
(To-hoku University School of Medicine, Japan), was main-tained in suspension in Iscove’s modified Dulbecco’s
erythropoietin and antibiotics For transfection, the cells
AMAXA Nucleofector system, using kit R and program
Trang 9T-24 (Amaxa Biosystems, Cologne, Germany) All the cells
PCR for the presence of endogenous TTV (generic
UTR-PCR, and genotype 6 specific PCR) [44]
Full-length cloning
The TTV genome was cloned in three overlapping fragments
(Fig 1A) The previously cloned 3.3 kb region of TTV
iso-late HEL32 [44] was completed to contain the full-length
gen-ome of the isolate The GC-rich part of the TTV gengen-ome was
amplified from the original serum [44] with seminested
prim-ers: forward TTVGCF (nucleotides 3206–3225) 5¢-CAGA
(nucleotides 216–199) 5¢-CGAATTGCCCCTTGACTG-3¢
and second reverse TTVGCR3 (nucleotides 157–140) 5¢-GG
GATCACCCTTCGAGGT-3¢ Both PCR reactions (volume
25 lL) contained 200 lm of each dNTP (Roche, Basel,
dimethylsulfoxide, 1 m betaine (Sigma, St Louis, MO, USA)
and 17.5 U of enzyme mix (‘Expand High Fidelity PCR
remain-ing 20 cycles The gel-purified PCR amplicon was cloned into
pSTBlue-1 AccepTor vector (Novagen, Madison, WI, USA)
in Escherichia coli DH5a cells
To facilitate the cloning of the entire TTV genome into a
single plasmid, a third overlapping piece of 396 bp was
amplified by PCR from the original serum with primers
GGGTTCCATT-3¢
Restriction enzyme digestions and ligations of the three
genomic parts resulted in a plasmid that contains the
entire TTV genome (pTTV), flanked by overlapping
175 bp areas, inserted between the EcoRI sites in the
pSTBlue-1 vector In addition, a BspEI restriction enzyme
can be used to excise from the TTV clone the complete,
single-unit (without overlaps) TTV genome in linear
form
Sequencing of the GC-rich region was carried out at the
DNA sequencing facility of the Institute of Biotechnology,
University of Helsinki, Finland All other sequencing
reac-tions in this study were done using the ABI Prism 3100
Genetic Analyzer (Applied Biosystems, Foster City, CA,
USA) in the sequencing core facility of the Haartman
Insti-tute, University of Helsinki, Finland
Plasmid constructs for transfection
For functional analysis of the TTV clone, the cells were
transfected with TTV plasmids of two different forms: an
uncut plasmid clone, pTTV, containing overlaps (Fig 1C),
and a linear construct, linTTV [full-length genome (without
overlaps) excised with BspEI, but not purified from the backbone plasmid; Fig 1B] The pSTBlue-1 backbone vector was used as a negative control The cells were collected for RNA and DNA analyses on days 1–11 post transfection
The transfection efficiencies were optimized with pEGFP-Luc vector (Clontech, Mountain View, CA, USA) encoding green fluorescent protein On day 1 post transfection, the percentage of green fluorescent protein-positive cells was estimated with a fluorescence microscope (‘Axioplan2’; Zeiss, Oberkochen, Germany)
RNA isolation and RT-PCR
Total RNA of the cells was extracted with TRIzol Reagent (Invitrogen Life Technologies) To remove residual DNA when necessary, the RNA samples were treated with DNase
RT-PCR was performed with a RobusT II RT-PCR Kit (Finnzymes, Espoo, Finland) in the presence of RNase inhibitor (‘RNaseOut’; Invitrogen Life Technologies, or
‘RNase Inhibitor’; Roche) For TTV-specific amplification, primers flanking the first common intron of 101 bp [11]
5¢-GCAGCGGCAGCACCTCGAA-3¢ and reverse RT1R (nucleotides 683–660) 5¢-GTCTAGCAGGTCCTCGTCTG CGAG-3¢ This separates the possible DNA-derived ampli-cons from the RNA-derived ones by size (555 bp and
454 bp, respectively; Fig 2A) The PCR program consisted
for 3 min RT-PCR for the cellular retinoblastoma mRNA was used as a control for RT-PCR and RNA isolation [45] RT-PCRs were carried out on DNase-treated and non-treated samples The RNA experiments were performed at least twice
DNA replication analyses
Total cellular DNA was isolated by cell lysis
Ontario, Canada) and by shearing through an 18G needle,
as previously described [28] The isolated DNA was extracted with phenol and chloroform, precipitated with ethanol and Na-acetate, and resuspended into water Total cellular DNA was also alternatively isolated with QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany) Low molecular weight DNA was alternatively extracted with the Hirt protocol [46]
The DNA samples (2.5–5 lg of Hirt-extracted or 30–
40 lg of total DNA) were digested with BamHI Subse-quently, half the digest was treated with DpnI (which cuts only prokaryotic DNA; New England BioLabs, Ipswich,
Trang 10MA, USA) The BamHI- and BamHI⁄ DpnI-digested DNA
samples were separated by gel electrophoresis The DNA
was transferred to nylon membranes (‘Hybond-N+’,
Amer-sham Biosciences, Piscataway, NJ, USA), and was
hybrid-ized overnight For hybridization, a DIG-labelled
TTV-specific probe, covering nucleotides 1803–2200 (Fig 1), was
prepared as described [44] using the outer primer pair of
genotype 6 PCR The DIG label was detected with
5-bromo-4-chloro-3-indolyl-phosphate (BCIP, Roche) and
4-nitro blue tetrazolium chloride (NBT, Roche) forming a
coloured precipitate For the circularization experiments,
another DIG-labelled probe was prepared to cover the
entire pTTV construct by DIG-Nick Translation Mix
consist-ing of a 560 bp fragment of the untranslated region
(NsiI-BspEI fragment from pTTV, Fig 1) was used The probe
the random primer method [47], and the signal was detected
by film exposure
(Sigma) was added to the medium, and 293T cells were
transfected by the calcium phosphate method The cells
were incubated in aphidicolin for 16 h, washed, and grown
further with aphidicolin On day 2 post transfection, the
DNA was isolated by the Hirt method, 10 lg of DNA was
were transfected with the Lipofectamine2000 method and
grown in the presence of aphidicolin On day 3 post
trans-fection, the total DNA was isolated, 30 lg of DNA was
hybridization with the DIG-probe
Acknowledgements
The authors thank Drs Ilkka Julkunen (National
Pub-lic Health Institute, Helsinki, Finland), Eiji Morita
and Kazuo Sugamura (Tohoku University School of
Medicine, Sendai, Japan), and Eiji Miyagawa
(Fujire-bio Inc., Tokyo, Japan) for cell lines, and Pa¨ivi Norja
and Heidi Bonde´n for excellent technical assistance
Dr Malcolm Richardson is acknowledged for revision
of the manuscript This study was supported by the
Finnish Technology Advancement Fund, the Finnish
Academy (project 76132), the Helsinki Biomedical
Graduate School, the Alfred Kordelin Foundation, the
Paulo Foundation, the Sigrid Juse´lius Foundation, the
Research and Science Foundation of Farmos, the Ella
and Georg Ehrnrooth Foundation, the Finnish
Kon-kordia Fund, the Helsinki University Central Hospital
Research and Education Fund, The Maud Kuistila
Memorial Foundation, The Finnish Foundation for
Research on Viral Diseases, and the Medical Society
of Finland (Finska La¨karesa¨llskapet)
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