Journal of Virological Methods 136 (2006) 83–92

Development of real-time PCRs for detection and quantitation of human MMTV-like (HML) sequences HML expression in human tissues
Shaman Muradrasoli 1 , Anna Forsman 1 , Lijuan Hu, Vidar Blikstad, Jonas Blomberg ∗
Section of Virology, Department of Medical Sciences, Uppsala University, Academic Hospital, 751 85 Uppsala, Sweden Received 18 November 2005; received in revised form 30 March 2006; accepted 4 April 2006 Available online 19 May 2006

Abstract The human genome contains around 1000 betaretrovirus-like copies, human mouse mammary tumour virus (MMTV)-like (HML) groups 1–10, also referred to as human endogenous retrovirus “HERV-K”. Despite many efforts, it is not established whether betaretroviruses, exo- or endogenous, are involved in the etiology of breast cancer, or other cancer diseases, in humans. Quantitative real-time PCR (QPCR) TaqMan® -based assays for HML groups 1–7, targeting the conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were designed. Plasmids containing the entire pol gene of HML1–7 were used as standards. The RT and IN based QPCRs could detect 100 –103 copies per PCR reaction of the plasmids. However, not all plasmids gave a signal in both RT and IN QPCRs, probably due to mismatches. Furthermore, RT and IN based HML6 specific QPCRs were developed. They were specific for amplification of transcripts for the whole HML6 group. The methods allow the monitoring in body fluids and tissues of expression of a wide range of betaretrovirus-like sequences. Betaretrovirus-like RNA was studied in normal human tissues and of HML6 in brains of multiple sclerosis (MS) patients. Brain, adrenal gland and testis had a high betaretrovirus-like expression. Multiple sclerosis plaques contained the same HML6 RNA concentration as control tissue. These assays are expected to enhance studies on involvement of betaretroviruses in physiology and disease. © 2006 Published by Elsevier B.V.
Keywords: HML; Endogenous retrovirus; Betaretrovirus; Real-time PCR; Expression; Transcription

1. Introduction Human endogenous retroviruses (HERVs) comprise up to 8% of the human genome. According to a recent bioinformatical analysis the human genome (version hg15, April 2003) contains 826 betaretrovirus-like copies (Blomberg J. et al., to be published). The betaretrovirus genus, as defined by the International Committee for Taxonomy of Viruses (ICTV) (Fauquet and Martelli, 1995) does not yet encompass endogenous retroviral sequences. The human betaretrovirus-like sequences were originally identified by their sequence similarity to mouse mammary tumour virus (MMTV), the reference virus for betaretroviruses. Some members of this group have intact open reading frames
Corresponding author. Tel.: +46 18 611 55 93; fax: +46 18 55 10 12. E-mail addresses: (J. Blomberg), (S. Muradrasoli). 1 Shaman Muradrasoli and Anna Forsman share the first authorship. 0166-0934/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.jviromet.2006.04.005

for the gag, pol or env genes and therefore are considered to be the HERVs with the greatest potential for biological activity. They are expressed in a tissue-specific way (Yin et al., 1997). Of particular interest is betaretrovirus-like expression in human breast cancer. The human betaretroviruses are classified into 10 human mouse mammary tumour virus-like (HML) groups (Andersson et al., 1999). One study, which used a semiquantitative technique, (Yin et al., 1997) found that all HML groups are expressed at lower levels in breast tissues than in placenta, and also that all HML pol genes are not more significantly expressed in malignant as compared with non-malignant breast tissues: However, expression of HML env gene transcripts was greater in human breast cancer than in non-malignant breast epithelial cells (Wang-Johanning et al., 2003). The role of these elements in breast cancer remains unclear. Attempts to associate HERVs with disease will be rather complicated, due to their ubiquity and great number. One potential way to establish an association is gene expression analysis in healthy

25 U Taq Gold polymerase (Applied Biosystems).loni. 56. while the more broadly targeted real-time PCR method allows quantitative detection of all or most HML sequences (in which HML6 is included). Total RNA was isolated using the Qiagen RNeasy Mini Kit according to the instructions of the manufacturer. The screening result showed that betaretrovirus-like HERVs are expressed to different degrees among tissues. Materials and methods 2. according to the instructions of the manufacturer. The recombinant plasmids were isolated using QIAprep® Spin Miniprep . The technique targets the pol gene of HML1–7. 59 ◦ C (HML7) for 30 s. HML3 Pol forward primer [HML3PolFw] 5 -AAGGTTTTGAGTGTCTTGATGG-3 .wi.5 ◦ C (HML2). Goteborg.. genome. Several reports have focused on a possible involvement of endogenous retroviruses in development of autoimmunity and multiple sclerosis (Perron et al. The primers were derived from the pol region of published HMLs and BAC www. 2.cgi) and analyzed by OLIGO Analyzer 2.5 ◦ C (HML6). HML5 (GenBank accession no. The observation that the MS predisposing haplotype DR2 contains an HML6 integration located close to the DRB1*1501 allele in the DRB6 gene complex motivated us to develop HML6-specific QPCRs to study the expression of HML6 in normal human tissues and in brain tissue samples of multiple sclerosis patients. All primers were purchased from Thermo Hybaid. its reverse primer [HML2PolRev] 5 -GCAGGCATAGGGAGACTTACC-3 . its reverse primer [HML6PolRev] 5 -TGGAAAGACAGAAAAAGATTG G-3 . HML4 (GenBank accession no. its reverse primer [HML5PolRev] 5 -CAAAGCGAGAGCATA AAA AGG-3 . Therefore. due to the high lipid content of brain tissue. 1993). A panel of RNA from normal tissues was screened to evaluate the method. AC092674) HML7 (GenBank accession no.1.43. Seifarth et al. HML2 Pol forward primer [HML2PolFw] 5 -ATAGCCCCA CGAGTCAA AA-3 .mit. DNA concentration was measured by the DyNA Quant fluorometer (Amersham Pharmacia Biotech. 57.2. UCLA.ucla. The technique also makes it possible to approximate the frequency of retroviral DNA sequences in the genome and to quantify RNA expression in normal and pathological tissues. HML6 Pol forward primer [HML6PolFw] 5 -CCAGTTATGGGGGTTGATGT-3 . its reverse primer [HML7PolRev] 5 -TGCCAGAGGAGGATGTGTG-3 . / Journal of Virological Methods 136 (2006) 83–92 and diseased tissue with quantitative real-time PCR (QPCR)..32.. 2003. Expression of the HML6 group in normal human tissues and in brain of multiple sclerosis (MS) patients was studied. Shih et al.2 ◦ C (HML5). which were healthy prior to death. AF020092). 56 ◦ C (HML3). HML7 Pol forward primer [HML7PolFw] 5 -CAGGAGGAG ACAGAGGTTGAG-3 . AC004536).. its reverse primer [HML3PolRev] 5 -CAATGGATGAAATGGGGTATG-3 . stained with ethidium bromide at 100 V for 1. Hilden. 1997) (Christensen et al. from the Human Brain and Spinal Fluid Resource center (c/o professor Wallace Tourtellotte. 600 nM forward primer. The pol gene is the most conserved sequence among retroviruses (Medstrand and Blomberg. HML3 (GenBank accession no. HML4 Pol forward primer [HML4PolFw] 5 -GTAGGTGGGGTTGAACGAGA-3 . following the manufacturer’s instructions. followed by a final elongation at 72 ◦ C for 20 min. Uppsala. 1989. as they were purchased. Amplification and cloning of Betaretrovirus-like pol sequences used as PCR standards Betaretrovirus-like pol genes (HML1–7) were amplified from human DNA with primers designed using primer3 (www. 5–10 ␮l of PCR-product was separated by electrophoresis on 1% agarose gel in 0. Isolation of total RNA from human brain tissue Human brain tissue samples were obtained from four patients with MS and from four individuals. Sweden).5 h and visualized by UV irradiation.2. Samples were from normal appearing white matter (NAWM).. The PCR reaction was carried out in an total volume of 20 ␮l containing 100 ␮M dNTP. 30 mg of tissue were disrupted in buffer containing guanidine isothiocyanate and homogenized using the Ultra-Turrax T18 with disposable dispersion tools (G¨ oteborgs termometerfabrik. 2005. normal appearing grey matter (NAGM) and for MS patients also from multiple sclerosis plaques. HML5 Pol forward primer [HML5PolFw] 5 -ATGGGAGGTCATTCTGTGGA-3 . annealing at 61 ◦ C (HML1). 1997). AC002464). Following amplification.5 (207. The band of expected size was excised from the agarose gel and DNA was extracted by QIAquick Gel Extraction Kit. Germany). Interactiva Division (Ulm. 600 nM reverse primer. Seifarth et al. 2.1. The amplified pol genes of HML1–7 were cloned into TOPO® TA vectors and transformed into competent Escherichia coli cells (TOPO 10) using the TOPOTM TA Cloning Kit (Invitrogen. AC003100). its reverse primer [HML1PolRev] 5 -GGGCTGTTAATGCTGTGACTC-3 . 1 mM MgCl2 and 1. The HML primers are 100% identical to the target sequences of the following published HMLs and BAC clones: HML1 (GenBank accession no.. Stockholm. The amplification reaction was subjected to one cycle of denaturation and activation of polymerase at 95 ◦ C for 10 min and 30 cycles of denaturation at 95 ◦ C for 45 s. 59. Tuke et al. Sweden). 2.84 S.5 ◦ C (HML4). broadly targeted real-time PCR methods that allow quantitative detection of HML sequences were developed. Isolation of genomic DNA DNA was extracted from blood-cells using the QIAamp DNA blood Mini Kit (Qiagen. its reverse primer [HML4PolRev] 5 -CTATTCGCCCCCTCATTA CA-3 . All reverse primers are written as the inverse complement. The HML6-specific QPCR detects transcripts from the whole HML6 group. Muradrasoli et al. HML6 (GenBank accession no. Twice the recommended volume of lysis buffer was used. AF074086). Germany).. 56. Primer sequences were as follows: HML1 Pol forward primer [HML1PolFw] 5 -GCCCTTTCCACTATTGCTTG-3 . HML2 (GenBank accession no.html). 5× TBE buffer (45 mM Tris-borate 1 mM EDTA. pH 8). Degenerate primers were used to enable amplification of many betaretrovirus-like sequences (Medstrand et al. 2002). and elongation at 72 ◦ C for 5 min. http://www. Sweden). AC064878).248).

AH013888). Hercules. Master Mix without UNG were used due to the low annealing www. and its probe [HML6RTProbe] 5 -ATWYGCCTTCTCTGTGCCTTCTRTTAATCAAAG-3 .mit. The program uses codon statistics. Serial dilutions of plasmids containing 106 –100 copies per PCR reaction were used as quantitative standards in the QPCRs that detect betaretroviruslike sequences and determination of sensitivity of the PCR. cDNA-synthesis was performed on an Cycler iQTM PCR-cycler (Bio-Rad Laboratories. 2.5. AH013885). Oligonucleotides synthesized were. CA). The TaqMan® probes were purchased from MWG Biotech.O. 50 U of reverse transcriptase (Stratascript RT. at 25 ◦ C for 10 min. Germany). with results accrued and completed by hand. Australia) with a total reaction volume of 50 ␮l containing 25 ␮l 2× TaqMan® Universal PCR Master Mix without UNG.O. Stratagene) and 2 ␮l cDNA. cDNA synthesis from human RNA cDNA was synthesized in 50 ␮l reactions with 2 ␮g of RNA from 20 human tissues (Human Total RNA Master Panel II. Madison. and Blomberg J. Foster City. Primer3. and a list of repetitive elements from Repbase. and processed according to the manufacturer’s instructions. Fig.3.5 s. AH013887). (Sperber G.0 Ready Reaction kit (Perkin Elmer) and on an ABI PRISM® 310 genetic analyzer according to the manufacturer’s recommendations (Applied Biosystems. Development of TaqMan PCRs targeting reverse transcriptase (RT).cgi and Net Primer (http://www. www. When designing the primers. annealing at 40 ◦ C for 1 min and elongation at 60 ◦ C for 20 s. The alignments were made using Clustal W and RetroTector© -derived pol sequences.genome. Both HML6 PCRs were carried out in 50 ␮l-reactions containing 1× TaqMan Universal PCR Master Mix (Applied Biosystems). Degenerate TaqMan® probes and primers were designed for the RT. 10 mM DTT (Promega). ABI PRISM® Big DyeTM Terminator Cycle Sequencing v2. its reverse primer [␤INRev] 5 -ACATGAACATATSTWARYYTNCCAAA-3 . HML4 (GenBank accession No. All primers were purchased from Thermo Hybaid. 500 nM of primers and 200 nM of each probe. its reverse primer [␤RTRev] 5 -CCTACAAAADKCTGACAWAWWGTDGG-3 .wi. care was taken to minimize the degree of primer self-complementarity. these programs were used to a certain degree. and Blomberg J. HML2 (GenBank accession No. HML5 (GenBank accession No. the HML6 IN forward primer [HML6INFw] 5 -ACCACCTYARWYAAAAYTACABTTA-3 . To avoid false positive results due to DNA or RNA contamination filtered pipette to be published). The HML6 clone sequencing was not successful. HML7 (GenBank accession No. and its probe [␤INProbe] 5 -HTHTGGCAAATGGATGTTACACATATWCC-3 .1 ␮g of RNA. 530 ng random hexamers (Pharmacia). a retroviral sequence detection software. Oligo Analyzer 2. the beta-IN forward primer [␤INFw] 5 -TAATCCHMGAGGTYTKKVHCC-3 . 207. the HML6 RT forward primer [HML6RTFw] 5 -TGGMCTCTTGTADBAATAGRTCTTA-3 . frequency of stop codons and alignment to known Betaretrovirus proteins to approximate the original ORF. Briefly. its reverse primer [HML6INRev] 5 -CTTTBCCTCTAACABTTGCCAVT-3 . 1(a–c). Interactiva Division (Ulm. As the mentioned software’s are not suitable for design of degenerate primers and probes. its reverse primer [HML6RTRev] 5 -CTTGRGSYAAAACTYTCCAYTG-3 . 2. 800 ␮M dNTPs (Applied Biosystems). Mortlake. The sequences were reported to GenBank: HML1 (GenBank accession No.premierbiosoft. although a certain degree of self-complementarity is difficult to avoid due to the degeneracy of the primers. AH013884).0. RetroTector© recognizes consensus motifs and constructs putative HERV proteins (“puteins”) from the different reading frames in the gene candidates. 37 ◦ C for 90 min and 70 ◦ C for 15 min in 50 ␮l reactions containing 1× Stratascript buffer (Stratagene).S. The TaqMan PCRs were carried out in a Corbett Research Rotor-Gene 2000 Real-time ThermoCycler (Corbett Research. The majority of the Alu sequences were removed prior to the analysis. and its probe [HML6INProbe] 5 -AYTCC- TGGTAYRGATGGTAAGACTCYAGCAGAA-3 . / Journal of Virological Methods 136 (2006) 83–92 85 Kit (Qiagen). This software was used to screen the human genome for all betaretrovirus-like elements. Clontech). All QPCRs were run both with cDNA and mock cDNA in parallel.. AH013886).248/.43. They were labeled with the fluorescent reporter dye 6-FAM (6-carboxyfluorescein) at the 5 -end and the quenching group TAMRA at the 3 -end position. HML3 (GenBank accession No. Several primer design programs were used: OLIGO 6. The number of cDNA copies detected in the PCR thus derived from approximately 0. 2. .. RNasin (160 U. and amplification profile was as follows: one cycle of initial denaturation and Taq polymerase activation at 95 ◦ C for 10 min and 70 cycles of denaturation at 95 ◦ C for 15 s. to be published). using similarity to Pol proteins. USA). (Applied Biosystems). Description of the RetroTector© retroviral sequence detection software RetroTector© is a program newly developed for detection of endogenous retroviruses and similar structures in genomes (Sperber G. The RetroTector© interpretations were then automatically matched with sequences in an annotated list of retroviral sequences from Genbank. and its TaqMan® probe [␤RTProbe] 5 TWTCAGTGGAAAGTKTTACCTCARGGAATGCTTAATAG-3 . The concentration of plasmid DNA was quantified by using the DyNA QuantTM 200 fluorometer. AH013889). All the inserts were further verified by sequencing using the fluorescent dye terminator method. Muradrasoli et al.32.4. integrase (IN) Sequences with structures corresponding to complete human betaretrovirus-like pol genes with a minimum of stops and frame shifts were identified using RetroTector© . 40 ◦ C which is the temperature-interval when UNG is most active. PCR hoods with ultraviolet light and separate rooms for PCR preparation and product analysis were used. CA. WI).and IN region of betaretrovirus-like pol sequences. the beta-RT forward primer [␤RTFw] 5 -ATTTGCCTTTACTRTDCCWKCHRTHAA-3 . Promega.

Note that probe and reverse primer areas overlap by two nucleotides. 1. (c) Alignment at the nucleotide level of the target portion of the integrase region of the pol gene for different human ␤-retroviral sequences. Alignment of target sequences. (b) Alignment at the nucleotide level of the target portion of the reverse transcriptase region of the pol gene for different human ␤-retroviral sequences. Nucleotides identical to the nucleotides of the master sequence (“Chr7 4384369” on top) are shown as a period (. / Journal of Virological Methods 136 (2006) 83–92 Fig.). . Nucleotides identical to the nucleotides of the master sequence (on top) are shown as a period (.86 S. Forward primer positions are indicated in grey. Nucleotides identical to the nucleotides of the master sequence (on top) are shown as a period (. Reverse primer and probe positions are indicated in grey and black. primers and probe positions are indicated in grey and black.). Muradrasoli et al. (a) Alignment at the nucleotide level of the target portion of the reverse transcriptase region of the pol gene for different human ␤-retroviral sequences.).

S. for each group (normal white matter.1. 1. and for MS patients also from MS plaques). The ratios are calculated by dividing the HML expression level (copies/␮l) in human tissues by the expression level of histone 3. Serial dilutions of Histone 3. its reverse [GAPDRev] 5 GGCATGGACTGTGGTCATGAG-3 . GAPD forward [GAPDFw] 5 TGCACCACCAATCGCTTAGC-3 . 5 ␮l 2× SYBR Green Master Mix buffer (Applied Biosystems) and 300 nM forward and reverse primers. normal grey matter. 3. (Continued ). and annealing and extension temperatures.3 (copies/␮l) in same tissue. / Journal of Virological Methods 136 (2006) 83–92 87 Fig.3 QPCR and GAPD. HPRT1 [HPRT1Fw] 5 TGACACTGGCAAAACAATGCA-3 . Reactions were run on a Corbett Research Rotor-Gene 2000 Real-time ThermoCycler (Corbett Research. HERV expression levels where normalized to the expression of histone 3. and because of an unknown degree of fit between the primer/probe combination and their target sequences. and it probe [HisProbe]: 5 -CTCTGGAAGCGCAGATCTGTTTTAAAGTCCTG-3 . Results 3. its reverse [HPRT1Rev] 5 -GGTCCTTTTCACCAGCAAGCT-3 .6. using similarity to the entire nucleotide sequence of the elements. Australia). Optimization and sensitivity of broadly targeted TaqMan® PCRs Optimizations included primer and probe concentrations. The thermal conditions comprised 10 min polymerase activation at 95 ◦ C followed by 40 cycles at 95 ◦ C for 15 s and 60 ◦ C for 60 s.3 plasmids containing 106 –100 copies per PCR reaction were used in the experiment as quantitative standards.. and UBC forward [UBCFw] 5 -ATTTGGGTCGCGGTTCTTG-3 . The primers were.D. The optimized primer/probe concentrations were as follows: ␤RTFw and . Mortlake. In the HPRT1 and UBC SYBR® Green PCRs A549 cell line cDNA were used as standards. Muradrasoli et al. 12. Amplification mixtures (25 ␮l) contained 25 ng template cDNA. both because of an unknown yield of cDNA from the RNA. 2002) were used. The term “RNA equivalents” denotes the copy number of a cDNA interpolated in a plasmid DNA standard curve. data are expressed as the average ± S. 104 –100 copies/PCR reaction. Such a copy number will deviate to some degree from the real copy number. resulting in varying degrees of amplification efficiency. its reverse [UBCRev] 5 -TGCCTTGACATTCTCGATGGT-3 . its reverse [HisRev] 5 -TGCCTCCTGCAAAGCACCGATA3. Histone forward [HisFw] 5 -CCTCTACTGGAGGGGTGAAGAA3 .3. For HML6 expression in the brain samples. HPRT1 and UBC SYBR Green QPCRs (Vandesompele et al. Housekeeping genes as controls As reference a real-time histone 3. 2.

7 0. The thermal conditions for all TaqMan® QPCRs were optimized to be the same. ␤INFw 1200 nM. and later against seven HML plasmid clones. and the number of mismatches (a) Number of mismatches with ␤RTFw/␤RTRev primers in combination with ␤RTProbe Number of mismatches between the HML2 variant of primer or probe with target sequence ␤RTProbe (38 b.87 22. The sensitivity of the method was increased considerably by optimization.7 – 24.53 – 27.71 0. which makes it possible to run all general beta PCRs at the same time. degeneracy 432) 3 0 1 0 3 2–3 1 ␤INRev (26 b. one targeting the reverse transcriptase domain and the other one targeting the integrase domain.31 – – 2 3 1 4 4–5 3–5 2 (−) No amplification. Table 2 Degenerated primers and probe. / Journal of Virological Methods 136 (2006) 83–92 Table 1 Sensitivity and detection limits of betaretrovirus-like PCRs with plasmids containing betaretrovirus-like pol genes Detection limit (number of HML plasmid DNA copies) ␤RT PCR HML1 HML2 HML3 HML4 HML5 HML6 HML7 a Amplification efficiencya – 0. It shows the result of a sensitivity test with ␤INFw/␤INRev in combination with the ␤INProbe. degeneracy 128) 4 1–2 3 6 1–3 2–5 4 24. The HML7 clone did not give a signal with the ␤IN QPCR. Muradrasoli et al.7 0.70 – 0. The number of degenerations in primers and probes required the low annealing temperature of 40 ◦ C instead of the usual 60 ◦ C.and ␤IN primer/probe systems were taken together. HML3 and HML5 clones gave no signal in the ␤RT QPCR.94 26. as shown in Table 2(a and b).88 S.76 0. Plasmid DNA with cloned pol gene was used as a standard to determine the sensitivity and dynamic range of the assay.85 – 0. The optimized amplification profile was as follows: one cycle of initial denaturation and Taq polymerase activation at 95 ◦ C for 10 min and 70 cycles of denaturation at 95 ◦ C for 15 s. ␤RTRev and ␤INRev 1200 nM and ␤RTProbe and ␤INProbe 400 nM probe.47 – – 100 – 1 – 10 10 (−) No amplification.27 28.8 36. annealing at 40 ◦ C for 1 min and elongation at 60 ◦ C for 20 s. A representative example of the many PCR reactions that were done during the project is shown in Fig.74 Detection limit (number of HML plasmid DNA copies) ␤IN PCR 100 10 1 1000 1000 1 – Amplification efficiencya 0.7 Ct (106 HML plasmid DNA copies)a (b) Number of mismatches with ␤INFw/␤INRev primers in combination with ␤INProbe Number of mismatches between the HML2 variant of primer or probe with target sequence ␤INProbe (29 b. degeneracy 432) 2–4 0–(3) 3–5 4 2–4 2–3 2–3 ␤RTRev (26 b.53 32. Amplification of 10-fold serial dilutions of the plasmid standard ranging from 100 to 106 copies per reaction was carried out in duplicates. degeneracy 8) HML1 HML2 HML3 HML4 HML5 HML6 HML7 7 0 8–9 7 7–8 4–7 4 ␤RTFw (27 b. degeneracy 144) 7 0 5 2 7 1–2 1 – 27. To determine the sensitivity of the betaretrovirus-like TaqMan® assays the primers and probes were tested against human DNA. A linear correlation between Ct values and the log10 of the initial concentration of the plasmid DNA quantities was observed in the range 101 –106 copies per reaction. . using 106 –101 copies of the HML2 clone as standard.81 0. 10 plasmid copies per reaction for each of the HML1–7 clones were detected (Table 1). When results of parallel analyses with ␤RT. 2. The sensitivity did not obviously depend on the number of mismatches. degeneracy 18) HML1 HML2 HML3 HML4 HML5 HML6 HML7 a Ct (106 HML plasmid DNA copies)a ␤INFw (21 b.7 0.

which nominally contained a constant amount of RNA.3 signal were 627-. Thus. with all HERV QPCRs and all housekeeping gene QPCRs. 521-.7 × 10−3 fold and 5. 446-. but with similar proportions as with ␤RT (Fig. 74-. No signals were obtained in the negative control. 103-. HPRT1 and UBC). To investigate the expression of HML6 in multiple sclerosis. the HML6 RT and IN methods were used with four MS patient and four non-MS control samples. from left to right. and 63-fold.9 × 10−3 fold. The HML6 RT and IN methods detected a preferential expression in placenta. To compensate for variable recovery of detectable RNA. The average ratios of the HML6 RT and IN signals to that of the histone 3.0 × 10−3 ± 1. respectively.7 × 10−3 ± 2. expression ratios was chosen to be normalized to histone 3.3 × 10−3 -fold for non-affected white matter (NAWM). (HIS. For non-affected gray matter (NAGM) they were 4. The ratios of expression of betaretrovirus-like RNA. Results with the four housekeeping genes were to some degree comparable. kidney. The mock cDNA signal was 0 or less than 1% of the cDNA signal. 4. testis. the expression of four housekeeping genes was assessed in parallel with the RNA containing betaretroviral reverse transcriptase and integrase domains in a RNA panel of human tissues. Muradrasoli et al. 66-.5 × 10−3 ± 2. 196-. Using the ␤IN system. respectively. 2. 103 .7 × 10−3 -fold and 5. cerebellum and fetal liver to the histone 3. 4(a–c)). gave very low signals. 3.S. 436-. lung and to a lesser degree also skeletal muscle and trachea. respectively. Info). heart. the same ratios were 144-. and 383-fold. 3(A)). As a control a panel of RNA from twenty human tissues and body fluid was used in the broadly targeted TaqMan® PCRs. (B) Standard curve describing sensitivity tests of primers ␤INFw/␤INRev in combination with ␤INProbe. Info. They were excluded from the presentation in Fig. / Journal of Virological Methods 136 (2006) 83–92 89 Fig. adrenal glands. The curves represent. 5.3 signals were most even among the samples of the RNA panel. Fig.4 × 10−3 ± 1.9 × 10−3 -fold. However. Further data are shown in Supplemental information. testis and brain (Fig. respectively. (A) Amplification plot describing sensitivity tests of primers ␤INFw/␤INRev in combination with ␤INProbe. 523-. in whole brain. The heart and lung results were judged to be severely compromised by RNA loss and therefore not reliable. using the ␤RT system. 105 .3 × 10−3 -fold. 102 . Betaretrovirus-like RNA expression in human tissues The QPCR assays were able to detect and quantify betaretrovirus-like RNA expression in human tissues.2.2 × 10−3 ± 1. indicating that HML6 expression is a minor portion of the betaretrovirus-like expression in brain. For MS plaque they were 4.2 × 10−3 ± 0. Template concentration is calculated plasmid copy number. The result from this panel showed betaretrovirus-like expression in a tissuespecific manner (Suppl. significantly lower.3 QPCR (Fig. However. 6 Suppl. GAPD. However. 3(A and B)) because histone 3. 101 copies of plasmid containing HML2 pol. Threshold cycle is plotted against template starting concentration. 3. there was no . 3(B)) (Fig. respectively. 106 . the number of RNA equivalents was considerably lower that those obtained with the ␤RT and ␤IN QPCRs.3 signal were 3. and was therefore not shown in these figures. Expression levels where normalized to the expression of selected control genes. 104 . Fig.

. 1999. 1999). 2004. Testis and some skin samples were the only normal tissues that expressed HERV-K(MEL. rec and Np9. 4. It is reasonable to expect retainment of some basic retroviral properties in those enzymatic proteins. a higher degree of expression does not necessarily mean etiological involvement. Muster et al. 1999.. HERV-K-MEL. (A) Expression of HML sequences in various human tissues by betaretrovirus-like real-time PCR broadly targeting the reverse transcriptase and integrase region. In a recent study.. 1997) (Blikstad et al. Mayer et al. (B) HML6 expression pattern in various human tissues. 1999. Muster et al. appreciable difference in HML6 expression level between the three groups. In addition to the structural proteins.HML6) according to the proposed nomenclature (Andersson et al. some of the HML groups may produce regulatory proteins. unpublished).. 3. or HERV-K(MEL. Results are summarized in Fig. 2003. Magin et al.. / Journal of Virological Methods 136 (2006) 83–92 Fig. Rec transcripts are generated from the env reading frame by alternative splicing. Cancers often lose some of their methylation control. Buscher et al.. Discussion The HML1–10 sequences include a few elements with open reading frames for the structural and enzymatic proteins Gag. However. Betaretrovirus-like sequences and particles were expressed in melanoma cells but not in melanocytes and normal lymph nodes (Muster et al.90 S... 2003). and may become a useful tumour marker.. using HML6RT and HML6IN specific primers and probe... Wang-Johanning et al. Evidence is emerging that this protein has a function in tumorigenesis (Armbruester et al.HML6) at a high level.. The protein is a functional homologue of the HIV-1 Rev protein (Lower et al. 2003. 1995.. Yin et al. 2005. Pro. using ␤RT and ␤IN system. the expression of the envelope gene of HERV-K in breast cancer was higher in human breast cancer than in control tissue (Buscher et al. 2003). testicular carcinoma and malignant melanoma (Wang-Johanning et al.3 (copies/␮l) in the same tissue. Muradrasoli et al.. A provirus encoding a tumor antigen recognized on melanoma cells by cytotoxic T lymphocytes was recently identified.. 2005. 2005). Pol and Env (Andersson et al. Higher expression of some HML sequences than in controls have been observed in breast cancer. 5(a and b) in Supplementary information. 1999). The ratios are calculated by dividing the HML RNA expression level (equivalents/2 ␮g RNA) in human tissues by the expression level of histone 3. Yang et al. which may . 2003). Galli et al...

The ␤RT and ␤IN systems gave similar results in all tissues. In a digital expression pattern study using ESTs.. the methods are quantitative.. Transcripts from some loci may therefore fit better to one of the two systems. cerebellum and fetal liver. skin and pancreas. and from MS plaques. To evaluate the QPCR assay. Yin et al. skin and brain (Stauffer et al. 1997. Several pathogenic mechanisms are possible. DQB1*0602. In a recent study (Stauffer et al.. Herbst et al. The probability of this result. 2005). like in the present study. 1999). Medstrand and Blomberg.. the fit of the target sequences to primers and probe differs. submitted for publication). protein expression encoded by the DRB6/HML6 complex on a cell surface has been demonstrated (Fernandez-Soria et al.. testis. / Journal of Virological Methods 136 (2006) 83–92 91 release expression of retrotransposons. In principle.. Yin et al. (Andersson et al. cf. breast. 2004). Conclusion The presented broadly targeted QPCRs should be useful for investigating the pathophysiological contribution(s) of human endogenous betaretrovirus-like sequences.3 expression did not show a significant difference in expression between the three types of brain tissue. 1998). 2001). The average ratios of the HML6 RT and IN expression to that of the histone 3. kidney. Christensen et al. The HERV RNA was both detected as such. The HML6 expression level did not differ between samples from MS patients and healthy controls. Thus.. 2004. 1996. using expressed sequence tags (ESTs). normal grey matter.. The assays were based on plasmids with inserts containing corresponding pol gene sequences. Muradrasoli et al. adrenal glands... Palmarini et al. the Swedish Society against Cancer (contract 4239-B00-02XBB). unpublished) indicated that the DRB6 gene and the HML6 provirus is in linkage disequilibrium with the MS predisposing haplotype HLA-DRB1*1501. Another study confirmed that. to validate previous observations.. 2003. 1998. thus disfavouring the HML6MS etiological connection.. This leads to different amplification efficiencies. 1996. which serve as quantification standards and demonstrate the broadness of detection. Medstrand et al... HERV-K elements belonging to several HML groups are active in human cells in a wide-spread but tissue-specific manner. 2004).S. They allow detection of a few molecules and are consequently more sensitive than previous broadly detecting methods (Shih et al. there are several reasons to quantitate human betaretrovirus-like RNA. or as particle associated RNA. 2001). placenta and uterus. 1992.. The expression pattern of HML sequences was tissue-specific. which obviates exact quantification of all target sequences. 1989). the expression of human betaretrovirus-like (HML) nucleotide sequences was analysed in tissues from healthy individuals. Acknowledgements We are grateful for the support from the Stanley Foundation (contract 02R-376).. amplifying from all betaretrovirus-like pol plasmids. Null hypothesis. Low expression levels were detected in skeletal muscle. HML6 expression was detected by QPCR of RNA from human brain tissue samples from four patients with MS and four apparently healthy individuals. by using a qualitative evaluation of chip hybridisation signals and quantitative analysis by real-time RT-PCR (Seifarth et al. 5. or from their transcripts. A recent study (Blikstad et al. Interestingly. Analysis of variance between the three groups were made by a one-way ANOVA. assuming the null hypothesis. High expression levels were detected in the following normal tissues: brain. The MS predisposing haplotype DR2 contains an HML6 integration located close to the DRB1*1501 allele in the DRB6 gene complex that generates a new promoter region and a new exon 1 in the truncated DRB6 pseudogene.. is 0.. thymus. Samples were from normal white matter. it is not yet known if HERVs represent causal factors. A higher level of expression was detected in whole brain. Sometimes RT. 1999)) were investigated using digital expression profiling. Wang-Johanning et al. muscle.77 for HML6 RT expression and 0. 1997. the MICMAN project of the Finnish . This may or may not be an epiphenomenon during tumour progression. Herbst et al. 1997).. it often is synonymous with HERV-K(HML2).. HERV-H and HERV-W have been associated with multiple sclerosis (Christensen et al.. One cannot expect amplification from all loci.82 for HML6 IN. The reason for setting up HML6specific QPCRs was the possible involvement of HML6 in MS-pathology. Some of these observations have been contested (Uzhameckis et al. This result is in concordance with previous findings in which it was described that the transcriptional activity of HML-2 elements was enhanced in breast cancer and germ line tumours compared to corresponding normal tissues (Herbst et al. and associates to approximately 60% of the MS patients versus 30% in the control group.. Together they may give a relatively unbiased estimate of betaretroviral expression in a tissue. WangJohanning et al. A similar pattern of tissue-specific expression was seen in previous work with a semiquantitative method (Andersson et al. the expression levels in testis. Human betaretroviruses are expressed to different degrees among individuals and cell lines (Yin et al. The inability to amplify some of the HML pol clones illustrates the difficulty when mutated HERV sequences are studied.. It did not support a difference of HML6 RNA expression between MS plaques and non-plaque tissue. and to explore a possible involvement of a betaretrovirus-like HERV in human carcinogenesis (Blomberg et al. HERV-K(HML2) expression has been reported in multiple cancer tissues as well as normal tissues such as muscle. sometimes IN targets will be damaged. the Swedish Scientific Council (contract 521-2001-6520). 1993. However. 2002. The differences probably are attributable to random substitutions or deletions in the primer or probe target sequences. as seen in the alignments and discussed above. The pol gene was selected since it is the most highly conserved retroviral gene. Hu et al. Moreover. submitted for publication. 2004) the tissue expression patterns of the HERV-K group (this is an inexact term. This situation occurred in HERV-H sequences (Jern et al. In any way.. 1997). The ␤RT and ␤IN system together had a high sensitivity and breadth. H0 : meanNAWM = meanNAGM = meanPLAQUE . head and neck cancerous tissues were higher than in the respective normal tissues.

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