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

Construction and biological activity of a full-length molecular clone of human Torque teno virus (TTV) genotype 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 Soderlund-Venermo1 ă Department of Virology, Haartman Institute and Helsinki University Central Hospital, University of Helsinki, Finland Department of Molecular Cell Biology, Leiden University Medical Center, the Netherlands 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 19126491 Tel: +358 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) 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 functional, 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 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 restriction 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 doi:10.1111/j.1742-4658.2007.06020.x TT-virus (TTV) recently named Torque teno virus [1] was found in 1997 in Japan from a patient with posttransfusion 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 structure, 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 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 distinct 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 FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS 4719 Biological activity of a full-length TTV DNA clone L Kakkola et al ORF1, 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 nonhuman 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 particularly 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 genotype 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 transcription and DNA replication) in a number of different cell lines in which two nucleotides differ from the in vivo genotype 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 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 amplicons 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 experiments substantiated that the 454 bp amplicon originated from the transcribed viral RNA and not from DNA The TTV RNA was shown by RT-PCR to persist in subcultured cells for at least 11 days Nontransfected 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 retinoblastoma mRNA yielded the expected amplicons (data not shown) demonstrating mRNA integrity The results were identical for all the cell lines Replication of TTV DNA Results Genotype cloning and sequence analysis A full length molecular clone, pTTV, in plasmid pSTBlue-1 was constructed of TTV genotype (Fig 1A) The cloned genome was sequenced (GenBank accession number AY666122; nucleotide numbering accordingly), 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 fulllength clone were used for transfection experiments; the excised linear genome (linTTV) and the intact plasmid pTTV (Fig 1B,C) The full-length plasmid clone contains at its left end the NG136 primer sequence [25] 4720 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 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 post transfection, a fulllength TTV DNA of 3748 bp (after BamHI digestion, FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS L Kakkola et al Biological activity of a full-length TTV DNA clone 3748/1 A * BspEI NsiI • TTV genotype6 BspEI tscrtlat BspEI • TTVGCF NG136 pSTBlue-1 C TTV genome 3748 bp 32P 3165 bp 2162 bp 32P DIG BamHI DIG BamHI 2162 bp BspEI 3004 bp BamHI BspEI B 32P pTTV total length 7775 bp 32P BamHI Fig The full-length clone and the constructs 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 indicated 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 transcription 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 pTTVderived 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 pSTBlue-1 3851 bp 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 linear construct had occurred Furthermore, this 3748 bp fragment was resistant to DpnI (Fig 3A,C, marked with an arrow), indicating TTV DNA replication The linearized 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 The DpnI-resistant DNA progressively accumulated in the transfected cells from day to day 3, as shown for 293T cells in Fig However, on days 3, and 8–10 post transfection upon cell passage, the amount of DpnIresistant (replicating) TTV DNA declined, and was detectable in Southern analyses for up to day Interestingly, in those cells that permitted replication of the excised linear construct, high molecular weight double bands (sensitive to DpnI) were visible (Fig 3) Singlestranded DNA (ssDNA) could, however, not be visualized by Southern analysis, suggesting that its production remained below the detection limit Of note, when comparing the 293 cells, with and without the SV40 large T antigen, the amount of DpnI-resistant replicating TTV DNA detected on day post transfection (relative to a standard amount of total cellular DNA) was invariably 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 post transfection, the same 3165 bp fragment (Fig 3A, marked with an asterisk) was DpnI resistant, indicating replication of the complete pTTV The results with the 32P-probe verified this: the input FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS 4721 Biological activity of a full-length TTV DNA clone A nt L Kakkola et al nt 112 nt 3748 DNA nt 112 nt 182 nt 284 poly-A RNA RT1F nt 129-147 B Mw day3 D day5 D C day10 D Mw day3 D day3 D Mw D pTTV day1 bp non-transf cells linTTV day1 bp 500 400 300 RT1R nt 683-660 pSTBlue-1 day6 D day10 D day3 D Mw D 600 500 400 linTTV day3 D bp Mw +RT -RT R Circularization of the linear construct D 600 500 400 input pTTV input linTTV bp Mw +RT -RT R D +RT -RT R D 600 500 400 Fig 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 transfected with (B) the linear excised TTV (linTTV) and (C) intact pTTV Nontransfected cells and cells transfected with the backbone plasmid (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 pTTV was seen (after BamHI digestion) as two DpnI-sensitive restriction fragments of 3165 bp and 4610 bp (Fig 3C) and, on day 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 4722 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 cytopathological changes were microscopically detected in the cells supporting TTV DNA replication The results were the same regardless of the DNA isolation 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 nontransfected 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 digestible with other restriction enzymes (data not shown) In the linTTV-transfected cells on day 3, the emergence of the 3748 bp TTV DNA after BamHI digestion (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 post transfection yielded additional fragments of approximately 1630 bp and 1520 bp, respectively (Fig 5B), indicating fusion of the linearization breakpoint 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 replication machinery, aphidicolin (an inhibitor of eukaryotic FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS L Kakkola et al Biological activity of a full-length TTV DNA clone 293T A bp 293 linTTV pTTV pSTBlue-1 day1 day3 day1 day3 day3 Mw B B/D B B/D B B/D B B/D B B/D 4899 3639 2799 bp linTTV pTTV pSTBlue-1 day1 day3 day1 day3 day3 Mw B B/D B B/D B B/D B B/D B B/D 4899 3639 2799 UT7/Epo-S1 KU812Ep6 pTTV linTTV cells day1 day3 day1 day3 Mw B B/D B B/D B B/D B B/D B B/D bp 4899 3639 2799 linTTV pTTV cells day1 day4 day1 day4 B B/D B B/D B B/D B B/D B B/D Mw bp 4899 3639 2799 Cos-1 Huh7 linTTV pTTV linTTV pTTV pSTBlue-1 pSTBlue-1 day1 day3 day1 day3 day3 day1 day3 day1 day3 day3 Mw B B/D B B/D B B/D B B/D B B/D Mw B B/D B B/D B B/D B B/D B B/D bp bp 4899 3639 2799 4899 3639 2799 Chang liver bp Mw 4899 3639 2799 linTTV pTTV pSTBlue-1 day1 day3 day1 day3 day3 B B/D B B/D B B/D B B/D B B/D B bp Mw input linTTV U B B/D bp 4899 3639 2799 C bp 4625 Mw input pTTV U B B/D 4899 3639 2799 input pTTV 293T pTTV linTTV 293 pTTV linTTV 3165 Fig 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) Southern 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 FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS 4723 Biological activity of a full-length TTV DNA clone bp Mw day0 B B/D day1 B B/D day2 B B/D L Kakkola et al day3 B B/D A - APC pTTV linTTV + APC pTTV linTTV 4899 3639 2799 B Fig Accumulation of DpnI-resistant full-length TTV DNA (marked with an arrow) in 293T cells from day to day post transfection 293T cells were transfected with linTTV, and DIG-labelled probe was used to detect the TTV DNA bp Mw B -APC B/D B + APC B/D 4899 3639 2799 III HI aI m oI nd Sc Ba Xh Hi BspEI A TTV genome 3748 bp BspEI SalI 887 bp 634 bp 2227 bp 1521 bp Sal I bp 3639 oI BamHI/SalI lin day3 Mw Xh HI pSTBlue-1 Bam Sal I 1631 bp B Fig The effect of aphidicolin (APC) on TTV DNA replication 293T cells were transfected with pTTV or the linear (linTTV) construct in the absence (–APC) or presence (+APC) of aphidicolin (A) Southern analysis with the 32P-probe of Hirt-extracted (BamHI ⁄ DpnI-digested) DNA (B) Southern analysis with the DIG-labelled probe of BamHI- (B) and BamHI ⁄ DpnI-digested (B ⁄ D) DNA on day post transfection Arrows are pointing to the sites of replicating, DpnI-resistant TTV DNA 744 bp 2117 bp XhoI/SalI Mw lin day3 pSTBlue-1 Discussion 2799 1953 1882 1515 1482 992 718 Fig Circularization of the linear construct in 293T cells (A) Schematic representation of the restriction enzyme cutting sites and the corresponding restriction-fragment sizes (B) The input linear construct (lin) and the total cellular DNA (from the linTTV-transfected 293T cells on day 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 performed with a probe prepared by nick translation (the band corresponding to the cloning vector pSTBlue-1 is indicated) The fragments from the circularized TTV DNA are marked with arrows 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 4724 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 activities 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, genotype 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 transcribed and proteins translated) in 293 cells was determined 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 FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS L Kakkola et al detected in Cos-1 cells transfected with a plasmid clone of TTV genotype [9] The cloning strategy used by this group was similar to ours: the plasmid clone contained overlapping genomic regions and the cloning breakpoint was in the noncoding region Their genotype clone and our genotype 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 has been shown to be active in many cell types, including Huh7 and cells of erythroid origin [5,8] Our results with genotype 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 demonstrated to be useful for the study of TTV DNA replication TTV DNA replication was detected in the majority of cell types When the excised linear genotype DNA was transfected into 293T, 293, UT7 ⁄ Epo-S1, Chang liver and Huh7 cells, circularized and replicating (DpnI resistant) forms of TTV DNA accumulated 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 replication 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 replication 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 replication (after transfection with linTTV), could theoretically 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 highest 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 Biological activity of a full-length TTV DNA clone 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, replicates 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 plasmid 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, recombination, and rolling circle replication) [31,32] In comparison 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 transfection 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 productive 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 erythroid origin, as are UT7 ⁄ Epo-S1, and could thus be thought to support TTV replication However, according 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 replication Indeed, our results with aphidicolin, which inhibits eukaryotic nuclear DNA replication and blocks the cell cycle at early S-phase, strongly suggest that TTV FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS 4725 Biological activity of a full-length TTV DNA clone L Kakkola et al utilizes the cellular DNA polymerase and S-phase for replication The requirement for rapid cell division, together with the diminished viability of the electroporated KU812Ep6 cells, could explain the lack of replication of our full-length clone in those cells The deficiency of TTV replication in monkey kidneyderived Cos-1 cells could represent species specificity However, because mRNAs were produced, the potential species barrier does not appear to affect transcription In previous studies, the sites of TTV replication in humans have been suggested to be the hematological compartment and the liver [15,16] Our results, demonstrating replication of cloned TTV genotype 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 transcription of genotypes and 6, which belong to the same genogroup, appears to be similar [9,11] The production of infectious virions from the transfected cells could not be verified unequivocally with the methods in use, and thus remains to be investigated In our experiments, upon cell subculture, even though the more sensitive RT-PCR continued to be positive, the levels of replicating TTV DNA in Southern analysis declined below detection limit These results suggest that, if infectious virions were produced, the infection did not spread efficiently to the neighbouring cells, and ⁄ or that the amounts of progeny 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 resembling many other viruses such as human parvovirus 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 chronically, 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 4726 TTV has been recently elucidated with this fulllength 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 biology, 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 significance of this recently discovered human virus Experimental procedures Cell lines and transfection methods Seven cell lines, Cos-1, Huh7, Chang liver, KU812Ep6, UT7 ⁄ Epo-S1, 293T and 293, were used in this study Cos-1 cells (African green monkey kidney; transformed with SV40) were maintained in DMEM containing 10% fetal bovine serum, 10 mm Hepes buffer (Gibco BRL ⁄ Invitrogen, Carlsbad, CA, USA) and antibiotics The cells were transfected day after subculture, at approximately 95% confluency, with 30 lL Lipofectamine2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) and lg DNA per 60 mm2 plate Human hepatoma cells, Huh7, were maintained in MEM, with 10% fetal bovine serum and antibiotics, 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 kidneyderived 293T cells (expressing the SV40 T antigen, originally 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, mL)1 erythropoietin (‘Eprex’; JanssenCilag, Berchem, Belgium) and antibiotics For transfection, the cells were harvested on days 3–4 For one reaction, · 106 cells were resuspended in 0.5 mL medium and lg DNA was added The cells were electroporated immediately at 300 or 350 V, and 960 lF (‘Gene Pulser’; Bio-Rad, Hercules, CA, USA) A human erythroblastoid cell line UT7 ⁄ Epo-S1 [42,43], kindly provided by Dr Morita (Tohoku University School of Medicine, Japan), was maintained in suspension in Iscove’s modified Dulbecco’s medium containing 10% fetal bovine serum, mL)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 FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS L Kakkola et al T-24 (Amaxa Biosystems, Cologne, Germany) All the cells were grown at 37 °C in 5% CO2; and were monitored with PCR for the presence of endogenous TTV (generic UTRPCR, and genotype 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 isolate HEL32 [44] was completed to contain the full-length genome of the isolate The GC-rich part of the TTV genome was amplified from the original serum [44] with seminested primers: forward TTVGCF (nucleotides 3206–3225) 5¢-CAGA CTCCGAGATGCCATTG-3¢, first reverse TTVGCR1 (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, Switzerland), 400 nm of each primer, 1.5 mm MgCl2, 10% dimethylsulfoxide, 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 °C for 30 s and extension at 72 °C for for the first ten cycles, with addition of sỈcycle)1 of extension time for the remaining 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 forward NG054 (nucleotides )2–18) [25] and reverse TTVGCR6 (nucleotides 394–375) 5¢-CGTTCGAGTT 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 reactions 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 Institute, 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 Biological activity of a full-length TTV DNA clone 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 pEGFPLuc vector (Clontech, Mountain View, CA, USA) encoding green fluorescent protein On day 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 (1.5 U ⁄ 15 lL; ‘RQ1 RNase-Free DNase’; Promega, Madison, WI, USA) for 70 at 37 °C 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: forward RT1F (nucleotides 129–147) 5¢-GCAGCGGCAGCACCTCGAA-3¢ and reverse RT1R (nucleotides 683–660) 5¢-GTCTAGCAGGTCCTCGTCTG CGAG-3¢ This separates the possible DNA-derived amplicons from the RNA-derived ones by size (555 bp and 454 bp, respectively; Fig 2A) The PCR program consisted of reverse transcription for 30 at 42 °C, followed by PCR: 94 °C for min, and 35 cycles at 94 °C for 30 s, 65 °C for 30 s, 68 °C for 45 s, with final extension at 68 °C for 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 nontreated samples The RNA experiments were performed at least twice DNA replication analyses Total cellular DNA was isolated by cell lysis (‘Proteinase 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] The DNA samples (2.5–5 lg of Hirt-extracted or 30– 40 lg of total DNA) were digested with BamHI Subsequently, half the digest was treated with DpnI (which cuts only prokaryotic DNA; New England BioLabs, Ipswich, FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS 4727 Biological activity of a full-length TTV DNA clone L Kakkola et al MA, USA) The BamHI- and BamHI ⁄ DpnI-digested DNA samples were separated by gel electrophoresis The DNA was transferred to nylon membranes (‘Hybond-N+’, Amersham Biosciences, Piscataway, NJ, USA), and was hybridized overnight For hybridization, a DIG-labelled TTVspecific probe, covering nucleotides 1803–2200 (Fig 1), was prepared as described [44] using the outer primer pair of genotype 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 (Roche) In some experiments, a 32P-labelled probe consisting of a 560 bp fragment of the untranslated region (NsiIBspEI fragment from pTTV, Fig 1) was used The probe was labelled to high specific activity with [a-32P]dCTP by the random primer method [47], and the signal was detected by film exposure For aphidicolin treatment, lgỈmL)1 of aphidicolin (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 post transfection, the DNA was isolated by the Hirt method, 10 lg of DNA was digested (BamHI ⁄ DpnI), separated on an agarose gel, and detected with the 32P-probe Alternatively, the 293T cells were transfected with the Lipofectamine2000 method and grown in the presence of aphidicolin On day post transfection, the total DNA was isolated, 30 lg of DNA was digested (BamHI ⁄ DpnI), and analyzed with Southern hybridization with the DIG-probe Acknowledgements The authors thank Drs Ilkka Julkunen (National Public Health Institute, Helsinki, Finland), Eiji Morita and Kazuo Sugamura (Tohoku University School of Medicine, Sendai, Japan), and Eiji Miyagawa (Fujirebio Inc., Tokyo, Japan) for cell lines, and Paivi Norja ă and Heidi Bonden 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 Juselius Foundation, the Research and Science Foundation of Farmos, the Ella and Georg Ehrnrooth Foundation, the Finnish Konkordia 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 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alternatively isolated with QIAamp DNA Blood... Nishizawa T, Kato N, Ukita M, Ikeda H, Iizuka H, Miyakawa Y & Mayumi M (1998) Molecular cloning and characterization of a novel DNA virus (TTV) associated with posttransfusion hepatitis of unknown... geminiviruses and plant circoviruses Arch Virol 143, 1723–1744 14 Okamoto H, Takahashi M, Nishizawa T, Tawara A, Sugai Y, Sai T, Tanaka T & Tsuda F (2000) Replicative forms of TT virus DNA in bone marrow