POWIC, Department of Psychiatry, Warneford Hospital, University of Oxford, Oxford OX3 7JX, UK1
Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama, Aichi 484, Japan 2
Author for correspondence: Timothy J. Crow.Fax +44 1865 244990. e-mail tim.crow{at}psychiatry.oxford.ac.uk
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Abstract |
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Introduction |
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Pathogenic influences of retroviruses may occur as a result of exogenous infection, as in the case of human immunodeficiency virus, but may also occur from activation of endogenous elements as in the case of mouse mammary tumour virus and murine leukaemia virus. Such models in the mouse raise the possibility that endogenous sequences play a role in human disease. A series of different types of HERV has been described (Martin et al., 1981 ; Mager & Henthorn, 1984
; Steele et al., 1986
) and classified as HERV-H, HERV-R, ERV-9 and HERV-K on the basis of their homologies (Löwer et al., 1996
). In general, there are homologues of these viruses in non-human primates and each class is likely to have a different evolutionary origin (Steinhuber et al., 1995
; Zs íros et al., 1998
). In the great majority of cases, the sequences are defective, although in a number of instances expression has been detected, e.g. HERV-E in placenta and various tumour cell lines, HERV- H, HERV-K and ERV-9 in teratocarcinoma cell lines and HERV-K in testicular tumours and at low levels in placenta and some normal tissues (for review see Löwer et al. , 1996
).
A retrovirus superantigen has been suggested as a candidate autoimmune gene in type I diabetes (Conrad et al., 1997 ; although see Lan et al., 1998
; L öwer et al., 1998
; Murphy et al., 1998
), and a general role for endogenous viral proteins in the aetiology of autoimmune disease has been discussed (Perron & Seigneurin, 1999
). Retroviral particles have been recovered from monocyte cultures from patients with multiple sclerosis (Perron et al., 1997
) and virion-associated RNA of multiple sclerosis-associated retrovirus (MSRV) has been reported in serum of patients with the disease (Garson et al., 1998
). Expression of MSRV sequences in normal placenta allowed the reconstruction of a 7·6 kb putative genomic retroviral RNA with RU5gagpolenvU3R organization, with a polypurine- binding site (PBS) showing similarity with avian retrovirus PBS used by tRNATrp (Blond et al., 1999
). This new family of endogenous retrovirus sequences has been named HERV-W. The gag and pol open reading frames (ORFs) were interrupted by frame-shifts and stop codons, whereas a complete ORF encoding an envelope was found. Homologies within the pol and env genes with murine type C and simian type D retroviruses, respectively, suggest a chimeric genome structure. In terms of its phylogenetic relationships, the HERV-W family is considered to be related to the ERV-9 and RTLV-H families and to belong to endogenous retrovirus class 1 (Boeke & Stoye, 1997
). From the sequence divergence of 3' and 5' LTRs, an integration event has been estimated at approximately 6 million years ago (Blond et al., 1999
).
The HERV-W family may have relevance to the MSRV particles associated with multiple sclerosis, with which the genomic mRNA sequences so far described share 8288% identity (Blond et al. , 1999 ). The possibility that they have relevance to other neuropsychiatric conditions is raised by the finding of sequences homologous to HERV-W in a representational difference analysis of DNA from monozygotic twins discordant for schizophrenia (Deb et al. , 1998
), for which an endogenous retrovirus aetiology has previously been proposed (Crow, 1984
). Our objective is to identify endogenous virus sequences that have relevance to psychotic illness (including schizophrenia). We hypothesize that, if such sequences have a pathogenic role, they will have been subject to change in recent primate evolution and will be associated with variations between individuals and with gene expression. Here, we report an investigation of endogenous retrovirus W family (ERV-W) from hominoids, Old World monkeys and New World monkeys.
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Methods |
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Cloning of PCR products.
PCR products were separated on a 1·8% agarose gel, purified with the QIAEX II gel extraction kit (Qiagen) and cloned into the T- khs307 vector (Kim et al., 1998 ). The cloned DNA was isolated by the alkaline lysis method by using the High Pure plasmid isolation kit (Boehringer Mannheim).
DNA sequencing and data analyses.
Individual plasmid DNAs were screened for inserts by PCR. Positive samples were subjected to sequence analyses on both strands with T7 and M13 reverse primers by using an automated DNA sequencer (model 373A) and the Dye Deoxy terminator kit (Applied Biosystems). DNA and deduced amino acid sequence analyses were performed by using the GAP, TRANSLATE, PILEUP and PRETTY programs from the GCG software (Genetics Computer Group, University of Wisconsin). The extents of synonymous and non-synonymous divergence were calculated by the method of Li et al. (1985) . A correction for superimposed substitutions at single sites was made by the two-parameter method of Kimura (1980)
. The neighbour-joining phylogenetic analysis (Saitou & Nei, 1987
) was performed with the MEGA program (Kumar et al., 1993
). Statistical significance evaluation of the branching pattern was performed with 100 replications. DNA sequences of the HERV-W family were retrieved from the GenBank database with the aid of the BLAST network server (Altschul et al., 1997
).
Nucleotide sequence accession numbers.
The accession numbers of HERV-W sequences from human were obtained from the GenBank database by using the BLAST network server (Altschul et al., 1997 ): MSRV (AF009668), HWX1 (AB021919), HWX3 (AB021920), HWX5 (AB021921), Pi5T (AF072502), 7A16 (AF072501), 45I4 (AL023581), B353C18 (AC004066), B153K6 (AC005187), U134E6 (Z83850), Q11M15 (AF045450), RG083M05 (AC000064) and BAC378 (U85196).
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Results and Discussion |
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Sequence analysis and synonymous/non-synonymous substitutions
Among 32 pol gene sequences of ERV-W from primates, no disruption by point mutations or insertions/deletions that inactivated the ORF by a frame-shift or termination codon appeared in six sequences, CHW1-1 from chimpanzee, GOW2-1 from gorilla, GIW4-1 and GIW4- 2 from gibbon and HBW6-5 and HBW6-6 from baboon, whereas the sequences of each of the clones identified from the pol gene of Japanese monkey and the orangutan were disrupted by several point mutations or insertions/deletions. This kind of phenomenon has been found in other HERV sequences, e.g. HC2, HC2-10, HC2-16, pCRTK1 and pCRTK6 (Haltmeier et al., 1995 ; Kabá t et al., 1996
; Kim & Crow, 1999
). We aligned the amino acid sequences of pol genes of ERV-W from the uninterrupted primate clones with human clones (HWX5, MSRV, Q11M15, RG083M05 and BAC378) (Fig. 2
). They had 8792% sequence identity with humans. Amongst primates, 8892% sequence identity has been shown (Table 1
). Notable substitutions were seen at positions 5 and 68. The variation at position 5 includes a cysteine residue; this is likely to have functional significance. Synonymous and non-synonymous substitutions within the pol gene of the HERV-W family in humans and primates were analysed to discover the evolutionary forces at work. As shown in Table 2
, the mean synonymous substitutions (K s) ranged from 3·3 to 12·1%, whereas mean non- synonymous substitutions (Ka) ranged from 5·5 to 11·7%, and the value of Ka/Ks ranged from 0·67 to 2·38. In terms of the Ka/Ks ratio, 62% showed higher values for non-synonymous substitutions than for synonymous substitutions in each of the comparisons. We suggest that the sequences of CHW1-1 from chimpanzee, HBW6-6 from baboon and HWX5 from the human X chromosome are under negative selective pressure, since at least 70% of Ka/Ks values in pairwise comparisons were <1·0. These pol gene sequences could therefore be associated with an active provirus in primates.
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In summary, 32 members of a novel ERV-W family were identified from hominoids and Old World monkeys by PCR amplification. In their pol genes, they showed a high degree of sequence identity to the HERV- W family. In six sequences, CHW1-1 from chimpanzee, GOW2-1 from gorilla, GIW4-1 and GIW4-2 from gibbon and HBW6-5 and HBW6-6 from baboon, as in MSRV and HWX5, there was no disruption of the pol gene by point mutation or insertions/deletions. Phylogenetic analysis by the neighbour-joining method of nucleotide sequences of pol fragments of the ERV-W family from humans and primates showed that MSRV is closely related to GIW4-3 and HBW6-2, suggesting that the ERV-W family has evolved independently in a number of different branches in humans and primates. The ratio of synonymous to non-synonymous substitutions showed that negative selection is acting on CHW1-1, HBW6- 6 and HWX5. This suggests that these sequences may represent an active provirus in primates. Our study of the ERV-W family during primate evolution contributes to the understanding of their biological roles in primate genomes.
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Acknowledgments |
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Footnotes |
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References |
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Received 8 April 1999;
accepted 15 June 1999.