Identification and phylogeny of new human endogenous retroviral sequences belonging to the HERV-H family.

Abstract

2055 HUMAN ENDOGENO US RETROVIRUSES (HERVs) comprise at least 1% of the human genome and are dispersed over the whole human genome.1 The HERVs have been inserted into the germ cells of primates and have remained an integral part of the primate genomes during evolution. In addition, HERVs present full-length or incomplete sequences with multiple stop codons, insertions, deletions, and frame shifts. Such retroviral elements have been described in primates and humans.2,3 The vast majority of these have no influence on gene function or relevance to pathology.4 A small minority of such sequences has acquired a role in regulating gene expression, and some of these may have been subject to recent change, e.g., retrotransposition or rearrangement. They could be relevant to differences between individuals, and to expression of disease.5 An approach to identifying this minority of elements is through phylogeny. Those viral sequences that are of recent origin or have changed most recently are more likely to be associated with instability and with differences between individuals. A number of families of retrovirus and related elements have been distinguished and their origins and genomic locations are increasingly recognized.1 The HERV-H class of elements, characterized by a histidine tRNA-binding site, was identified in relation to the breakpoints of three large deletions involving the b-globin gene cluster.6 Analysis of several genomic isolates of HERV-H elements has revealed that the majority of elements are approximately 5.8 kb in length and contain gag-, pol-, and env-related sequences that are flanked by 400to 500-bp long terminal repeat (LTR) elements.6,7 The HERV-H sequences are expressed in placenta8 and T cells9 and one such sequence has been shown to influence expression of the PLA2L gene.10 Here we have identified three new HERVH env sequences and analyzed them phylogenetically with other HERV-H family sequences in the nucleotide sequence database. Human genomic DNA was subjected to polymerase chain reaction (PCR) amplification. New HERV-H family sequences were amplified by the primer pair HS45 (59-CCCATTCTTATGTCATCCTCTA-39, bases 728–749) and HY75 (59-GTACCCAGACTCCTAGGGAT-3 9, bases 1562–1581) from the human endogenous retroviral element HERV-H (DDBJ/EMBL/ GenBank accession no. AF108843). The PCR conditions followed were those of Kim et al.,11 with an annealing temperature of 56°C. PCR products were separated on a 1.5% agarose gel, purified with a QIAEX II gel extraction kit (Qiagen, Chatsworth, CA), and cloned into the T-khs307 vector.12 The cloned DNA was isolated by the alkali lysis method, using a High Pure plasmid isolation kit (Boehringer Mannheim, Indianapolis, IN). Individual plasmid DNAs was screened for inserts by PCR. Positive samples were subjected to sequence analyses on both strands with T7 and M13 reverse primers, using an automated DNA sequencer (model 373A) and a DyeDeoxy terminator kit (Applied Biosystems, Foster City, CA). Nucleotide sequence analyses were performed with GAP, PILEUP, and PRETTY (GCG software; Genetics Computer Group, University of Wisconsin, Madison, WI). Neighbor-joining phylogenetic analysis13 was performed with the MEGA program.14 Statistical significance evaluation of the branching pattern was performed with 100 replications. Nucleotide sequences of the HERV-H family were retrieved from the GenBank database with the aid of the BLAST network server.15 We identified three HERV-H env sequences from human genomic DNA and compared them with sequences retrieved from the GenBank database. Figure 1 presents our new env elements (HERV-H5, HERV-H21, and HERV-H22) in relation to existing homologies in the nucleotide sequence database. They showed a similarity of 89.7–92.7% with 13 different HERV-H elements. HERV-H21 and HERV-H22 were 91.7% similar to HERV-H19, while HERV-H5 was 93% similar to HERVH19.16 HERV-H21 and HERV-H22 revealed deletions of 4 bp (CCAT) and 10 bp (CTAGGTAATC) in comparison with HERV-19. In HERV-H5, deletions of 1 and 2 bp were noted in comparison with the HERV-19. As the results of such events, the env sequences of the three new elements had stop codons and frameshifts (Fig.1), whereas HERV-H19 had an intact open reading frame.16 The translated env genes of the three new elements, HERV-H10, HERV-H18, and HERV-H19,16 and RGH1 and RGH2,17 were aligned with the HERV-H family in