Quantification of human T-cell lymphotropic virus type 1 proviral load by quantitative competitive polymerase chain reaction

Journal of Virological Methods 75 (1998) 123 – 140

Quantification of human T-cell lymphotropic virus type 1 proviral load by quantitative competitive polymerase chain reaction Bjo¨rn Albrecht a,b, Nathaniel D. Collins b, Garret C. Newbound b, Lee Ratner d, Michael D. Lairmore a,b,c,* a Molecular, Cellular and De6elopmental Biology Program, The Ohio State Uni6ersity, Columbus, OH 43210, USA Center for Retro6irus Research and Department of Veterinary Biosciences, The Ohio State Uni6ersity, 1925 Coffey Road, Columbus, OH 43210 -1092, USA c Comprehensi6e Cancer Center, The Arthur James Cancer Hospital and Research Institute, The Ohio State Uni6ersity, Columbus, OH 43210, USA d Departments of Medicine, Pathology, and Molecular Microbiology, Washington Uni6ersity School of Medicine, St. Louis, MS 63110, USA b

Received 15 December 1997; received in revised form 26 May 1998; accepted 26 May 1998

Abstract The polymerase chain reaction (PCR) has been established as a highly sensitive technique for detection of viral DNA or RNA. However, due to inherent limitations of PCR the amount of amplified product often does not correlate with the initial amount of template DNA. This is particularly true for PCR detection of viral infections that are characterized by low in vivo viral copy numbers in certain stages of the infection, such as human T-cell lymphotropic virus type 1 (HTLV-1) and simian T-cell lymphotropic virus type 1 (STLV-1). Therefore, we developed a quantitative competitive polymerase chain reaction (qcPCR) for detection of HTLV-1 and STLV-1 proviral DNA. The assay was optimized using an infectious HTLV-1 clone, ACH, HTLV-1 infected cell lines, MT-2.6 and HUT-102 and STLV-1 infected lines Kia and Matsu. Applicability of this system was demonstrated by determining HTLV-1 proviral load in peripheral blood mononuclear cells (PBMC) of human subjects with HTLV-1 associated diseases and an asymptomatic carrier as well as rabbits infected experimentally. This qcPCR method, the first designed specifically for HTLV-1 and STLV-1, will provide an important tool for pathogenesis studies of HTLV-1 and for evaluating the efficacy of antiviral drugs and vaccines against the viral infection using animal models. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: HTLV-1; Quantitative competitive polymerase chain reaction; Pathogenesis; Animal model; Diagnosis; Retrovirus

Abbre6iations: PCR, polymerase chain reaction; qcPCR, quantitative competitive polymerase chain reaction; RT-qcPCR, reverse transcriptase quantitative competitive polymerase chain reaction; PBMC, peripheral blood mononuclear cells; STLV-1, simian T-cell lymphotropic virus type 1. * Corresponding author. Tel.: + 1-614-2924819; Fax: + 1-614-2926473; e-mail: [email protected]. 0166-0934/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 9 8 ) 0 0 0 8 7 - 1

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1. Introduction Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL) and is associated with a neurodegenerative disorder designated tropical spastic paraperesis/HTLV-1 associated myelopathy (TSP/HAM) (Poiesz et al., 1980; Gessain et al., 1985; Osame et al., 1986). Endemic regions of HTLV-1 infection occur throughout the world and include Japan, the Caribbean basin, Africa, Central and South America as well as the USA, where the infection occurs mainly in at-risk groups such as intravenous drug users (reviewed in Franchini, 1995). Viral infection may be detected indirectly by a variety of serologic assays including enzyme linked immunosorbant assays (ELISA) or immunoblots. These assays utilize lysates or recombinant viral proteins to monitor production of viral antigens or antibodies directed against the virus (reviewed in Lairmore and Lal, 1992). Alternatively, nucleic acid-based methods such as PCR and Southern blots are used for detection of proviral DNA and analysis of viral integration patterns in infected cells. Assays specifically designed to test for HTLV-1 proviral load include various semiquantitative PCR techniques (Kira et al., 1991; Wattel et al., 1992; Kubota et al., 1993; Ono et al., 1995; Feuer et al., 1996) and slot blot hybridization (Abbott et al., 1988). However, these techniques are not genuinely quantitative, since they rely on coamplification of a heterologous internal standard such as the b-globin housekeeping gene or a template dilution series with subsequent qualitative PCR, respectively. Semiquantitative PCR methods cannot measure accurately proviral copy numbers in infected cells due to differences in wild-type and standard template abundances, variation in priming efficiencies for different primer– target combinations or alterations in amplification efficiencies between the internal standards and target sequences of different size. A tax specific quantitative competitive PCR for assessment of HTLV-2 proviral load has been reported (Cimarelli et al., 1995). This assay was also used to detect HTLV-1 from an HTLV-1 infected cell line (MT-2) and two patients as well

as in squirrel monkeys (Kazanji et al., 1997). Despite considerable sequence homology in the tax gene between HTLV-1 and HTLV-2, no data were presented in the original report to verify the sensitivity of the assay or the derived HTLV-1 proviral copy number. We report the development of a qcPCR for detection of a conserved region in the gag gene of HTLV-1 and STLV-1. This region was chosen to assure applicability of this procedure for a wide variety of HTLV-1 molecular clones. The primers chosen are specific for HTLV-1 and certain strains of STLV-1 and therefore enable distinction from HTLV-2 infections. Two different competitor DNA templates, DAluI (with a deleted AluI restriction site) and StyID28 (with a 28-bp insertion) were evaluated in competition with a wild-type infectious clone of HTLV-1, designated ACH (Kimata et al., 1994). In support of previous results (Gilliland et al., 1990; Bagnarelli et al., 1992; Cimarelli et al., 1994), the competitor differing in size (StyID28) appeared to be more consistent in amplification. This is due most likely to inconsistencies related to DNA isolation and restriction digest of PCR products obtained with DAluI after amplification. Therefore, StyID28 was used subsequently to monitor the proviral load in HTLV-1 infected cell lines (MT-2.6 and HUT-102). A higher proviral load was demonstrated in HUT102 cells (17 copies/cell) than in MT-2.6 cells (three copies/cell). Peripheral blood mononuclear cells (PBMC) derived from rabbits infected either with the wildtype clone of HTLV-1, ACH, or a mutant of this clone, ACH.p12, which cannot express the putative regulatory protein p12I of the pX region of the HTLV-1 genome were also assayed for proviral load using the qcPCR. While 0.3–2.5% of wild-type infected PBMC cells showed integrated proviral copies, PBMC from rabbits inoculated with ACH.p12 had no detectable proviral copies. HTLV-1 proviral load was also determined in human ATL and TSP/HAM patients as well as an asymptomatic carrier. The asymptomatic carrier had proviral loads comparable to that determined in the asymptomatic rabbits (0.7%), whereas the symptomatic patients had :100-fold higher proviral loads, which is comparable to

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Fig. 1. Specificity of the primer pair used in qcPCR study. (A) Primer recognition sequence alignment between several strains of HTLV-1, HTLV-2 and STLV-1. All HTLV-1 strains showed homology to the primer sequences, whereas one of the two STLV-1 strains did not show homologous sequences to either of the primers used. HTLV-2 cannot be detected by our primers due to a lack of complementarity to the downstream primer. The primers used are therefore specific for HTLV-1 and certain strains of STLV-1. The cut-off for positivity for homology in sequence comparisons was set at 80% according to Lee and Caskey (1990). (B) Qualitative PCR on two STLV-1 cell lines and one HTLV-2 cell line. STLV-1 cell lines Kia and Matsu and the HTLV-2 immortalized line Mo-T were used as representative samples. A 500 ng sample of genomic DNA was amplified as described in Section 2. One of the two STLV1 lines (Kia) is detected strongly whereas Matsu is only weakly positive. Mo-T is not amplified at all. Lanes: (1) 100 bp ladder, (2) Kia, (3) Matsu, (4) Mo-T, (5) MT-2.6 (HTLV-1 positive control, (6) no template control.

previous reports (Kira et al., 1991; Ono et al., 1995). This qcPCR for HTLV-1 will be particularly important for monitoring proviral load during the course of the infection and for clinical management of infected patients undergoing antiviral therapy (Sheremata et al., 1993; Gill et al., 1995; Izumo et al., 1996). It will also be invaluable for evaluating the efficacy of HTLV-1 antiviral drugs and vaccines currently under development (Lairmore et al., 1995; Kaumaya et al., 1995) and the analysis of infectivity of different mutant viral strains of HTLV-1, such as ACH.p12I (Collins et al., 1998). Furthermore, this assay will be a

powerful tool to determine proviral loads in animal model systems using either STLV-1 or HTLV-1.

2. Materials and methods

2.1. Specificity of qcPCR primer pair To evaluate the specificity of the primer pair used for qcPCR, primer recognition sequence alignments were carried out between several strains of HTLV-1, HTLV-2 and STLV-1 (HTLV-1ACH (Kimata et al., 1994), HTLV-1EL

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Fig. 2. Megaprimer-based PCR mutagenesis for construction of competitive standard for HTLV-I qcPCR. (A) General outline of the procedure showing primer binding sites in the gag region of the full length infectious HTLV-I clone ACH. Also shown are the targeted and an additional AluI restriction site as well as the StyI restriction site subsequently used for insertion of a 28 bp linker. Primers sequences are shown in Table 1. PCR reactions contained 2.5 U Pfu-polymerase in the appropriate buffer (Stratagene), 0.12 fmol ACH-template (1 ng of a 12 kb plasmid), 50 mM dNTPs and 25 pmol of both the downstream and mutagenic primers. Ten amplification cycles/step were performed at 94°C for 1 min, 60°C for 1 min, 72°C for 1.5 min, followed by a 5 min extension at 72°C, and holding at 4°C. A total of 10 ml of the reaction were separated on a 1% agarose gel, and the amplified full-length product was excised for subsequent cloning. (B) AluI digest of PCR products from a standard amplification with SG166H3 and SG167X1 and the mutagenesis products. While AluI still cuts the PCR products of the control reaction, the AluI restriction site is deleted in the mutagenesis product. Lane 1: molecular weight marker (100 bp ladder, GIBCO BRL), 2: uncut control, 3: control amplification:, 4: PCR mutagenesis.

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(Ratner et al., 1985), HTLV-1CH (Ratner et al., 1991), HTLV-1ATK (Seiki et al., 1983), HTLV1MT-2 (Gray et al., 1989), HTLV-1TSP1 (Evangelista et al., 1990), HTLV-1HS35 (Malik et al., 1988), HTLV-1RD1 (Mukhopadhyaya and Sadaie, 1993), HTLV-1BOI (Bazarbachi et al., 1995), HTLV-1MEL5 (Gessain et al., 1993), HTLV-2Gu (Salemi et al., 1996), HTLV-2Mo (Shimotohno et al., 1985), HTLV-2G12 (Pardi et al., 1993), STLV-1TE4 (Ibrahim et al., 1995) and STLV-1PH969 (Van Brussel et al., 1997) (Fig. 1A). Viral genome sequences were obtained from GenBank or from published reports. The cut-off for positivity for sequence homology was set at 80% according to findings of Lee and Caskey (1990) in order to ensure sufficient primer binding and amplification efficiency. Furthermore, qualitative PCR was undertaken on representative HTLV-2 (Mo-T) (Saxon et al., 1978) and STLV-1 (Kia, Matsu) (Lazo et al., 1994) infected cell lines (Fig. 1B). Cells were maintained in RPMI 1640 (GIBCO, BRL) supplemented with 15% FBS, 1% glutamine, 1% penicillin and 1% streptomycin. Genomic DNA from 1× 107 cells was isolated according to the manufacturer’s protocol (Qiamp Blood Kit, QIAGEN) and 500 ng were amplified per reaction using primers SG166 and SG296 (Ehrlich et al., 1990). Amplification was conducted as follows: preheat cycle: 94°C for 10 min in order to activate the polymerase; 37 cycles: 94°C for 1 min, 60°C for 1 min and 72°C for 45 s followed by final extension at 72°C for 5 min. Reactions were held at 4°C.

products generated in each step of the protocol are shown in Table 1. The mutagenic primer 166M167 carried an internal C“ G substitution at position 12 (equivalent to position 1661 of ACH) causing the deletion of the AluI restriction site. As wild-type plasmid template the full length clone of HTLV-1, ACH (Kimata et al., 1994) was used. Optimal yield of full-length product was obtained by deviating from the original protocol which suggested a five fold higher concentration of upstream and downstream primer in steps 2 and 3 (Picard et al., 1994). Instead we used equimolar concentrations of all primers (data not shown). To demonstrate deletion of the AluI restriction site in the PCR product, aliquots of a standard amplification using SG166H3 and SG167X1 alone and of the mutagenesis reaction were digested with AluI for 1hr at 37°C and separated on a 1.5% agarose gel (Fig. 2B). Final PCR products were gel isolated (Millipore MC filter units), double digested with XbaI and HindIII and ligated directionally into pGEM4z under standard conditions (Fig. 3A). Transformation into DH5a competent cells (Gibco BRL) was carried out as described by Maniatis et al. (1989). This construct, designated DAluI, was confirmed by restriction analysis with XbaI/HindIII and subsequent AluI digestion of the isolated insert, P6uI digestion (Fig. 3C), as well as automated laser fluorescent SANGER-sequencing (Applied Biosystems) (data not shown).

2.2. Construction of competitor DAluI

Two synthetic oligonucleotides 5%-GGTATCGATCGATACAGGTCCTGTC-3% and 5%-CATAGCTAGCTATGTCCAGGACAGGTTC-3% were annealed according to standard procedure (Maniatis et al., 1989) forming a 28 bp adapter containing internal P6uI and DraII restriction sites and StyI adhesive ends. The adapter was ligated into StyI-linearized DAluI and the resulting plasmid (StyID28) transformed into DH5a competent cells as decribed (Fig. 3A). StyID28 was confirmed by XbaI/HindIII and P6uI digests (Fig. 3C), as well as SANGER-sequencing as above (data not shown).

DAluI was constructed using a refined PCRbased site-directed mutagenesis designated megaprimer method (Picard et al., 1994). The mutagenesis procedure leads to a single basepair substitution to generate a competitive standard that differs from the wild type in the deletion of an AluI restriction site. The identical size assures equal amplification efficiency of the two templates and is one advantage over competitors that differ in molecular weight. The procedure is outlined in Fig. 2(A). Primer sequences and

2.3. Construction of competitor StyID28

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Table 1 Primers used for construction of internal standard DAluI and products generated by each step of the megaprimer mutagenesis method and primers used for qcPCRa

Primer sequences Upstream primer SG166H3 Mutagenic primer 166M167 Downstream primer SG167X1

5%-ATA TAA GCT T6 CT GCA GTA CCT TTG CTC CTC CCT C-3% 5%-CGT AGA AAC GCC TCA ACG TAG GTC TTG AC-3% 5%-ATA TTC TAG ACC CGG GGG GAG GAC GAG GCT GAG T-3%

PCR mutagenesis Step

Primers used

Product created

Product size

1 2 3

166M167-SG167X1 Megaprimer-SG166H3 SG166H3-SG167X1

Megaprimer Full-length product Amplified full-length product

292 bp 588 bp 588 bp

qcPCR primers Upstream primer SG166 Downstream primer SG296

5%-CTG CAG TAC CTT TGC TCC TCC CT-3% 5%-TTC TAC GAA GGC GTG GTA AG-3%

a Bold/italic letters indicate unpaired nucleotides; bold underlined letters indicate restriction sites; the C “ G substitution in 166M167 is indicated in bold and the deleted AluI restriction site is underlined. SG166H3 and SG167X1 contained HindIII and XbaI restriction sites at their 5% ends, respectively, used for subcloning.

2.4. Cell lines and infected rabbit PBMC HTLV-1 infected human lymphocyte lines HUT-102 (Gadzar et al., 1980) and MT-2.6 (Miyoshi et al., 1981) and the human acute T-cell lymphoma cell line Jurkat (ATCC, Cat. No: TIB152) were cultured under standard conditions at 37°C in RPMI 1640 supplemented with 1% glutamine and 1% penicillin/streptomycin and 10% fetal bovine serum (complete RPMI). Genomic DNA was isolated according to the manufacturer’s protocol (QIamp Blood Kit, QIAGEN) or by phenol:chloroform extraction (DNA STAT-60, Tel-Test). Genomic DNA from HTLV-1 positive MT-2.6 or HUT-102 cells was serially diluted with HTLV-1 negative Jurkat DNA. Dilutions were carried out using DNA to avoid inconsistent results from variations in the ploidy of the transformed cell lines. Ratios used were typically 10:0 (500 ng HTLV-I positive DNA:0 ng HTLV-I negative DNA), 5:5, 2:8 and 1:9. DNA mixes were subsequently digested with EcoRI and quantified by spectrophotometry (Pharmacia Genequant). Proviral copy numbers per cell were determined as described previously (Piatak et al., 1993a). In brief, the amount of DNA per cell (7.4 pg) wascalculated based on the number of bases per

diploid genome (1.32× 1010). Therefore, 500 ng of genomic DNA used per qcPCR reaction correspond to 6.73 ×104 cells. Equivalency points derived in qcPCR reactions indicate directly the amount of competitor plasmid in moles. This is converted to copy numbers based on the weight of the competitor plasmid and divided by 6.73× 104 resulting in proviral copy numbers/cell. Quantitative competitive PCR was also undertaken on genomic DNA from the STLV-1 infected cell lines Kia and Matsu. Cells were cultured and genomic DNA isolated as described above. 500 ng genomic DNA were used per qcPCR reaction with serially diluted competitor as discussed below (data not shown). Twelve-week-old specific pathogen free New Zealand White rabbits (Hazelton) were inoculated as described (Collins et al., 1996). Rabbits were infected with heterologous human PBMC transfected with the wild-type molecular clone of HTLV-I, ACH (R52–54), or the mutant clone ACH.p12 (R55–58) which cannot express the p12I message due to deletion of the splice acceptor of the last exon of the p12I gene (Collins et al., 1998). At 10 weeks post-inoculation PBMC genomic DNA was prepared and 500 ng were amplified as described below. This amount of PBMC

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DNA was determined to be optimal in preliminary trials (data not shown). Rabbit 50 was a mock infection with the pKS plasmid only. Rabbits were monitored for viral expression by p19 ELISA, and qualitative PCR as described elsewhere (Collins et al., 1998) and the week 10 bleed was used for quantitative analysis of proviral load via qcPCR. Proviral copy number per PBMC was calculated as above. Percentage of infected PBMC is based on the assumption that infected cells carry one proviral copy only. Infected PBMC/ml blood is derived by multiplication of derived percentage with number of PBMC/ml blood (: 5.31× 106).

2.5. Human PBMC clinical specimens PBMC from HTLV-1 associated disease patients and an asymptomatic carrier were obtained from samples taken for diagnostic purposes. Genomic DNA was extracted and qcCPR was carried out using 500 ng of genomic DNA per reaction as described below.

2.6. Quantitati6e competiti6e PCR

Fig. 3. Construction of competitive standards DAluI and StyID28. (A) Cloning procedure for construction of DAluI and StyID28, respectively. (B) Restriction map of the competitor plasmid StyID28. P6uI and DraII restriction sites engineered into the 28 bp oligonucleotide linker allow confirmation of correct ligation and absence of concatamers. DAluI lacks the 28 bp insertion. (C) Restriction analysis of DAluI and StyID28. In order to confirm correct ligation, both plasmids were subjected to XbaI/HindIII double digests (X/H) and P6uI digests (P). In order to confirm that DAluI still retained the deleted AluI site, the XbaI/HindIII-generated fragment was further digested with AluI. Lane 1: 100 bp ladder, 2: DAluI (X/H), 3: DAluI (P), 4: StyID28 (X/H), 5: StyID28 (P), 6: 100 bp ladder, 7: DAluI X/H-fragment digested with AluI, 8: AluI digest of the X/H fragment obtained from a clone constructed with the product of a control amplification as in Fig. 1(B), lane 2.

2.6.1. qcPCR setup StyID28 proved to be the more consistent competitor in pre-trials (data not shown). The observed inconsistencies with this competitor are most likely due to PCR product isolation and subsequent isolation and subsequent A1uI restriction digests necessary in the application of DAluI. Therefore, StyID28 was used for further optimization of the assay. Hot-start PCR (AmpliTaq Gold, PerkinElmer) was conducted in the appropriate buffer with 2.5 U AmpliTaq Gold/reaction in a total reaction volume of 50 ml. EcoRI-linearized ACH or genomic DNA was kept constant, whereas ScaI-linearized competitor (StyID28) was varied over a range of two orders of magnitude in the following molar ratios to the wild-type template: 1:10, 1:7.5, 1:5, 1:2.5, 1:1, 2.5:1, 5:1, 7.5:1 and 10:1. The total amount of DNA was kept at 500 ng/reaction. Primers SG166 and SG296 (Ehrlich et al., 1990) are shown in Table 1 and were used at 250 nM. dNTPs were kept at 50 mM each and MgCl2 at 1

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mM. Reactions were performed in a multiblock thermal cycler (MJR Tetrad Cycler, MJR Research) under the following conditions: preheat cycle: 94°C for 10 min in order to activate the polymerase; 30 cycles: 94°C for 1 min, 60°C for 1 min and 72°C for 45 s followed by final extension at 72°C for 5 min. Reactions were held at 4°C. For PBMC samples the cycle number was increased to 37 to ensure greater sensitivity.

2.6.2. Sample analysis and quantification Aliquots of the qcPCR reactions (10 ml) were separated on 10% polyacrylamide gels in 1 × TBE. Gels were stained with ethidium bromide for visualization under UV light and photographed using a Gel Print 2000i gel documentation system (Bio Photonics). Quantitative analysis of the images was undertaken with Image Quant® software (Molecular Dynamics). Fluorescent intensity of the competitor band (IC) was corrected (I %C) for statistical analysis by multiplication with the size ratio of wild-type (bpWT): competitor (bpC) templates: I %C =ICbpWT/bpC. Equivalence points were derived by plotting regression curves of log (IWT/I%C) against the log10 of the initial concentration of competitor template (logC(0)C). 3. Results

3.1. Specificity of qcPCR primer pair The primer pair SG166 and SG296 which was used in this study is reported as HTLV-1 specific (Ehrlich et al., 1990). To test this and to evaluate further the applicability of our assay to quantification of STLV-1 proviral loads, primer recognition sequence alignments were undertaken with genomic sequences of several strains of HTLV-1, HTLV-2 and STLV-1 obtained from published reports or GenBank (Fig. 1A). All HTLV-1 strains compared showed nearly 100% sequence homology for both primers, whereas no homologous region (\ 80%) for the downstream primer could be detected in HTLV-2. One of the two STLV-1 sequences analyzed also showed sequence homology with both primers (STLV-1TE4). No homology with either primer could be de-

tected in the other STLV-1 strain (STLV-1PH969). Qualitative PCR conducted on the prototypic HTLV-2 infected cell line Mo-T and two STLV-1 cell lines (Kia, Matsu) showed strong reactivity of the primer pair with the cell line Kia, very weak reactivity with Matsu and none with Mo-T indicating that the primer pair used in this study is specific for HTLV-1 and some strains of STLV-1 (Fig. 1B).

3.2. Optimization of plasmid system The basic principle of qcPCR is that competitor (C) and wild-type (W) template have identical thermodynamics and amplification efficiency. Therefore, the ratio between the amplification products (C/W) reflects the initial amounts of both template species (C% and W %) according to C/W=C%(1+e)n/W %(1+e)n, with n being the number of cycles (Clementi et al., 1994). In order to demonstrate equal amplification efficiency and therefore reliability of our system, equimolar amounts of ACH and StyID28 were amplified over a range of six orders of magnitude. Plots of the band intensities for wild-type and competitor illustrate equal amplification efficiency for the two templates over the whole range of detection (Fig. 4A). The sensitivity of the system was analyzed by serial dilution and determined to be 0.113 fg or 5× 10 − 5 aM of entire competitor plasmid (data not shown). For calibration of quantification of HTLV-1 DNA a standard qcPCR with StyID28 serially diluted into ACH was carried out. ACH was kept constant at 5 amol and competitor was added from 0.5 to 50 amol (Fig. 4B). Equivalence is shown in lane 6 (sample 5) in which equimolar amounts of competitor and wild-type template were added (5 amol: 3×106 copies). At the equivalence point the log of the ratio of the fluorescent intensity of the wild-type PCR product to the corrected fluorescent intensity of the competitor PCR product equals roughly one (log 1=0) as shown in Fig. 4(C). Thus, we established a reliable system that allowed analysis and further calibration in HTLV-1 infected cell lines.

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Fig. 4. Optimization of plasmid system. (A) Lanes: (1) 100 bp ladder, (2) 0.02 aM ACH and 0.02 aM StyID28 were amplified under conditions described in Section 2, (3) 0.2 amol, (4) 2 amol, (5) 20 amol, (6) 200 amol, (7) 2 fmol. Results show equal amplification efficiency for both wild-type template (ACH) and competitor StyID28. (B) Quantitative competitive PCR using wild-type template ACH and competitor StyID28. Competitor concentrations were varied and are indicated above each lane. ACH was kept constant at 5 aM. (C) Plot of the log of fluorescent intensities of wild-type over competitor PCR products [log(IACH/IStyID28)] against the log of initial amounts of StyID28 plasmid DNA [log C0(StyID28)]. Equivalence after correction of competitor product band intensity is approximately shown for lane 6 as expected.

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3.3. HTLV-1 cell lines and qcPCR analysis To show applicability of the assay for HTLV-1 infected cell lines and to test assay sensitivity with proviral HTLV-1 DNA, genomic DNA was isolated from HTLV-1 positive cell lines MT-2.6 and HUT-102 and serially diluted with genomic DNA from HTLV-1 negative Jurkat cell DNA at four different ratios. QcPCR was then conducted for each of the four dilutions. Competitor plasmid was varied over a range of three orders of magnitude (Figs. 5 and 6). As expected, for the HTLV-1 positive cell lines, MT-2.6 and HUT-102, equivalence points shifted according to the dilution ratio of HTLV-1 positive genomic DNA in Jurkat DNA. MT-2.6 was determined to have approximately three integrated viral copies per cell, whereas HUT-102 had :17 proviral copies/cell. This :5.5-fold difference in proviral load was consistent and was demonstrated using two different DNA-isolation procedures (reversed phase column chromatography versus phenol:chloroform). The determined copy numbers for the MT-2.6 cells is slightly higher than that determined in a previous report (1.9 copies/cell) (Cimarelli et al., 1995).

3.4. Pro6iral load in PBMC of rabbits infected with HTLV-1 molecular clones ACH and ACH.p12 Previous reports have shown that the regulatory protein p12I from the open reading frame I (ORF I) of the pX region of the HTLV-1 genome is not necessary for viral infectivity in vitro (Derse et al., 1997). However, recent findings suggest that p12I plays an essential role for viral infectivity in vivo (Collins et al., 1998). Therefore, the assay described above was used to determine the proviral load in PBMC derived from rabbits infected with different molecular clones of HTLV-1. Wildtype (ACH) infected animals had detectable levels of virus that varied roughly between 0.003 and 0.025 proviral copies/cell. Assuming that infected cells only have one integrated copy, this would correspond to 0.3–2.5% of total PBMC or approximately 18 000– 133 000 PBMC/ml blood infected. No proviral sequences were detectable in

ACH.p12 infected animals (Table 2). These results are consistent with a previous report in which HTLV-1 proviral load in ACH versus ACH.p12 infected PBMC was measured by serologic methods and semiquantitative PCR (Collins et al., 1998). The low proviral loads observed in ACH infected animals are also in accordance with previous determinations of HTLV-1 proviral load in asymptomatic patients (Richardson et al., 1990; Kubota et al., 1993) and correlate with data described below.

3.5. Quantification of HTLV-1 pro6iral load in clinical specimens To evaluate the applicability of the qcPCR to clinical samples, PBMC from two ATL patients, two TSP/HAM patients and one asymptomatic carrier were obtained and qcPCR was carried out on 500 ng genomic. Proviral copy numbers/ PBMC were derived as described above and are listed in Fig. 7A. No differences were apparent between the proviral load in ATL and TSP/HAM patients. Approximately 0.7% of the PBMC in the asymptomatic patient were infected, which correlated with our data of proviral copy numbers determined for asymptomatic, ACH infected rabbits and previous reports of asymptomatic human carriers (Richardson et al., 1990; Kubota et al., 1993). Proviral loads in patient 4 did not appear to be reduced significantly 4 weeks after start of 3TC therapy (4-II).

4. Discussion To date PCR is the most sensitive assay for detection of specific DNA sequences or gene transcripts (RT-PCR) and is commonly used for virological diagnosis. However, inconsistencies inherent to the technique make it unreliable for quantitative analyses. Tube-to-tube differences in temperature or any of the PCR reaction components, such as dNTP, MgCl2, primers etc. make PCR only a qualitative technique. Quantitative competitive PCR obviates these typical problems of inconsistency by the coamplification of internal competitors that serve as standards in each

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Fig. 5. Detection and quantification of HTLV-I proviral DNA from MT-2.6 cells serially diluted into HTLV-I negative Jurkat DNA. (A) Genomic DNA from MT-2.6 [MT-2] cells diluted into Jurkat (JUR) DNA. In all four dilutions competitor was varied from 5 × 10 − 3 to 5 ×10 − 1 aM (3 000–300 000 copies) as indicated above each lane. Dilution ratios were (i) 50ng [MT-2]: 450 ng [JUR], (ii) 100:400, (iii) 250:250 and (iv) 500:0. Ethidium bromide stained gels are shown for each of the four dilutions. (B) Plot of the equivalence points determined in each of the four dilutions over the amount of MT-2.6 genomic DNA used. Calculations: (I) number of cells/reaction= 500 ng DNA/reaction= 67 300 cells (with 7.4 pg DNA/cell), (II) viral copy number at equivalency point=moles of competitor plasmid × 6.023× 1023 and (III) copies/cell=% PBMC infected =(II)/(I).

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Fig. 6. Detection and quantification of HTLV-I proviral DNA from HUT-102 cells serially diluted into HTLV-I negative Jurkat DNA. (A) Genomic DNA from HUT-102 cells diluted into Jurkat DNA. In all four dilutions competitor was varied from 5 × 10 − 2 to 5 aM (30 000 – 3×106 copies) indicating an approximately 5.5-fold higher proviral load for HUT-102 cells than for MT-2 cells (Fig. 4). Dilution ratios were as in Fig. 4(B) Plot of the equivalence points determined in each of the four dilutions over the amount of HUT-102 genomic DNA used. Calculations were as in Fig. 5.

qcPCR reaction. These internal competitors, when bearing the same primer binding sites and having nearly identical length as the wild-type template, share the same thermodynamics, primer binding efficiencies and therefore amplification

efficiencies as the wild-type template. Use of these internal standards therefore makes qcPCR the most sensitive and accurate quantitative analysis of proviral load currently available. Adaptations of standard PCR assays have been

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Table 2 Quantification of proviral DNA in ACH and ACH.p12 infected New Zealand White rabbits (week 10 post inoculation)a Rabbit

Inoculum

HTLV-1 copy Number/PBMC as per qcPCR

Infected PBMC/ml bloode

p19 antigen ELISAf

Qualitative and semiquant PCR

50 52 53 54 55 56 57 58

pKSb ACHc ACH ACH ACH.p12d ACH.p12 ACH.p12 ACH.p12

− 3.4×10−3 2.9×10−3 2.5×10−2 − − − −

− 18 054 15 370 132 750 − − − −

− + + + − − − −

− + + + − − − −

a

A detailed description of the conditions for the p19 antigen ELISA and the qualitative and semiquanitative PCR can be found in Collins et al. (1998). +, seropositive or PCR positive; −, seronegative or PCR negative. Sensitivity of the qcPCR noted in proviral copy numbers/infected PBMC can roughly be estimated at 4.5×10−4 copies/cell or, assuming infected PBMC only having one integrated proviral copy, one in 45 000 cells, or :28 infected PBMC/ml blood. b Negative control. c Wild-type. d Mutant. e Based on 5.31×106 PBMC/ml blood (Quesenberry, 1994). f Detection was performed in cell culture supernatants. The cut-off for positivity was 25 pg p19/ml.

developed to analyze quantitatively proviral load (qcPCR) and proviral transcription (RT-qcPCR) for a number of retroviral systems (Piatak et al., 1993b; Desforges et al., 1996; Vahlenkamp et al., 1996; Watson et al., 1997). In developing these techniques the design of the internal competitor is critical for the reliability of the assay. Competitors must contain identical primer recognition sequences as the wild-type target, but can be of two different kinds. They either differ in molecular weight or contain or lack an analytical restriction site (Clementi et al., 1995). The use of internal competitors that differ from the wild-type template in molecular weight circumvents the need for repeated isolation and potentially incomplete digestion of either wild-type or competitor PCR products. However, the difference in base pairs should ideally not exceed 10% to ensure similar thermodynamics and amplification efficiency for both templates (Tang et al., 1996). We developed a quantitative competitive approach for HTLV-1 proviral DNA. The p24 gag gene was used as the target region for two reasons. Firstly, the gag gene of HTLV-1 is highly conserved among viral strains as shown by the

sequence alignments in Fig. 1(A). This ensures applicability of this assay to a wide variety of HTLV-1 strains. Moreover, it has been shown that HTLV-1 proviruses in TSP/HAM patients often show deletions or mutations in the 3% end of the viral genome especially the pX region which encodes Tax, Rex and all accessory gene products of HTLV-1 (Saito et al., 1995, 1996). Quantification of HTLV-1 proviral load in TSP/HAM patients might therefore be problematic if either one of these genes was the target region for qcPCR. We acknowledge that ATL cells sometimes show deletions in the gag gene (Korber et al., 1991; Ohshima et al., 1991). Although this might present a limitation to this study, we were able to amplify and quantitate provirus in two out of two ATL patients (Fig. 7A). In addition, it must be remembered that the cells with deleted gag regions represent clonal outgrowths of tumor cells, whereas these patients still remain persistently infected with full-length HTLV-1. Moreover, a pX defective provirus in an ATL patient has been reported, as well (Sakurai et al., 1992). For clinical evaluation it might therefore be helpful to carry out qualitative PCR with primer pairs spe-

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Fig. 7. Quantification of HTLV-1 proviral load in clinical specimens. Four human symptomatic patients (two ATL and two TSP/HAM) and one asymptomatic carrier were analyzed for HTLV-1 proviral load using quantitative competitive PCR. (A) Summary of results for all patients. Note that patient 4 had a 20% decrease in viral copy number 4 weeks after commencing 3TC treatment (4-II). (B) Two representative samples showing gel electrophoretic separation of amplified patient genomic DNA and qcPCR competitor. Note the difference in the range of detection noted above the lanes and therefore the dramatic change in proviral load from asymptomatic (patient 5) to symptomatic patients (patient 4-II).

cific for both the 5% and the 3% end of the provirus before applying the method described above for quantification of proviral load. The specificity of our assay for HTLV-1 and STLV-1 was suggested by sequence comparison of the primer recognition sequences between several strains of HTLV-1, HTLV-2 and STLV-1 as well as by qualitative PCR analysis of an HTLV-2 infected cell line and two STLV-1 infected cell lines. HTLV-1 strains representing all phylogenetic groups were nearly 100% homologous with the sequences of the primers used in this study, whereas none of the HTLV-2 strains listed had considerable sequence homology to the downstream primer SG 296. One of the two STLV-1 strains was also homologous. According to work by Koralnik et al. (1994), HTLV-1 viral strains can be divided into three major clads (cosmopolitan, Melanesia, Zaire). Based on the geographical origin of the strains compared in this study HTLV-1EL belongs to the Zaire clad, HTLV-1CH, HTLV-1TSP1, HTLV-1HS35, HTLV-1RD1, HTLV1BOI (all Caribbean) and HTLV-1ATK, HTLV-

1MT2 (both Japanese) all belong to the Cosmopolitan clad, and HTLV-1MEL5 is a representative of the Melanesia clad. Therefore, the primer pair used will amplify HTLV-1 proviral sequences of representatives of any of the three current phylogenetic clads. Two competitors were developed and evaluated. Our internal competitor distinguishable from the wild-type based on size (StyID28) proved to be more reliable due most likely to the elimination of DNA isolation and PCR product restriction digest steps that were necessary for the application of DAluI. In this regard, the data are consistent with previous reports (Gilliland et al., 1990; Bagnarelli et al., 1992; Cimarelli et al., 1994). The assay was optimized for use in pathogenesis studies of both cell lines as well as evaluation of proviral loads of HTLV-1-infected PBMC in vivo. First, equal amplification efficiency was shown for the wild-type and competitor templates using the HTLV-1 infectious clone ACH and our competitor construct StyID28. QcPCR was then carried out with known concentrations of both

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templates for calibration of equivalence points. These results illustrated the applicability of the assay to a plasmid system. However, genomic DNA amplifies differently than short plasmid templates, since the chances of mispriming are far greater due to the increased length of the template molecule and variety and amount of non-specific sequences. Therefore, the assay was applied to quantitate HTLV-1 proviral load in HUT-102 and MT-2.6 cell lines. As expected, it was demonstrated that equivalence points shifted according to the dilution ratio of MT-2.6 or HUT-102 DNA to Jurkat DNA. Moreover, the results showed consistently a higher proviral load in HUT-102 than in MT-2.6. Therefore, this assay offers an accurate and reliable means for the quantification of proviral load in different HTLV-I infected cell lines. In order to apply the assay to an in vivo model system, HTLV-1 proviral load was determined in PBMC derived from rabbits infected with the wild-type infectious clone of HTLV-1, ACH, and a mutant clone, ACH.p12, showing different proviral loads in wild-type infected animals and non-detectable levels of virus in ACH.p12 infected rabbits. These findings support recent results that demonstrate a reduction of HTLV-1 viral infectivity in vivo upon ablation of p12I (Collins et al., 1998) and suggest an essential role for this regulatory protein in HTLV-1 replication and pathogenesis. Whereas the percentage of infected PBMC in the ACH infected animals (0.3 – 2.5%) was consistent with values reported previously for asymptomatic HTLV-1 patients (Richardson et al., 1990; Kubota et al., 1993), ACH.p12 infected animals had no detectable virus at all. Furthermore, we have quantified HTLV-1 proviral loads in human PBMC derived from four diseased patients and an asymptomatic carrier. Two of these patients (1 [ATL] and 3 [TSP/HAM]) had comparable proviral loads whereas patients 2 (ATL) and 4 (TSP/HAM) differed 6-fold in proviral copies/cell (Fig. 7A). Previous reports have shown higher proviral copy numbers in TSP/HAM patients than in ATL patients (Renjifo et al., 1996). Several re-

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ports have quantified HTLV-1 proviral load in total leukocytes or PBMC of asymptomatic carriers or symptomatic patients by semiquantitative measures and have shown differences in proviral load between these two groups of up to 100-fold (Kira et al., 1991; Ono et al., 1995). Our data are consistent with this trend. Furthermore, proviral load was quantified in patient 4 (TSP/HAM) before 3TC treatment and 4 weeks after start of treatment. Proviral copy numbers decreased by 20% after treatment. More dramatic changes were not observed most likely because reverse transcriptase inhibitors are less effective in the treatment of HTLV-1 infections than in HIV infections. Viruses from the HTLV/ BLV group replicate mainly through host cell proliferation rather than through the cell free virus stage, which makes the role of reverse transcriptase in the replication of the HTLVs less critical and would explain the low efficacy of 3TC in the treatment of HTLV-1 infections. Thus, using the combination of the in vivo rabbit model system (Cockerell et al., 1990; Lairmore et al., 1992) and qcPCR, this assay will allow the accurate and reliable evaluation of the efficacy of potential HTLV-1 peptide vaccines and antiviral drugs. In summary, an accurate and consistent method was developed to quantitate the proviral load of HTLV-1 and STLV-1 using qcPCR. This method is applicable to a wide variety of studies of the pathogenesis of the viral infection or for evaluation of antiviral therapies to abate HTLV-1 associated disease.

Acknowledgements The authors would like to thank Yasuko Rikihisha for providing the Bio Photonics gel documentation system, Patrick L. Green for helpful discussion and critical reading of the manuscript, and Tim Vojt for preparation of the Figures. This work was supported in part by National Institute of Health grants NCI CA55185, NIAID AI40302 and NIAID AI01474.

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