Isolation of a human endogenous retroviral HERV-H element with an open env reading frame.

About 100 elements of the human endogenous retroviral HERV-H family have full-length env genes potentially coding for Env proteins with sequences highly similar to the immunosuppressive peptide CKS-17 from the MLV transmembrane protein p15E. However, previously sequenced HERV-H env genes have contained stop codons or framehifts. To isolate elements with open env reading frames, we first tried to assess the diversity of HERV-H env genes by comparing PCR-generated env sequences from genomic DNA with published HERV-H sequences. A region at the beginning of env displayed a similarity of 84-98% among 15 different elements. We then used a probe from one of the PCR-generated clones, 98% similar to the consensus sequence in this region, to screen a human genomic lambda library. Three HERV-H elements displaying ca. 98% identity in the env gene were isolated and were shown to have integrated relatively recently, after the divergence of the orangutan and the african great ape lineages. One of these elements, HERV-H19, had a 1752-bp open env reading frame, producing a 77-kDa Env protein in in vitro translation reactions. This is the first demonstration of a coding competent member of the HERV-H family. These findings raise the possibility that HERV-H Env proteins may play a biological role in human cells.

Diversity of human endogenous retrovirus class II-like sequences.

Class II human endogenous retroviruses (HERVs), often referred to as mouse mammary tumour virus (MMTV)-like or HERV-K elements, have similarities to several animal infectious retroviruses. Single clones from each of nine class II HERV groups (NMWV 1 to NMWV 9), isolated from a human breast cancer cell genomic library, were sequenced over a 244 bp stretch of the conserved reverse transcriptase region. These sequences were aligned to related exogenous and endogenous retroviruses and a phylogenetic tree was constructed. Sequences with more than 80% identity were considered as members of one group and we report here that the class II HERV family consists of at least ten groups. Three of the sequenced clones, from groups NMWV 3, 7 and 9, could not be related to any other previously identified elements and constituted their own groups. NMWV 8 had no similarity to any retroviral sequences in the sequenced region and is so far considered to be non-retroviral.

Coamplification and dispersion of adjacent human endogenous retroviral HERV-H and HERV-E elements; presence of spliced hybrid transcripts in normal leukocytes.

In an RT-PCR study of HERV-H spliced subgenomic transcripts, we found transcripts with HERV-H leader and protease-encoding sequences spliced to HERV-E integrase-encoding sequences in lymphocytes from healthy blood donors. In other cell types, including two T-cell leukemia cell lines, these transcripts were absent. The PCR fragments of the hybrid transcripts contained two open reading frames (ORFs). One was a hybrid HERV-H protease/HERV-E integrase ORF and the other was the HERV-E envelope surface glycoprotein ORF. Alternative splice products were also identified. The genomic DNA origin of the hybrid transcripts was shown to be a HERV-H element with a large 3'-end deletion, adjacent to a HERV-E element lacking the 5'-LTR. This hybrid structure was shown to be amplified and dispersed to six different human chromosomes. Thus, a relatively large part of full-length HERV-E elements (15-20%) is potentially under the transcriptional control of HERV-H LTRs. The HERV-H/HERV-E junction was present in multiple copies also in the chimpanzee and gorilla, but not in the orangutan or old world monkeys.

Spliced human endogenous retroviral HERV-H env transcripts in T-cell leukaemia cell lines and normal leukocytes: alternative splicing pattern of HERV-H transcripts.

The majority of human endogenous retroviral HERV-H elements in the human genome have large deletions in pol and lack most of env, 5-10% are more or less complete with a potentially immunosuppressive transmembrane protein-encoding env region. Spliced HERV-H env transcripts were detected in T-cell leukaemia cell lines and lymphocytes from healthy blood donors by using RT-PCR. The transcripts all contained a splice donor in the leader region downstream from the primer-binding site and a previously unreported splice acceptor in the integrase-encoding region of pol, absent in the HERV-H deletion elements. In singly spliced transcripts the leader and integrase regions were joined directly whereas in multiply spliced transcripts they were joined with an alternative exon from the protease-encoding region located between the two regions. env transcripts from three different HERV-H elements were identified: one element similar to a HERV-H consensus sequence was primarily amplified from the T-cell leukaemia cell lines and two other more defective elements were amplified from normal lymphocytes. One of these elements was shown to be a reintegrated spliced transcript where the protease and integrase regions were joined, removing most of pol but leaving gag intact. Other spliced transcripts, joining the protease region and the 3'-LTR, were also amplified. The fact that HERV-H elements with an intact env splice acceptor also use the splice sites in the protease-encoding region suggests that this unusual multiple splice pattern could have a biological function in the intact HERV-H.

Sequence variation of human endogenous retrovirus ERV9-related elements in an env region corresponding to an immunosuppressive peptide: transcription in normal and neoplastic cells.

Evolutionarily conserved sequences corresponding to an immunosuppressive region in retroviral transmembrane proteins were amplified by the polymerase chain reaction from human genomic DNA and reverse-transcribed RNA from one glioma, three pieces of macroscopically normal brain tissue, kidney, lymphocytes, cultured embryonic lung cells, and a rhabdomyosarcoma cell line. Amplification products (125 bp) from DNA and RNA from the glioma and RNA from one normal piece of brain tissue were cloned and sequenced (45 clones). A variety of sequences similar to ERV9 (75 to 93%) were identified. Amplification products were immobilized on nylon filters and hybridized to four different synthetic oligonucleotides derived from the sequenced clones. Sequences without the stop codon seen in ERV9 in this region, possibly encoding functional immunosuppressive proteins, were present in RNA amplificates from all samples. The various cell types showed different hybridization patterns with the four probes. The open reading frame sequences were identified in genomic Southern blots, one probe detecting about 10 copies and another detecting a single copy. Northern (RNA) blots of mRNA from various normal human tissues revealed 2.5-kb (e.g., lung) and 10-kb (e.g., placenta) transcripts hybridizing to one of the probes.

Expression of human endogenous retroviral sequences in peripheral blood mononuclear cells of healthy individuals.

The polymerase chain reaction was used to detect expression of retroviral sequences with oligonucleotide primers derived from conserved regions of the retroviral genome. Four primer pairs derived from gag and one from pol were used in amplification of reverse-transcribed total RNA prepared from peripheral blood mononuclear cells of seven blood donors. The amplification pattern was the same from each of the seven samples. Sequencing of cloned amplification products revealed that at least three subclasses of sequences related to the human endogenous retroviruses (HERV) RTVL-H, HERV-E and HERV-K, are expressed in peripheral blood mononuclear cells of healthy individuals. This has not been previously reported.