AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 15, Number 17, 1999, pp. 1529–1533Mary Ann Liebert, Inc.

Short Communication

Phylogeny of a Novel Family of Human EndogenousRetrovirus Sequences, HERV-W, in Humans and

Other Primates

CÉCILE VOISSET,1 ANTOINE BLANCHER,2 HERVÉ PERRON,1 BERNARD MANDRAND,1

FRANÇOIS MALLET,1 and GLÁUCIA PARANHOS-BACCALÀ1

ABSTRACT

A novel human endogenous retrovirus, HERV-W, has been characterized on the basis of multiple sclerosis-associated retrovirus (MSRV) probes. We have analyzed the phylogenetic distribution of HERV-W in humansand other primate species. As HERV-W presents a C/D chimeric nature and is largely composed of deletedelements, Southern blots were performed using gag, pol, env, and LTR probes. The relative complexities ob-served for gag, pol, env, and LTR regions were similar in humans, apes, and Old World monkeys, the mini-mal number of bands observed after Southern blot analysis being 25, 50, 10, and at least 100, respectively.The HERV-W family entered the genome of catarrhines more than 25 million years ago.

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RET RO VIR US SEQ UEN CES have been found in the genomicDNA of many eukaryotic species, including primates. As

the endogenous retrovirus (ERV) sequence characterization hasrevealed similarities with infectious retroviruses, it has beenpostulated that ERVs are derived from infectious retrovirusesthat entered the germ line, and were fixed in the population dur-ing evolution. 1 HERVs (human endogenous retroviruses) pre-sent full-length or incom plete genomes with multiple stopcodons, frameshifts, and/or deletions. Such complete endoge-nous retrovirus genom es have been described in monkey andin human DNA.2,3 Most HERV families encompass a relativelylow copy number per haploid genome,4 compared with othersthat are either high copy number,5 or single-copy retroviral el-ements.6 Although no conclusive evidence has been found inhumans, HERVs are suspected to be associated with certain au-toimmune disorders 7 and possibly in cancer pathogenesis. 8 Ina previous report,9 new retrovirus pol sequences were isolatedfrom lymphoblastoid cell cultures and plasma derived from pa-tients with multiple sclerosis (MS) and referred to as multiplesclerosis-associat ed retrovirus (MSRV). Southern hybridiza-tions using MSRV probes allowed characterization of a multi-copy MSRV-related human endogenous retrovirus family

named HERV-W.10 Sequence analysis of the gag and pol re-gions of HERV-W members has shown similarities to C-typeretroviruses, while HERV-W env sequences are most closelyrelated to type D retrovirus elements. The molecular charac-terization of HERV-W reveals that, in the human genom e, it ispresent as complete nonreplicative as well as mostly deletedretroviral sequences. 10 Moreover, HERV-W is phylogeneticallyclose to, but distinct from, the ERV-9 family,4 particularly inpol regions. 10 Here, we analyze the phylogenetic distributionof HERV-W within mammals, and more particularly amongprimates, by Southern blot, using nucleic acid probes derivedfrom gag, pol, env, and long terminal repeat (LTR) sequencesof the extracellular MSRV-related HERV-W family.

The MSRV probes were derived from polymerase chain re-action (PCR) amplification products (Table 1) using gag CL2,pol 68/1, env CL5, and LTR CL6 clones (GenBank accessionnumbers AF123881, AF009668, AF127228, and AF127229, re-spectively) as templates. These clones were obtained from theplasma of an MS patient and from supernatants of synoviocytecultures of a rheumatoid arthritis patient, as described else-where.11 The MSRV probes were labeled with [ a -32P]dCTP bythe random-primi ng method, according to the user’s manual

1Unité Mixte de Recherche 103 CNRS-bioMérieux, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France.2Laboratoire d’Immunologie Moléculaire de l’Université Paul Sabatier, Hôpital Purpan, 31059 Toulouse Cedex, France.

(Ready-to-Go DNA-labeling kit; Pharmacia Biotech, Piscat-away, NJ). To check Ppol-68/1 and Pgag-CL2 probe specificitywith ERV-9 gag and pol regions, the pol-ERV-9 sequence(GenBank accession number AF072495) was derived by PCRamplification of a B lymphocyte cDNA library of an MS pa-tient.10 Gag-ERV-9 (clone 8pTGAG) was amplified by PCRfrom a cloned sequence kindly given by G. La Mantia (Uni-versity of Naples, Naples, Italy).

Samples are described in Fig. 1. Additional mammal sam-ples were obtained from cells of mouse (Mus, 3T6), pig(IBRS2), cow (MDBK), hamster (BHK), rat (C6P165), dog(CF2), rabbit, and sheep. The cat (cat, PG4) and Pongo pyg-maeus (or EB185JC) cell lines were purchased from the Euro-pean Collection of Cell Cultures (ECCAC). Ten different hu-man samples were obtained from blood donors (ETS, Lyon,

France). Genomic DNA was prepared according to a standardprocedure described elsewhere, 12 using RNase A and proteinaseK treatm ents, and basic phenol extraction.

Eight micrograms of each genomic DNA, and 4 m g of gib-bon DNA, were single-digested with 40 U of EcoRI or Hind/III(Boerhinger Manheim , Indianapolis, IN) or double-digestedwith 40 U of EcoRI and PstI, overnight at 37°C. The hydrolyzedDNA was fractionated by 0.8% agarose gel electrophoresis andalkaline blotted on a nylon membrane (Hybond-N 1 ; Amer-sham, Arlington Heights, IL), using 0.4 N NaOH.12 After 4 hrof prehybridization (in 5 3 SSC, 1 3 Denhardt’s, 0.1% sodiumdodecyl sulfate [SDS], 50% formamide, 20 mM Tris-HCl [pH7.5], herring sperm DNA [0.1 mg/ml]), nylon membranes werehybridized (in 5 3 SSC, 1 3 Denhardt’ s, 0.1% SDS, 50% for-mamide, 20 mM Tris-HCl [pH 7.5], herring sperm DNA [0.1

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TABLE 1. PRIM ER SEQ U EN CES USED TO OBT AIN MSRV PROBES AND ERV-9 GEN ES

Probe sizeProbea Primer Sense primer Primer Antisense primer (bp)

Ppol-68/1 A 5 9 -GAC TTG AGC CAG TCC B 5 9 -CTT TAG GGC CTG GAA 438TCA TAC CT-3 9 AGC CAC T-3 9

Pgag-CL2 C 5 9 -CCT AGA ACG TAT TCT D 5 9 -TGG CTC TCA ATG GTC 1100GGA GAA TTG GG-3 9 AAA CAT ACC CG-3 9

Penv-TM-CL15 E 5 9 -CTT CCT TTT GTT ATC F 5 9 -TGG GGT TCC ATT TGT 521GAG CAG GAG TGC-3 9 AAG ACC ATC TGT AGC-3 9

PLTR-CL6 G 5 9 -CCT CCC CAA CAG CAC H 5 9 -CAG TTG CAA GAT TTA 367TTA G-3 9 ATA GAG TG-3 9

pol ERV9 I 5 9 -TCA GGG ATA GCC CCC J 5 9 -AAC CCT TTG CCA CTA 649ATC TAT-3 9 CAT CAA TTT C-3 9

gag ERV-9 K 5 9 -GAA ACA CTC AGA CAT L 5 9 -GCT GGC GCT TGC CCC 1473CAA CAG-3 9 AGG CAC C-3 9

aMSRV probes were derived from PCR amplification products using pol 68-1 (AF009668), gag CL2 (AF123881), env CL15(AF127228), and LTR CL6 (AF127229) clones as template.

FIG. 1. Distribution of the HERV-W gag region in different primate species. Southern blot analysis of mammalian DNA: hu,human (Homo sapiens); ch, common chimpanzee (Pan troglodytes ); go, gorilla (Gorilla gorilla ); or, orangutan (Pongo pygmaeus);gb, pileated gibbon (Hylobates pileatus ); rh, rhesus monkey (Macaca mulatta); ba, guinea baboon (Papio papio); sq, black-cappedsquirrel (Saimiri boliviensi ); ta, cottontop tamarin (Suinus oedipus ); mar, common marmoset (Callithrix jacchus); le, black lemur(Eulemur macaco); cat, cat; mus, mouse. Eight micrograms of mammalian DNA (and 4 m g for gb genomic DNA) were digestedwith EcoRI (A) or HindIII (B) and probed with radiolabeled Pgag-CL2. After Southern hybridization, the blots were washed un-der high-stringency conditions (0.2 3 SSC–0.1% SDS, twice for 15 min, at 65°C; 0.1 3 SSC–0.1% SDS, twice for 15 min, at65°C; and 0.1% SDS, twice for 30 min, at room temperature). After washing, blots were exposed to X-ray film (Kodak) at 2 80°Cuntil development. The blots containing EcoRI-digested DNA or HindIII-digested DNA were exposed for 3 and 2 days, respec-tively. Size markers are shown on the left.

mg/ml], 5% dextran sulfate) overnight at 42°C, with a 32P-la-beled DNA probe. Washings were performed under low-strin-gency conditions (2 3 SSC–0.2% SDS, twice for 15 min at45°C) and under high-stringency conditions as described in Fig.1. After washing, blots were exposed to X-ray film (Kodak,Rochester, NY) at 2 80°C until developm ent. Twenty nano-grams (5 3 1010 copies) of ERV-9 pol and 20 ng of pol-68/1PCR products were separated and blotted on a nylon membraneas described above. The membrane was hybridized with Ppol-68/1. Likewise, 1 m g (9 3 1011 copies) of ERV-9 gag and 2 ngof MSRV gag-CL2 PCR products were hybridized with Pgag-CL2.

To check the specificity of the MSRV gag (Pgag-CL2) andpol (Ppol-68/1) probes, Southern blot membranes were hy-bridized with ERV-9 gag and pol fragments. After high-strin-gency washes, we did not observe any hybridization of thePgag-CL2 probe with ERV-9 gag fragment, while Ppol-68/1probe gave a faint, nonsignificant, hybridization signal onERV9 pol fragment (data not shown). This result was consid-ered nonsignificant because the ERV-9 gag and pol PCR prod-ucts correspond to an excess of 18,000 times and 200 times, re-spectively, compared with the copy numbers of ERV-9 gag(5 3 107 copies) and pol (2.5 3 108 copies) regions present in8 m g of mammalian DNA used in our experiments.

The similarity between MSRV probes used in this study andthe HERV-W genom ic DNA (RG083M05; GenBank accessionnumber AC000064) prototype previously identified 10 rangedfrom 90% (for gag, pol, and env probes) to 99% (for LTRprobes).

All Southern analyses performed under low- and high-strin-gency conditions displayed the same hybridization patterns. BySouthern analysis, all probes used here did not hybridize withnonprimate mammalian DNA (cat and mouse; Figs. 1–3).Moreover, no hybridization signal was observed with othersamples of mammalian genomic DNA (rat, cow, pig, rabbit,dog, and sheep; data not shown).

Whatever the MSRV probe and wash stringency used, hy-bridization signals were observed only for humans, apes, andOld World monkeys, and not for New World monkeys andprosimian (lemur) samples (Figs. 1–3). Indeed, a complex hy-

bridization pattern was observed for all probes used on positiveprimates. Moreover, no additional bands could be observedwhen the films containing Southern blot data were overexposed.

The EcoRI patterns revealed by Ppol-68/1, Pgag-CL2, andPenv-TM-CL5 probes were characterized by a great variety ofrelative band intensities (Figs. 1A, 2A, and 3A). In contrast, theHindIII restriction patterns displayed a weak variation in bandsintensity (Figs. 1B, 2B, and 3B). The MSRV probes used re-vealed interspecies RFLPs (restriction length polym orphism s),which are easier to distinguish after EcoRI digestion. EcoRIgag and pol Southern blot patterns (Figs. 1A and 2A) are char-acterized by bands of high intensity, which are present in allpositive species. For example, the Pgag-CL2 probe revealedtwo intense and constant bands of about 3 and 1.5 kb (Fig. 1A).On the other hand, some bands are intense, but are not observedin all positive species. For example, only baboons possess a 2-kb band revealed by the Pgag-CL2 probe on EcoRI-digestedDNA (Fig. 1A). EcoRI and HindIII env Southern blots revealedsimpler patterns than did gag and pol probes (Fig. 3A and B).The LTR (PLTR-CL6) probe revealed numerous bands, repre-senting a complex pattern of the HERV-W family in all posi-tive species DNA (data not shown). The hybridization patterndid not reveal any particular polymorphism among the 10 dif-ferent human DNA tested with MSRV probes (data not shown).However, the sensitivity of detection of polym orphism bySouthern blot analyses is limited because of the complexity ofthe HERV-W family.

The minimal number of bands of HERV-W elements esti-mated by Southern blot analysis was approximately 25, 50, 10,and at least 100 fragments, using the Pgag-CL2, Ppol-68/1,Penv-TM-CL15, and PLTR-CL6 probes, respectively, in hu-man, ape, and Old World monkey samples and using EcoRIand HindIII restriction enzymes.

As shown in Fig. 4, the HERV-W family certainly enteredthe germ line, as retrovirus entities, more than 25 million yearsago.

A novel HERV-W family has been molecularly character-ized10 on the basis of MSRV probes. In this article we have de-termined the integration time of members of the chimericHERV-W family into the mammalian genomes, using probes

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FIG. 2. Distribution of HERV-W pol region in different primate species. Eight micrograms of mammalian DNA (and 4 m g forgb genomic DNA) were digested with EcoRI (A) or HindIII (B) and probed with radiolabeled Ppol-68/1. The Southern blotscontaining EcoRI-digested DNA or HindIII-digested DNA of primate species were exposed for 5 and 7 days, respectively. Themammalian DNA samples and the wash conditions were as described in Fig. 1. Size markers are shown on the left.

representing the gag, pol, env, and LTR regions of the MSRV-related HERV-W sequences. 11 Despite the fact that the HERV-W family is phylogenetically close to ERV-9, Southern blotanalysis has clearly shown that high-stringency washes weresufficient to eliminate all of the cross-hybridizatio n events be-tween the Pgag-CL2 and Ppol-68/1 probes and the corre-sponding regions of ERV-9. Thus, all of the hybridizations ob-tained in this study with MSRV probes revealed only membersof the HERV-W family. The high-stringency washes used andthe dense pattern suggested that the sequences detected withMSRV probes in humans, apes, and Old World monkey DNArepresented members of the HERV-W family.

To determine when HERV-W sequences entered the mam-malian genome during evolution, we checked the presence ofHERV-W sequences in several mammals. We have observedthat no HERV-W sequences were detected in the DNA of non-

primate mammals, which diverged from the primate lineage ap-proximately 90 million years ago, or in prosimian and NewWorld monkey genomic DNA, which diverged from the an-cestor of catarrhines approximately 60 and 40 million years ago,respectively. 13 HERV-W sequences were detected only in theDNA of humans, apes, and Old World monkeys. HERV-W in-fection of a catarrhine ancestor would therefore have occurredafter the geographic separation between the Old and New Worldanthropoid lineages, approximately 40 million years ago,13 butbefore the species radiation and geographic dispersal (outsideAfrica) of the ancestors of the Old World monkeys, apes, andhominoids estimated at 25 million years ago.13 Consequently,and as shown in Fig. 4, the HERV-W family has probably beeninserted as endogenous retroviral entities in the germ line of theprimate lineage for more than 25 million years.

The phylogenetic characterization of most HERVs, by South-

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FIG. 3. Distribution of the TM region of the env gene of the HERV-W family in various primate species. Eight microgramsof mammals DNA (and 4 m g for gb genomic DNA) were digested with EcoRI (A) or HindIII (B) and probed with radiolabeledPenv-TM-C15. The Southern blots containing EcoRI-digested DNA or HindIII-digested DNA of primate species were exposedfor 3 and 6 days, respectively. The mammalian DNA samples and the wash conditions were as described in Fig. 1. Size mark-ers are shown on the left.

FIG. 4. Phylogenetic schematic tree representing the approxim ate radiation times of the primate species analyzed. The arrowsindicate the probable insertion times of several endogenous retroviruses into the germline.

ern blotting, has shown that these elements have persisted inthe primate genome for different periods of time, ranging from10 to more than 60 million years.14 Moreover, it is interestingto note that, like HERV-W elements, most of the HERV fam-ilies examined arose early in primate evolution, and are foundin humans, apes, and Old World monkeys as class I and classII HERVs.14 Some other HERVs have also been reported inapes and Old World and New World monkeys, such as HERV-K10a,15 and even in nonprimate mammals, as HERV-L.16 Somemembers of the HERV family entered later into the primate lin-eage, and are present only in humans, chimpanzees, and goril-las, such as HERV-H type Ia LTRs.17

The number of fragments that we observed certainly repre-sents an underestimation of the real copy number of eachHERV-W gene. The HERV-W family is highly complex, whichdid not allow us to readily sequence members of this familyamong positive primates. The various copy numbers of the gag,pol, and env HERV-W regions may reflect a transposition ac-tivity, as previously described for LINE elements.18 However,it is difficult to determine whether HERV-W sequences wereintroduced in the genome by a single or low number of inser-tion, or by multiple insertions. The Southern blot analysisshowed a comparable complexity among human, apes, and OldWorld monkey genomic DNA using pol (C-type) and env (D-type) MSRV probes. Thus, these results suggested that theHERV-W ancestor active element entered the primate lineagemore than 25 million years ago, and has stayed relatively sta-ble until now.

ACKNOWLEDGMENTS

This work was supported by bioMérieux SA. We thank C.Guillon (Erasmus University, Rotterdam, The Netherlands) andD. Spencer (UMR103 CNRS-bioMérieux, Lyon, France) forcritical reading of the manuscript. We also thank G. La Man-tia (Department of Genetics, University of Naples, Naples,Italy) for providing gag ERV-9 clone. We are grateful to J.Y.Madec (CNEVA, Lyon, France) and A. Morgan (Departmentof Virology, University of Bristol, Bristol, UK) for providingsome mammalian samples.

C. Voisset was supported by a doctoral fellowship from theFondation Mérieux.

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Address reprint requests to:Gláucia Paranhos-Bacc alà

Ecole Normale Supérieure de LyonUnité Mixte CNRS-bioMérieux

46 allée d’Italie69364 Lyon Cedex 07, France

E-mail: gbaccala@ ens-bma.cnrs.fr

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