Báo cáo khoa học: Construction and biological activity of a full-length molecular clone of human Torque teno virus (TTV) genotype 6

Construction and biological activity of a full-length molecular 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

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

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.

(Received 11 April 2007, revised 10 July 2007, accepted 11 July 2007)

doi:10.1111/j.1742-4658.2007.06020.x

virus (PCV) and the beak and feather disease virus of birds. The human TT-virus is currently classiﬁed as a member of a new, ﬂoating genus, Anellovirus [1].

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-

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,

Abbreviations CAV, chicken anemia virus; DIG, digoxigenin; PBMC, peripheral blood mononuclear cells; PCV, porcine circovirus; TTV, Torque teno virus.

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ORF1, is assumed to encode a capsid protein that may also participate in DNA replication [3,12] as is the case with CAV [13].

in which two nucleotides differ from the in vivo geno- the BspEI-excised linear type 6 sequence. However, 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.

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].

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 transfected 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.

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 replication) in a number of different cell lines.

Replication of TTV DNA

Results

Genotype 6 cloning and sequence analysis

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 with 32P 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.

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 sequence ATTAAA at nucleotides and a poly(A) 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 plasmid clone contains at its left end the NG136 primer sequence [25]

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,

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A

BspEI 3748/1 *

(cid:129)

NsiI TTV genotype6

tscr

BspEI

tlat

BspEI (cid:129)

TTVGCF

NG136

pSTBlue-1

B

C

3165 bp 3004 bp

2162 bp 2162 bp

I E p s B

32P

I H m a B

32P

I E p s B

I H m a B

DIG

I H m a B

TTV genome 3748 bp

32P

pTTV total length 7775 bp

pSTBlue-1 3851 bp

I H m a B

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 the 32P-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.

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 the amount of DpnI-resistant replicating T antigen, 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,

indicating replication of

detected 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- (replicating) TTV DNA declined, and was resistant detectable in Southern analyses for up to day 5. Interest- ingly, in those cells that permitted replication of the

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, the complete pTTV. The results with the 32P-probe veriﬁed this: the input

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A

nt 1

nt 112

nt 3748

TTV genome from the backbone plasmid) remained absent.

DNA

nt 112 nt 182 nt 284

poly-A

RNA

RT1F nt 129-147

RT1R nt 683-660

B

linTTV

non-transf. cells

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.

day1

day3

day5

day10

day3

bp 500 400 300

C

pTTV

pSTBlue-1

day1

day3

day6

day10

day3

600 500

400

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-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).

linTTV day3

D

Circularization of the linear construct

+RT

-RT

600 500 400

the

that

input

indicated

(Fig. 3A)

input pTTV

input linTTV

-RT R

-RT

+RT

bp Mw

600 500 400

transfection yielded additional

1630 bp and 1520 bp,

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.

seen (after BamHI digestion) as

In the linTTV-transfected cells on day 3, the emer- gence of the 3748 bp TTV DNA after BamHI diges- linear tion 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 fragments of respectively approximately (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

two pTTV was 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

To reconﬁrm that TTV DNA replication had occurred and to investigate whether it utilizes the cellular replica- tion machinery, aphidicolin (an inhibitor of eukaryotic

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293T

293

A

linTTV pTTV linTTV pTTV day3 day3 pSTBlue-1 day3 day3 day3 pSTBlue-1 day3 Mw Mw day1 day1 B B/D B B/D B B/D B B/D B B/D day1 day1 B B/D B B/D B B/D B B/D B B/D bp bp

UT7/Epo-S1

KU812Ep6

4899 3639 2799 4899 3639 2799

pTTV linTTV pTTV linTTV cells cells day1 day3 day1 day3 day4 day4 B/D Mw B B/D B B/D B B/D B B/D B day1 day1 B B/D B B/D B B/D B B/D B B/D Mw bp bp

Cos-1

Huh7

4899 3639 2799 4899 3639 2799

linTTV pTTV linTTV pTTV day1 day3 day1 day3 pSTBlue-1 day3 day3 day3 pSTBlue-1 day3 Mw Mw day1 day1 B B/D B B/D B B/D B B/D B B/D B B/D B B/D B B/D B B/D B B/D bp bp

Chang liver

linTTV

pTTV

4899 3639 2799 4899 3639 2799

B

day1

day3

pSTBlue-1 day3

day1 B B/D B B/D B B/D B B/D B B/D

input linTTV input pTTV Mw U B B/D Mw U B B/D bp bp

4899 3639 2799

4899 3639 2799 4899 3639 2799

C

293

293T

linTTV input pTTV pTTV linTTV pTTV

bp 4625

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 32P- (C) labelled probe was used.

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Biological activity of a full-length TTV DNA clone

day0

day1

day2

day3

A

- APC

+ APC

B B/D B B/D B B/D B B/D

pTTV linTTV pTTV linTTV

4899 3639 2799

-APC

+ APC

B

B/D

4899

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.

3639

2799

H

I

BspEI

SalI

III BspEI

acS

dniH

I ohX

maB

A TTV genome 3748 bp

634 bp

887 bp

2227 bp

744 bp

2117 bp

1521 bp

1631 bp

X

B

h

I

a

o

I

m

laS

H

I

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 32P-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.

BamHI/SalI

B

lin day3 Mw

Mw

XhoI/SalI lin

day3

bp

pSTBlue-1

3639

Discussion

2799

1953 1882 1515 1482

992

718

Most of the data published on TTV are from PCR to studies of clinical patient materials. However, 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- this virus. The cloned viral genome was ties of 3748 nucleotides shorter in length; 105 nucleotides 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.

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.

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

nuclear 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.

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detected 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 genotype 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.

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.

raises

Interestingly,

amount

the

[35–37].

With PCV, it has been shown that the mere origin of replication, cloned into the plasmid, can lead to if transfected into replication of the entire plasmid, PCV-infected cells the question [33]. This 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. 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 therefore with swine papova virus possible that TTV indeed might beneﬁt from some helper functions of other viruses for efﬁcient produc- tive infection.

By contrast to the other cell

formed without

cellular

the

to our unpublished data,

the

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 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 replicating) DNA was (i.e. detected. However, rescued forms of the TTV genome

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- viability of ing 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

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In the present

full- TTV has been recently elucidated with this length TTV clone [11]. 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 the mechanisms pertaining to the examination of molecular biology, the cell tropism and the clinical sig- niﬁcance of this recently discovered human virus.

Experimental procedures

Cell lines and transfection methods

(African green monkey kidney;

utilizes 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].

that,

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 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 virus and human papillomavirus. 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.

lines, Cos-1, Huh7, Chang liver, KU812Ep6, Seven cell UT7 ⁄ Epo-S1, 293T and 293, were used in this study. Cos-1 cells transformed with SV40) were maintained in DMEM containing 10% fetal bovine serum, 10 mm Hepes buffer (Gibco BRL ⁄ 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 60 mm2 plate. Human hepatoma cells, Huh7, were 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, origi- nally described as 293 ⁄ tsA1609neo [38]), and 293 cells [39] 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 bovine serum, 6 UÆmL)1 erythropoietin (‘Eprex’; Janssen- Cilag, Berchem, Belgium) and antibiotics. For transfection, the cells were harvested on days 3–4. For one reaction, 2 · 106 cells were resuspended in 0.5 mL medium and 5 lg 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 UT7 ⁄ Epo-S1 [42,43], kindly provided by Dr Morita (To- hoku University School of Medicine, Japan), was main- tained in suspension in Iscove’s modiﬁed Dulbecco’s medium containing 10% fetal bovine serum, 2 UÆmL)1 erythropoietin and antibiotics. For transfection, the cells were harvested on days 3–4 and transfected by the AMAXA Nucleofector system, using kit R and program

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

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T-24 (Amaxa Biosystems, Cologne, Germany). All the cells were grown at 37 (cid:2)C in 5% CO2; and were monitored with PCR for the presence of endogenous TTV (generic UTR- PCR, and genotype 6 speciﬁc PCR) [44].

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.

Full-length cloning

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

ﬁrst

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 (1.5 U ⁄ 15 lL; ‘RQ1 RNase-Free DNase’; Promega, Madi- son, WI, USA) for 70 min at 37 (cid:2)C.

forward RT1F (nucleotides

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 genome was ampliﬁed from the original serum [44] with seminested prim- ers: forward TTVGCF (nucleotides 3206–3225) 5¢-CAGA reverse TTVGCR1 CTCCGAGATGCCATTG-3¢, (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, Swit- zerland), 400 nm of each primer, 1.5 mm MgCl2, 10% dimethylsulfoxide, 1 m betaine (Sigma, St. Louis, MO, USA) and 17.5 U of enzyme mix (‘Expand High Fidelity PCR System’; Roche). The PCR comprised annealing at 55 (cid:2)C for 30 s and extension at 72 (cid:2)C for 2 min for the ﬁrst ten cycles, with addition of 5 sÆcycle)1 of extension time for the 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.

394–375)

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 forward NG054 (nucleotides )2–18) [25] and reverse TTVGCR6 5¢-CGTTCGAGTT (nucleotides GGGTTCCATT-3¢.

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] were designed: 129–147) 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 of reverse transcription for 30 min at 42 (cid:2)C, followed by PCR: 94 (cid:2)C for 2 min, and 35 cycles at 94 (cid:2)C for 30 s, 65 (cid:2)C for 30 s, 68 (cid:2)C for 45 s, with ﬁnal extension at 68 (cid:2)C 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

Restriction enzyme digestions and ligations of the three genomic parts resulted in a plasmid that contains the (pTTV), ﬂanked by overlapping entire TTV genome inserted between the EcoRI sites in the 175 bp areas, pSTBlue-1 vector. In addition, a BspEI restriction enzyme can be used to excise from the TTV clone the complete, in linear (without overlaps) TTV genome single-unit 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

Total cellular DNA was isolated by cell lysis (‘Protein- ase K’, 2.4 U ⁄ 200 lL reaction; Fermentas, Burlington, 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].

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

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,

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