Freie Universität Berlin, Medizinische Klinik III, Hindenburgdamm 30, 12200 Berlin, Germany1
Author for correspondence: Thomas BurmeisterFax +49 30 8445 4468. e-mail tbu{at}gmx.net
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Abstract |
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Introduction |
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Methods |
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DNA preparation.
In all samples mononuclear cells had been isolated by either Ficoll gradient centrifugation or red blood cell lysis. DNA was prepared from the cells using an alkaline lysis-based method with subsequent isopropanol precipitation (Puregene, Biozym Diagnostik). DNA was dissolved in TrisEDTA buffer at a photometrically determined concentration (GeneQuant II DNA/RNA calculator, Pharmacia) of approximately 100 ng/µl.
PCR.
PCR was performed in a Perkin Elmer 9600 thermocycler using the Expand 20kB PLUS system (Boehringer Mannheim) with a 25 µl reaction mix containing 400 nM primers and 500 µM deoxyribonucleotides as well as polymerase mix (containing a mixture of proofreading Pwo polymerase and Taq polymerase). Buffer conditions and mineral oil overlay followed the recommendations of the supplier. The reaction mix was always prepared as a minimally 5-fold master mix to compensate for pipette inaccuracies and was used immediately after preparation. Two-hundred ng of genomic sample DNA was added to each tube. A negative control (leukocyte DNA from two healthy individuals) and a positive control (virus-infected DNA, see below) were included in every PCR. Cycling conditions were as follows: modified hot-start technique (Chou et al., 1992 ) (putting the vials in the cycler at >80 °C), denaturation at 92 °C for 2 min, 15 cycles for 10 s at 92 °C, for 30 s annealing at the PCR-specific temperature (Table 2
), and for a product-specific extension time (Table 2
) at 68 °C, 20 additional cycles as described but with a 10 s increment per cycle in extension time, a 7 min final extension at 68 °C, and cooling to 4 °C. Aliquots of the PCR mixture were analysed on a 1% agarose gel and visualized under UV illumination after ethidium bromide staining.
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Sequence alignments.
Sequence alignments were done using Clustal X 1.6 (Thompson et al., 1997 ) on an Apple Macintosh PPC computer. All nucleotide sequences were obtained from the EMBL/GenBank/DDBJ database. The accession numbers are given in Table 3
.
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Results |
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Only complete viral genomic sequences (containing the entire viral sequence between the two LTRs plus at least one of the U3, R and U5 regions) were taken into consideration, since incomplete sequence fragments and partial clones could originate from viruses that are somehow defective or truncated and therefore replication-incompetent. Endogenous retroviruses (ERVs) were not included, even if complete in the sense of possessing gag, pol and env regions with flanking LTRs, because ERVs usually have non-functional genes due to premature stop codons, frame-shift mutations and defective splice sites. Thus, for effective replication, they may rely on help obtained from endogenous or exogenous retroviruses via trans-complementation. The inclusion of such defective or truncated sequences would have distorted the final alignment and complicated the characterization of conserved regions. The accession numbers of all sequences thus obtained are listed in Table 3. The following sections discuss each group of viruses separately and then summarize the results obtained with the constructed primers.
Alpharetroviruses
Two complete isolates of avian leukosis virus (ALV) could be retrieved from the database (Table 3). The alignment showed very high overall nucleotide sequence similarity (>95%) with only slight differences, especially in the regions encoding the env proteins. Thus no single highly conserved genome region for primer design was readily identifiable. One primer (W-tRNA) was designed to be complementary to the tRNA binding site of ALV (coding for tryptophan-tRNA), since the nucleotide sequence of tryptophan-tRNA in humans is nearly identical to that of those avian species under consideration. The second primer was constructed by additional alignments of the two avian isolates with proviral nucleotide sequences of the most closely related animal retroviruses from other groups, i.e. type B (MMTV) and simian type D viruses. Alignment with mammalian type C viruses discussed above yielded no genome regions of significant homology. The mixed ALVMMTVsimian type D alignment disclosed one sufficiently conserved pol region that was used to construct the second primer (primer POL-Ca) (Fig. 2a
). The PCR was tested and optimized with serial dilutions of ALV-infected avian DNA as a positive control.
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Gammaretroviruses
Type C retroviruses have been reported in a variety of mammals and birds. Three well-characterized exogenous retroviruses whose complete viral genomes have been published were included in this study: murine leukaemia virus (MuLV), feline leukaemia virus (FeLV) and gibbon ape leukaemia virus (GaLV). Reticuloendotheliosis viruses (REVs) cause infected birds to develop lymphoproliferative disorders in rare cases. REVs were not included in this study, because no complete isolate was available in the EMBL/GenBank/DDBJ database. Altogether twelve complete isolates of MuLV, two of FeLV and one of GaLV were retrieved from the EMBL/GenBank/DDBJ database (Table 3). Global alignment of the sequences showed several homologous regions in the gag and pol genes. Two regions turned out to be suitable for primer design: the binding site for proline-tRNA (primer P-tRNA) and a region from the 3' part of the pol gene (primer POL-Cm) (Fig. 2c
). The proline-tRNA binding site is universal and characteristic for all viruses of this group. Its integrity and conservation is necessary for efficient virus replication, since proline-tRNA is used as a primer for the synthesis of the complementary DNA strand during the virus replication cycle. The latter region harbours the catalytic centre of the integrase, and its high degree of conservation is thus understandable. PCR conditions were optimized using PA317-DNA and serial dilutions of pAMS as positive controls.
Deltaretroviruses
This is the only group of viruses under investigation here that includes members pathogenic to humans. HTLV-1 infection is endemic in parts of Japan, the Caribbean, South America and Central Africa. With 95% nucleotide sequence identity, STLV is the simian counterpart that causes T-cell neoplasms in a variety of Old World monkeys or apes (Gessain & de Thé, 1996 ). Bovine leukaemia virus (BLV) has a genetic organization similar to that of HTLV/STLV and causes lymphoproliferative disorders in cattle. Twenty-one nucleotide sequences of proviruses available in the EMBL/GenBank/DDBJ database were collected (Table 3
), and the entire sequences were aligned. The 14 human isolates included isolates from different parts of the world and different ethnic groups (see Table 3
). Five simian isolates and two complete isolates of BLV were included. The alignment showed several gag, pol and even env regions of moderate homology, but only two regions proved to be sufficiently conserved to allow the creation of highly stringent primer sequences with low degeneracy (Fig. 2
). One region is the primer binding site for proline-tRNA (Primer P-tRNA). The other highly conserved region is located in the 3' region of the viral protease gene (primer POL-3). This sequence TYYCCKTTAAACYDGARCGCCTCCAGGCCY corresponds to the site in the HTLV/STLV/BLV protease (prt) where ribosomal frameshifting (ribosomal slippage) can occur. This ribosomal frameshifting takes place in an estimated 5% of the cases and leads to translation of the complete viral gag/prt/pol precursor polyprotein. If no frameshifting occurs, the translation is terminated some nucleotides downstream at stop codons, resulting in a gag/prt precursor polyprotein. For correct synthesis of viral proteins, it is very important for ribosomal frameshifting to always occur at the same position, since too early an occurrence could prevent correct synthesis of the protease and polymerase. The high conservation level of this sequence motif is thus understandable. The PCR was tested and optimized with DNA from an HTLV-1-infected human cell line (MT-2) and with a plasmid containing the entire BLV provirus as a positive control (Fig. 3
).
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Discussion |
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Some previous investigators also used PCR-based methods in their search for retroviruses but the primers they used were either not generic enough to also include distantly related virus strains or had many mismatches in the primer binding regions or did not discriminate between sequences of endogenous and exogenous retroviral origin (Shih et al., 1989 ; Donehower et al., 1990
; Medstrand & Blomberg, 1993
; Li et al., 1996
; Dube et al., 1997
). For example, the universal primers for detecting retroviruses developed by Donehower et al. could not distinguish between sequences of exogenous and endogenous origin and relied on concentrated and well-purified retrovirus preparations. In addition, the stringency of the PCR conditions had to be quite low (annealing temperature of 37 °C for the first 10 cycles) because of the many possible nucleotide mismatches in the primer regions.
The detection method was based on DNA analysis. It could be argued that RNA-based analysis would circumvent some of the difficulties caused by endogenous retroviral elements, since most HERVs are not expressed on the RNA level. However, a large number of HERV mRNA transcripts have been observed in human cells, and especially those with close nucleotide sequence similarity to known exogenous viruses are most likely to be expressed, as exemplified by HERV-K (Tönjes et al., 1999 ). It must also be pointed out that the expression of retroviral proteins or RNA is not absolutely necessary for malignant transformation. Type B and type C viruses are known to act oncogenically by transactivation of genes near their integration site through promoters located in the viral LTRs. The U3 region of the type C LTR contains sequences for control and regulation of viral transcription with binding sites for several factors influencing tissue-specific expression and regulation of expression of both virus-encoded and cellular proteins (Fan, 1997
; Barat & Rassart, 1998
). Even in those cases where viral proteins are known to act as oncogenic transactivators, e.g. the HTLV-1 Tax protein, their expression may vary considerably during a cellular lifetime and can reach very low levels that are difficult to detect. Thus, DNA-based analysis appears to be more reliable and suitable for our purpose.
There has been speculation regarding a direct involvement of animal retroviruses in the causation of human malignant diseases (Johnson, 1994 ; Spiegelman et al., 1974
). Currently, there is little epidemiological or experimental evidence pointing to zoonotic viral causes of human malignancies but few investigations have been published on this topic. It should be noted that many animal retroviruses are capable of infecting human cells at least in vitro (Sommerfelt, 1999
). GaLV and FeLV subtype B use the same cell surface receptor (Takeuchi et al., 1992
), and this receptor is present on at least a subset of human bone marrow cells (Morgan et al., 1993
). The recently identified receptor for xenotropic MuLVs is present on human cells derived from various tissues (Levy, 1999
). JSRV also seems to be capable of infecting human cells (Rai et al., 2000
). The results of this study, however, rule out an involvement of known exogenous oncogenic animal retroviruses or related endogenous counterparts in the aetiopathology of the diseases investigated here. HTLV-1 is a special case because it is the only known oncogenic human retrovirus. Since its first description, many investigations have focussed on the possible role of this virus in other malignant T-cell disorders. This study confirms previous reports that showed no association of HTLV-1/-2 with any disease under investigation here.
In summary, the results of this study suggest that none of the human diseases investigated here are caused by a known oncogenic animal retrovirus or a related but hitherto undiscovered human retroviral counterpart. The PCR system developed here has proven useful and reliable in searching for human oncoretroviruses related to known animal ones, since it is both generic, i.e. based on conserved consensus sequence motifs, and specific, i.e. capable of discriminating between exogenous and human endogenous retroviruses. Though applied to human haematological diseases in this study, the system is in no way limited to that pathological spectrum and may be applied to any human disease suspected of retroviral involvement. Though not developed and optimized for this purpose, it could even be useful in the search for unknown exogenous or endogenous animal retroviruses.
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Acknowledgments |
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References |
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Received 2 January 2001;
accepted 13 April 2001.
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