(A) Capture and detection probes used in ELOSA to detect PCR-amplified ERV-9 and MSRV pol-related sequences. Discrimination between ERV-9 and MSRV cloned PCR fragments was performed at the capture step with CpV1A and CpV1B-D, respectively. Colorimetric detection was achieved with the DpV1A-D peroxidase-labelled probe located within the most conserved region of ERV-9 and MSRV pol genes. The selected PCR fragments were called Ppol-MSRV and Ppol-ERV-9 according to ELOSA characterization. The reference (ref) ERV-9 (gb X57147, gb M85205, and gb M37638) and MSRV (gb AF009668) sequences are indicated as well as the consensus (cons) sequences. These consensus sequences resulted from RT-PCR amplification of the conserved region of the pol gene present in all retroviruses (40, 50); the RNA material was obtained from MS patients. (B) Northern blot analysis of poly(A)⁺ cellular RNAs from eight human tissues with the Ppol-MSRV and Ppol-ERV-9 probes characterized by ELOSA.

(A) Clones isolated from a placental cDNA library first with the Ppol-MSRV probe and probes derived from the resulting clones for additional screening. The length of the clone is indicated in parentheses. Black, white, and grey boxes represent regions showing extensive homology with gag, pol, and env genes, respectively; AAAAAAAA indicates poly(A) tail; flanking striped boxes represent UTRs; and repeats are indicated by black arrowheads. (B) Percentages of similarity between the overlapping regions of placental cDNA clones and between the cDNA clones and the probes used in this study. For the cDNA clone analysis, percentages are indicated in boldface and italics for overlapping fragments larger than 1 kb and 490 bp, respectively. The smallest overlapping fragment is 205 bp long. For the comparison between cDNA clones and probes, percentages labelled with asterisks indicate a partial overlap. Probes used for the placental cDNA library screening are underlined.

Phylogenetic analyses were performed at the nucleic acid level for LTR and pol regions and at the protein level for Env. The included sequences were selected as described in Materials and Methods. Experimental clones are indicated in shaded boxes, and MSRV sequences are underlined. LTR, POL, and ENV neighbor-joining trees based upon 123-nt LTR (nt 6 to 143 of clone cl.6A2), 575-nt pol (nt 2309 to 2933 of clone cl.6A1), and 113-aa Env (translation of nt 1852 to 2211 of clone cl.PH74) fragments are presented from left to right. Pairwise gap removal was used for the LTR tree, and all-gap removal was used for POL and ENV trees. Bootstrap values for 500 replicated trees are indicated. “c-” means that the sequence is presented in reverse orientation. Chromosomal assignments are indicated in brackets. Upstream (5) and downstream (3) LTRs are indicated when required. Sequences used in the LTR tree are as follows: clone RG083M05 (AC000064); IMMDL, DNA sequence of the human immunoglobulin D segment locus (X97051 S64822); cosmid U221F2 (Z75893); cosmid cU96H11 (Z70042); PAC 107N3 (Z75741); cosmid E110C7 (Z68223); P135H5C8, H. sapiens DNA sequence on chromosome 21 (L35660); cosmid cN74G7 (Z69715); cosmid U101D3 (Z85998); BAC clone CIT987SK-29B12 (U95738); PAC clone H74 (AC000352); cosmid 315B17 (Z73967); cosmid U61F10 (Z75895); cosmid U85B5 (Z69724); cosmid U116E9 (Z95333); human DNA sequence from clone J428A131 (Z82209); PAC 49C23 (Z93019); PAC 162C6 containing endogenous retrovirus pHE.1 (ERV-9) (Z84475); ERV-9LTR3 (M92648); ERV-9LTR2 (M92647); and ERV-9LTR-ZNF80, H. sapiens DNA for ZNF80-linked ERV-9 LTR (X83497). Additional sequences included in the POL tree are as follows: MSRV-pol reconstructed consensus (AF009668); Ppol-MSRV probe; ERV-9, HERV pHE.1 mRNA sequence (X57147, M85205, and M37638); Ppol-ERV-9 probe; PAC pDJ239b22 (AC003969 and U90583); and RTVL-H, HERV type H (D11078). Sequences appearing in the ENV tree are as follows: SNV, spleen necrosis virus (EMBL M87666); REV-A, reticuloendotheliosis virus strain A (X01455 K02537); SMRV-H, simian sarcoma virus (M23385); SRV-1 (L47.1), simian SRV-1 type D retrovirus (M11841); MPMV, simian Mason-Pfizer type D retrovirus (M12349); SRV-2, simian SRV-2 type D retrovirus (M16605); SRV-1, simian type D virus 1 (U85505); RD114, cat endogenous retrovirus RD114 env gene (X87829); BaEV, baboon endogenous virus virion (M16550); GALV, gibbon ape leukemia virus (M26927); MERV-Fv4, mouse endogenous retrovirus in Fv4 locus (M33884); Cas Br E MuLV, murine leukemia virus (Cas-Br-E MuLV) (M14702); MoMLV, Moloney murine leukemia virus (J02255, J02256, J02257, and M76668); MuLV, murine leukemia virus (M93052); and human sequences RTVL-H (NBRF B44282), ERV-9 (M85205 and M37638), and RAB7 (H. sapiens mRNA) (X93499).

(A) Southern blot analysis of human genomic DNA digested separately with EcoRI (E), HindIII (H), and PstI (P) and probed with Pgag-LB19, Ppro-E, and Penv-C15. The number of bands detected on the blot is indicated at the bottom for each restriction enzyme. The absolute copy numbers deduced from dot blot analysis, as well as confidence intervals, are also indicated at the bottom. Un., undigested. (B) Alignment of the placental cDNA clones (cDNAs; schematic representation of the 7.6-kb region defined with the overlapping cDNA clones) with the four sequences showing the higher scores with BLASTN query. Shown are human BAC clone RG083M05 (gb AC000064), human BAC378 (gb U85196 and gb AE000660), H. sapiens cosmid Q11M15 (gb AF045450), and human DNA sequence from cosmid U134E6 (EMBL Z83850). Locations of the aligned region of each clone are indicated. Chromosomal assignments are indicated in brackets. The percentages of similarity (without gaps) between the four sequences and the consensus sequence (cDNAs) are given (values for individual clones are indicated in the text). The presence of repeats at both ends of the genome is stated.

Definition of a putative LTR. (A) The 2,364- to 2,720-nt region of the cl.PH74 clone, identified as a putative promoter region with Signalscan 4.05, was PCR amplified and subcloned into pCAT3 reporter vector. The resulting pCAT3-3′LTR plasmid and pCAT3 control plasmid were used to transfect HeLa cells prior to CAT activity determination. Cells, HeLa cell extract; pCAT3 and pCAT3-3′LTR, extract of HeLa cells transfected with control plasmid and promoter plasmid, respectively. (B) Alignment of 5′ and 3′ UTRs of placental experimental clones with the 5′ (5-RG-28000-28872) and 3′ (3-RG-37500-38314) repeated sequences of the 28,000- to 38,314-nt fragment of human DNA sequence RG083M05. The end of the 3′-most ORF is indicated (env orf). The tandemly repeated CAAC flanking sequences are doubly underlined on DNA sequences. The 783-bp LTR consensus sequence is positioned at the bottom. The polypurine tract (PPT) upstream from the 5′ end of the LTR and the tRNA^Trp PBS downstream from the 3′ end of LTR are indicated. U3, R, and U5 subparts are indicated, except for the U3-R junction. Transcription factor sites determined with Signalscan 4.05 are underlined and labelled from I to VI. The sites V and VI may correspond to CCAAT box and TATA box, respectively. The 3′ region of clone cl.PH74 used in the CAT assay is underlined on the cl.PH74 sequence. ∗ and ▴, potential cap sites obtained in 5′ RACE experiments with human placental poly(A)⁺ RNA and total RNA extracted from pCAT-3′LTR-transfected HeLa cells, respectively. [polyA], polyadenylation signal.

(A) Northern blot analysis of poly(A)⁺ RNAs from placental tissues with the U5(g) (nt 115 to 717 of cl.6A2) and U5(e) (nt 1 to 491 of cl.24.4) probe, gag (Pgag-LB19), pro (Ppro-E), pol (Ppol-MSRV), and env (Penv-C15) probes as described in Materials and Methods and the U3(e) (nt 732 to 1116 of cl.C4C5) probe. (B) Hypothetical splicing strategy. The RG083M05 prototype DNA clone as well as six experimental clones (cl.6A2 and cl.44.4, cl.24.4 and cl.PH74, cl.PH7, and cl.Pi5T) isolated from the cDNA library is depicted. The nucleotide sequences of regions overlapping putative splice sites are boxed. Nucleic acid sequences of RG083M05 and all placental cDNA clones are indicated with lowercase and capital letters, respectively. AG and GT bordering splice sites are in boldface. Splice donor (DS) and acceptor (AS) sites are indicated by rightward and leftward arrows, respectively. U3 (hatched from lower left to upper right), R (light grey), U5 (hatched from upper left to lower right), 2-kb insert (vertically striped), Gag (black), Pol (white), and Env (dark grey) are shown as boxes. The positioning of the seven probes used in the Northern blot analysis and the placental region spanned by the cDNAs (cDNAs) are illustrated at the bottom of the figure.

(A) Sequence of the putative HERV-W envelope polypeptide derived from three different placental cDNA clones. The leader peptide (L), the surface protein (SU), and the transmembrane protein (TM) are indicated between arrows. The hydrophobic fusion peptide and the carboxy-transmembrane region are singly and doubly underlined, respectively. The immunosuppressive region is indicated by italics. Potential glycosylation sites are indicated by dots. Divergent amino acids are indicated on the lower line. (B) In vitro transcription-translation assay for HERV-W envelope gene product, without (RR−) or with (RR+) canine microsomes, and control (Cont) without template.