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Virology 260, 1–9 (1999)Article ID viro.1999.9792, available online at http://www.idealibrary.com on

RAPID COMMUNICATION

Molecular Cloning and Characterization of MSRV-Related SequencesAssociated with Retrovirus-like Particles

F. Komurian-Pradel,* G. Paranhos-Baccala,* F. Bedin,* A. Ounanian-Paraz,† M. Sodoyer,* C. Ott,*A. Rajoharison,* E. Garcia,* F. Mallet,* B. Mandrand,* and H. Perron*,1

*Ecole Normale Superieure de Lyon, UMR 103 CNRS–bioMerieux, 46, Allee d’Italie, 69364 Lyon Cedex 07, France; and †Laboratoire de Virologie,Faculte de Medecine/Universite Joseph Fourier, Domaine de La Merci, 38700 Grenoble, France

Received February 16, 1999; returned to author for revision March 24, 1999; accepted May 5, 1999

New sequences have been obtained by successive overlapping RT-PCR extensions from the pol region of a retroviral RNA(multiple sclerosis-associated retroviral element, MSRV) amplified in retrovirus-like particles from patients with multiplesclerosis. gag and pol sequences are related to type C oncoviruses, whereas the env sequence is closer to type D. Atryptophan-like (W) tRNA primer-binding site was identified downstream of the RU5 region in the 59LTR, and the U3R regioncloned in the 39LTR exhibited potent promoter activity. MSRV clones define a novel family of endogenous elements, HERV-W.From our data, HERV-W RNAs are copackaged in extracellular particles which might be produced by replication-competent

or transcomplemented HERV-W copies or by an exogenous member of the HERV-W family. © 1999 Academic Press

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Introduction. Retrovirus-like particles associated witheverse transcriptase (RT) activity have been describedy several groups in cell cultures from patients withultiple sclerosis (MS) (1–3). We have recently reported

he molecular characterization of a retroviral pol regionbtained from extracellular particles from MS samples

4). These results were first generated by using a “pan-etro” RT-PCR protocol based on consensus sequencesn the reverse transcriptase region of retroviral polenes. From the prototype sequence (named MSRV-cpol,

or multiple sclerosis-associated retroviral element–con-ensus pol) identified by this approach, a larger MSRVol region encoding the protease and the reverse tran-criptase was obtained by RT-PCR extension. In order toharacterize the sequences flanking MSRV pol in extra-ellular RNA, we have performed RT-PCR extension onNA extracted from different sources of retroviral parti-les such as B-cell lines immortalized by Epstein–Barrirus (EBV) or plasma from MS patients. Though quanti-ies of viral material were always very limited, overlap-ing clones extending toward the 59 and 39LTRs werebtained from extracted RNA and allowed us to defineag, pol, and env genes. These sequences were found toe strongly homologous to a novel family of humanndogenous retroviruses (ERVs) named HERV-W (5),

1 To whom correspondence should be addressed at present ad-ress: bioMerieux SA, Chemin de L’Orme, 69280 Marcy L’Etoile,

qrance. Fax: 33.4.78 87 20 90. E-mail: HPerron@ml.bioMerieux.fr.

1

hich is phylogenically related but genetically differentrom the ERV9 family. Identical sequences were obtainedrom various MS samples, but also in a patient withhumatoid arthritis (RA). Clones representing differentetroviral genes in RNA associated with retrovirus-likearticles are described here.

Results. In a previous study, RT-PCR extension on RNAxtracted from particles pelleted from MSRV pol-positiveulture supernatant had provided overlapping se-uences in continuity with the reference MSRV pol se-uence (4). Primers derived from these data were used

o amplify larger fragments in MS plasma or in viralarticles from cell cultures. The overlapping MSRVlones described in this study represent a putative630-nucleotide-long reconstituted retroviral organiza-

ion (Fig. 1a).

MSRV LTR Regions. A RT-PCR amplification was per-ormed with an antisense 39 primer located in the gagegion and with a 59 sense primer defined from the Requence previously obtained in the 39 end (clone 6,escribed below). The clone LB15 (Fig. 1b) encompass-

ng R (87 bp), U5 (456 bp), primer-binding site (PBS), and9 gag regions, was thus obtained from concentratedulture particles. A polyadenylation site is compatibleith the “CA” dinucleotide motif located at the junction of

he R and U5 regions and a putative poly(A) downstreamignal is located at a distance of 24 nucleotides down-tream of the poly(A) signal, with the consensus se-

uence “YGTGTTYY” (6). The putative PBS, identified

0042-6822/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

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2 RAPID COMMUNICATION

FIG. 1. MSRV RU5, gag, integrase, env, and U3R regions. (a) Schematic representation of MSRV genome with representative RT-PCR clonesescribed in the text and primer location. The thick bar represents previously described clones encompassing the protease and reverse-transcriptaseomains (4). The following sequences have been deposited with the GenBank database, LB15, CL2, CL17, C187-23, C15, CL6, and CL4, under the

ollowing accession numbers: AF123880, AF123881, AF12388, AF123882, AF127228, AF127229, and AF127227, respectively. (b) 59 nucleotide sequenceorresponding to regions encompassed by clones LB15 (represented in nt 1 to 623) in RU5 region and clones CL2 (represented in nt 624 to 1680)nd CL17 (represented in nt 1681 to 2052) in gag. The amino acid translation is shown under the gag nucleotidic sequences. The polyadenylationite in R is boxed and the polyadenylation downstream signal in U5 is underlined. (//) Frameshift and (●) stop codon. Amino acids in boldfaceorrespond to the major homology region in the capsid. Underlined amino acids correspond to the conserved positions in the nucleocapsid. (c)ucleotide and deduced amino acid sequence of the integrase region obtained from 87-23 clone. (//) The frameshift which is suppressed here tobtain the putative orf. Underlined boldface letters represent conserved amino acid residues among retroviral integrases. (d) 59 env region wasbtained from clone C15 (nt 1 to 1479) and 39 env and LTR sequences were obtained from clone CL6 (nt 1480 to 2030). Vertical arrows indicate thenv region and the U3 and R substructures. In the env region, the peptide signal and the putative immunosuppressive peptide are underlined, the-linked glycosylation sites are boxed, and the two putative cleavage sites are indicated by vertical arrows. In the U3R region, the CAAT regulatory

lement and the TATA box are underlined; the cap site and the poly(A) signal are also indicated.

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3RAPID COMMUNICATION

ownstream of the U5 region, has proved to be related tohe 39 end of avian tRNATrp complementary sequence (7).

Two rather short in-frame orfs (76 aa, MQLH-SGPCFLWLKLSFCSPSTTAVCHRHRPAADFHPFGSSR-SAVLLIQHRRPLPLPIGLKACHCSCTAKCLGSS, and 64a, MASNRAITLTAWSKIPFLGIRETKNPRSENTRLATML-AAHHHFGSSPPLSWELWEQGPQVTIW) were found in

he RU5 region. Since a coding capacity in such LTRegion has never been reported (even the uncommon

MTV superantigen orf in LTR is located in the U3egion), this should be noticed.

The 39LTR U3R region was identified in the CL6 cloneFig. 1d) obtained by amplification on DNase-treatedNA extracted from MS plasma. By comparison with the9LTR sequences obtained by 59 extension on MSRVNA, we could identify a 299-bp-long U3 region and an4-bp-long R region in the CL6 LTR region, located down-tream of the env orf and ending with the poly(A) tail. The

ypical regulatory element CAAT box was observed inwo locations in the putative U3 region, but other putativeinding sites for several transcription factors were alsoetected with the Transcription Factor database (8). AATA box (TATAAA) was observed in the U3 region asescribed for numerous retroviruses. Finally, a poly(A)ignal (AATAAA) was found 83 bp downstream of theATA box.

In order to evaluate the promotor activity of LTR clonesrom different origins, we have performed CAT assays

ith subcloned U3R regions from LTR clones obtained

FIG. 1

rom MS plasma RNA (CL6) and from related HERV-W g

opies in non-MS DNA (5M6) and in non-MS placentaNA (PH74). As shown in Fig. 2, a potent activity wasssociated with the LTR sequence in the CL6 clone fromS plasma, whereas a weak and moderate promotor

ctivity was found with 5M6 and PH74 clones, respec-ively. Similar results were obtained with different cellypes (data not shown).

MSRV gag Region. After the obtainment of severalverlapping sequences, reamplification of larger frag-ents was performed with MSRV primers on DNase-

reated RNA from cultured particles and/or from MSlasma. One of such clones from MS plasma, CL2, hasn orf of 359 aa interrupted by a stop codon in the 39 partf the capsid region. The coding capacity of this cloneas confirmed by the production of a 42-kDa recombi-ant protein in Escherichia coli (data not shown). As

epresented in Fig. 1b, the coding region correspondingo the MA and CA subdomains displays the characteris-ic “major homology region” located at the carboxy ter-

inus of the CA subdomain of most retroviruses (9) withhe three conserved residues of this motif, as well as thexact spacing between them (Q-X3-E-X7-R). An ATG initi-ting the MA subdomain in the gag protein followed bynother in-frame ATG, both in frame with the gag orf oflone CL2, could be identified in the different clonesncompassing this region. Another clone, CL17 (Fig. 1a),mplified in plasma RNA from a patient with “possible”S, has sequences encompassing a complete retroviral

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4 RAPID COMMUNICATION

hifts. The C-X2-C-X4-H-X4-C nucleic acid binding motifCCHC box) was identified in the deduced amino acidequence within the nucleocapsid region of CL17. Inter-stingly, the MSRV Gag polyprotein has three potentialites of N-glycosylation located in aa positions 217 to19, 300 to 302, and 314 to 316 (Fig. 1b).

A stop codon at amino acid position 487, correspond-ng to the possible end of the gag-encoded polyprotein,

as observed at the same position in CL2 and CL17lones encompassing the gag–protease junction.

The deduced amino acid sequence of this putativeSRV gag protein is shown under the nucleotide se-

uence in Fig. 1b. Figure 3a illustrates its relationshipith retroviral gag proteins and shows that MSRV gag ishylogenically close to type C oncoviruses as already

eported for pol (4). Figure 3b illustrates the phylogenicelationship between MSRV and HERV-W gag nucleotide

FIG. 1

equences, and sequences from human genomic data- r

ase. MSRV CL2 clusters with HERV-W sequences (Cl4.1 and Cl 6A1), whereas a significantly distant familylusters with ERV9. At the nucleotide level, 86% of ho-ology in the 59LTR/gag region was calculated betweenL2 and placental HERV-W RNA sequences (5).

MSRV integrase Region. The complete integrase re-ion presented here (clone 87-23) was amplified from alasma DNA-free RNA of a patient with possible MS. The

esulting clone (87-23) encodes the 39 end of the RTegion (774 bp), the IN region (1197 bp), and the 59 end ofhe env region (480 bp). Sequence analysis of this clonehowed that the integrase open reading frame was pre-aturely interrupted by two stop codons and one frame

hift. The corresponding nucleotide sequences andmino acid residues are presented in Fig. 1c. Blast

nterrogation showed that the putative integrase open

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eading frame of clone 87-23 was related to BaEV and

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5RAPID COMMUNICATION

uLV integrases and shared the structural motifs com-on to several retroviral integrases: a zinc finger-likeotif (HHCC) was found in the N-terminus domain (10)

nd a second highly conserved region, referred to as “D,(35) E” (11), was observed in the central region.

MSRV env Region. Clones were obtained by 39 exten-ion from MSRV pol primers and allowed us to define annv gene. CL6 and CL4 clones presented here werebtained from an MS plasma. C15 clone was selected for

he expression of a recombinant MSRV env protein sincet encompassed a large orf presenting 95–100% homol-gy with other shorter MS PCR clones, though obtained

n a parallel study from extracellular particles containingSRV RNA isolated from a patient with RA (12). C15

lone encodes most of the env sequence with a surfacerotein (SU) and a part of the transmembrane protein

TM), whereas CL6 encompasses the 39 end of the TMrotein and the complete U3R region as previously de-cribed. Figure 1d shows the complete env sequenceeconstituted by using two overlapping clones, whichhare about 92% of nucleotide homology with theERV-W env (5). Hydrophobic cluster analysis of itsmino acid sequences showed a putative signal peptide

amino acid positions 1 to 27) and an anchored trans-embrane domain (amino acid positions 441 to 461).

wo putative cleavage sites between SU and TM pro-eins were located in positions 870 and 948 bp, accord-ng to the presence of either hydrophobic or basic aminocids. In C15 clone, the env reading frame is interrupted

FIG. 2. Promotor activity of U3R clones obtained from MS plasmaNA (CL6), normal placenta RNA (PH74, Accession No. AF072506), anduman cell DNA (5M6, Accession No. AF127226). U3R sequences wereloned into pCAT3 Enhancer reporter vector. CAT activity was evalu-ted after 48 h of incubation and represents the promotor efficiency of

he corresponding sequences. The values presented correspond to theeans of three independent experiments.

y a premature stop codon which would thus generate s

n Env protein without signal peptide. Six potential N-inked glycosylation sites, the majority of which are lo-ated in the SU protein, were found. The TM regionhowed a number of conserved peptide tracks, such as

he highly conserved immunosuppressive peptide de-cribed for the p15E of murine leukemia virus (13). In Fig.c, phylogenic analysis based on the conserved regionncompassing the immunosuppressive peptide indi-ates a proximity with type D retroviral Env proteinsepresented by BaEV and MPMV Env proteins. In Fig. 3d,he phylogenic relationship of MSRV env nucleotide se-uence (CL6) with human genomic clones comprisingRV sequences in databases shows that MSRV CL6lusters with HERV-W sequences.

Discussion. The molecular characterization of theSRV-like sequences reported in this study confirmed

hat the first MSRV-pol sequences previously describedelong to a retroviral genome closely related to a noveluman ERV family which we named HERV-W (5).

Many clones obtained by RT-PCR extension in minuteuantities of RNA extracted from extracellular particlesave only partial orf due to the presence of stop codonsr frame shifts. Deletions were also observed in the 39art, particularly when the expected amplified retroviral

egion exceeded 4 kb in length (RT-PCR from pol to9LTR). But the use of internal primers derived from theselones allowed us to amplify the previously deleted re-ions. This could also result from the presence of co-ackaged defective/endogenous RNAs (14), which arereferentially amplified since they are much shorter than

he regular RNA targets, or from the existence of crypticplicing sites in MSRV RNA as shown in HERV-W (5).nder these conditions, the sequences illustrated by thelones described here cannot be attributed to a singleeplication-competent genome since they were not ob-ained from a single genomic clone from a uniqueource. This, however, does not preclude the existencef a “wild-type” genome with similar LTR, gag, pol, andnv sequences which may generate privileged copack-ging, transactivation, and/or recombination withERV-W sequences. Indeed, the clones presented hererovide tools (probes and, further, recombinant proteinsnd antibodies) which will help in isolating such anntity, if present in low percentages within the studiedaterial. They have already been used to characterize

he endogenous HERV-W family (5) which comprises aomplete proviral structure in a unique copy on chromo-ome 7q (contained in human BAC clone RG083M05).his HERV-W provirus encodes a transcriptionally activenv gene (5), but has no orf in gag and pol and cannot by

tself account for particle production.The MSRV sequences obtained are related to the

ncovirinae subfamily; gag and pol sequences are phy-ogenically close to type C retroviruses, whereas env

equences are significantly much closer to primate type

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6 RAPID COMMUNICATION

retroviruses. A recombinatorial origin of type D retro-iral envelope can explain such divergences. Interest-

ngly, a PBS specific for tryptophan tRNA was found inlones encompassing the U5–gag junction. Such tRNA,ever described in human retroviruses, is usual in avianncoviruses.

As shown with CAT assays, the U3R clone derivedrom an MS plasma (CL6) has potent promotor activity,

hereas the HERV-W equivalent from a placental RNA (5)as significantly much lower effect. Further studieshould discover whether specific motifs present in theTR sequence (over)expressed in a pathological contextould be involved in a strong promotor activity, but theorresponding DNA sequence itself is not necessarilyiscriminating an MS-associated genome.

In clones encompassing the gag–pol junction, the gagrf is in frame with the protease–RT orf but separated bystop codon, as it is the case for oncoviruses such asLV (15). Interestingly, the putative MSRV gag polypro-

ein has potential glycosylation sites. This observationas already been reported for MLV gag glycoprotein

FIG. 3. Phylogenic analysis of MSRV nucleotide and amino acid seqL2 orf and gag proteins of other retroviruses. The tree was calculat

epresent the real distances which are represented by the numbers oaLV, gibbon ape leukemia virus; HERV-W, human endogenous retrovi

ovirus; HTLV1, human leukemia virus type 1; IAP, intracisternal A-typonkey virus; MSRV, multiple sclerosis-associated retrovirus eleme

equences were obtained from human genomic databases, with the foRV-9 (M85205, M37638), 265J14 (Z95333), 162C6 (Z84475), HSU90583SRV gag nucleotides based on clone MSRV CL2 orf region and cor

equences from databases. c- means that the sequence is presentedave been described previously (5). (c) Phylogenic tree based on the and D retroviruses. (d) Phylogenic analysis of MSRV env nucleotides (M

elated human genomic sequences from databases. c- means thatAF072506) and C4C5 (AF072508) have been previously described (5).

haracterized as a nonstructural antigen expressed at r

he cell surface, necessary for tumorigenicity or involvedn neurovirulence of neurotropic MLV strains (16).

All retroviral regions identified in particle-associatedNA have endogenous homologs (HERV-W) in normaluman DNA which hybridized in Southern blot withSRV probes (5). The copy number of each retroviral

ene of this HERV-W family varied from about 20 copiesor env to over a hundred for the protease gene (5). Underhese conditions, the limited nucleotide differences ob-erved between particle-associated RNA sequences andERV-W homologs cannot be used to distinguish a par-

icular MSRV provirus from HERV-W copies. Indeed, theercentage of homology between sequences obtained

rom extracellular particles and equivalent regions fromERV-W placental RNA ranges from 83 to 94%, whereas

equence homology between placental HERV-W cDNAlones themselves was 89–100%. It may therefore bessumed that most RNAs packaged into these retrovirus-

ike particles are produced by transcriptional entitiesiffering from HERV-W copy(ies) expressed in placenta.

Further studies are required to understand if observed

. (a) Phylogenic analysis of MSRV gag protein based on clone MSRVthe ClustalW algorythm of GENEWORKS software. The lines do notbranch. BaEV, baboon endogenous virus; FLV, feline leukemia virus;

HIV1, human immunodeficiency virus type 1; HSRV, human spumaret-icle; MLVMO, Moloney murine leukemia virus; MPMV, Mason–PfizerRV-H, squirrel monkey retrovirus H; VISNA, visna retrovirus. HERV

GenBank Accession Nos.: RG083M05 (AC000064), U116E9 (Z95333),3), 49C23 (Z93019), and HS172K2 (Z84814). (b) Phylogenic analysis of

ding region of HERV-W and ERV9 as well as related human genomicrse orientation. HERV-W clones 44.4 (AF072504) and 6A1 (AF 072499)cid sequence of the conserved immunosuppressive domain of type CL6 region) and corresponding region of HERV-W and ERV9 as well asquence is presented in reverse orientation. HERV-W clones PH.74

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FIG. 3—Continued

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8 RAPID COMMUNICATION

ous member of the HERV-W family, as in, e.g., theaagsiekte retrovirus model (17), interfering with endog-nous elements (HERV-W and, possibly, other HERVs); (ii)

replication-competent or trans-complementing mem-er of the HERV-W family; or (iii) a particular HERV-Wopy transactivated and copackaged in particles en-oded by a helper genome, as in, e.g., the murine immu-odeficiency syndrome model associating a pathogenicefective genome and a replicative helper genome inP-BM5 virus (18). The genetic polymorphism of particu-

ar HERV-W copies in human population should also beonsidered in any case.

Recently, the presence of another HERV family (19) haseen reported in retrovirus-like particles from MS cell

ines. These additional observations, if confirmed, woulduggest that different endogenous retroviruses may betimulated and copackaged in retroviral particles in theontext of an autoimmune disease such as MS, but theoundaries and significance of a such a phenomenoneed to be evaluated (14). Nonetheless, the retroviralarticles packaging these RNAs and the associated RTctivity produced in MS cell cultures (1–3, 20) should bencoded by a functional genome, even if resulting from

ecombination events, which remains to be molecularlysolated.

In conclusion, the MSRV-related sequences character-zed by these clones should be considered mainly aselpful tools for the search of a full-length MSRV ge-ome, for the study of pathogenic properties and antige-icity of the corresponding proteins, and for the evalua-

ion of a possible association between MSRV/HERV-Wnd MS and/or other diseases such as RA (12). As an

llustration of such applications, recombinant proteinsorresponding to the MSRV orfs defined by clones en-

T

Primer Sequences Us

Clone Sense primers

LB15 E: 59-GCA ACA GCA ACC CCC TTT GGG T-39

CL2 A: 59-CGG ACA TCC AAA GTG ATG GGA AAC G-39C: 59-CCT AGA ACG TAT TCT GGA GAA TTG GG-39

CL17 I: 59-TCA TGC AAC TGC ACT CTT CTG GTC CG-39K: 59-ATC CCC CAG TAA CAA TTT GGT GAC CAC G-39

87-23 T: 59-TTA CGC AGG TCT CAG GGA TGA GCT T-39U: 59-CGG CAG TAG CAG TCT TAG TAT CTG AAG CAG

C15 M: 59-GCC ATC AAG CCA CCC AAG AAC TCT TAA CTT-3N: 59-CCA ATA GCC AGA CCA TTA TAT ACA CTA ATT-3

CL6 M: 59-GCC ATC AAG CCA CCC AAG AAC TCT TAA CTT-3N: 59-CCA ATA GCC AGA CCA TTA TAT ACA CTA ATT-3

CL4 Q: 59-CGC CTG GTG AGC CTA TTT AAT ACC ACC-39

Note. Some primarily designed primers (e.g., F and G in gag) presexpected region.

ompassing retroviral regions described here have been D

roduced and were used to raise monoclonal and poly-lonal antibodies which will also be helpful in, e.g., im-unohistochemical studies.

Material and Methods. Sera, plasma, and peripherallood cells from MS and control patients were obtained

rom the neurological departments of “Centre Hospitalierniversitaire” in Grenoble, “Hopital Neurologique P. Wer-

heimer” in Lyon, “Centre Hospitalier” in Bourg enresse, and “Centre de Transfusion Sanguine” in Lyon,rance.

B cell lines were obtained by immortalization with theBV B95 strain of PBLs from definite MS patients oron-MS controls. Particle purification from cell cultureupernatant and sucrose density gradients were per-

ormed as previously described (20).Total RNA was extracted from MS plasma or purified

articles by standard acidified guanidium thiocyanaterocedure or by the Viral RNA Extraction kit (Boehringerannheim). After a DNase I treatment, RNA was reverse

ranscribed (Expand RT; Boehringer Mannheim) with ran-om hexanucleotide primers, specific MSRV primer, ornchored oligo(dT) primer and was amplified by nestedr seminested PCR (Long Expand PCR kit—Boerhingerannheim). For each assay, a no-RT control was per-

ormed. 59 and 39 extension from the MSRV pol regionas performed with the primers indicated in Table 1 inrder to obtain the seven overlapping clones presentedere.

PCR fragments were cloned into pCR2.1 of the TAloning kit (Invitrogen) and were sequenced in both di-ections using the Prism Ready Reaction Kit Dye Deoxy-erminator Cycle Sequencing Kit (Applied Biosystems),

ith the Applied Biosystem 377 and 373A automated

btain MSRV Clones

Antisense primers

F: 59-CTT GGA GGG TGC ATA ACC AGG GAA T-39G: 59-CTA TGT CCT TTT GGA CTG CTG TTT GGG T-39B: 59-GGA CAG GAA AGT AAG ACT GAG AAG GC-39D: 59-TGG CTC TCA ATG GTC AAA CAT ACC CG-39J: 59-TCT TGC ACT AAC CTC CAC TGT CCG TTG G-39

L: 59-TCG GGT CTA AGA GGG TAC TTC CTT TGG TAG G-39V: 59-GGT ACG GAG GGT TTC ATG TAG TTT TGA G-39

O: 59-TGG GGT TCC ATT TGT AAG ACC ATC TGT AGC TT-39

P: 59-GAC TCG CTG CAG ATC GAT TTT TTT TTT TTT TTT T-39

R: 59-GAT TTT GAA GGA CTA CTG CTG CTA GGG AG-39S: 59-GAG TAG AGG TTG TGA TAC TGC CAA TGC C-39

ismatches with overlapping clones presented here, but amplified the

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9RAPID COMMUNICATION

ut using the MacVector 4.5 software (Kodak) or Gene-orks software. The sequence was also compared, both

t the nucleotide and at the protein level, to all majorequence databases (GenBank, EMBL, PIR, SWISS-ROT, Dbest), using BLAST similarity search softwares

BLASTN, BLASTP, TBLASTN). We used the databasesnr” provided by the NCBI GENINFO Experimental BLAST

etwork Service. Phylogenetic trees were calculatedith the UPGMA tree algorithm from Geneworks software.U3R regions from CL6, PH74, and 5M6 clones (respec-

ively obtained from MS plasma RNA, placenta RNA, anduman DNA) were cloned into pCAT3 (Promega–Biotech,adison, WI) for CAT assays. Transfection experimentsere performed on HeLa, PG4, BeWo, or Jurkat cellssing the Superfect Transfection Kit (Qiagen GmbH, Ger-any) with 2 mg of purified recombinant plasmid. After

8 h of incubation, the cells were harvested in order tovaluate CAT activity by the use of the CAT Enzymessay System (Promega–Biotech). For this purpose the

iquid scintillation counting protocol was followed asecommended by the manufacturer. A positive controlonsisted of cells transfected by 2 mg of pCAT3 Control

Promega–Biotech).

ACKNOWLEDGMENTS

F. Komurian-Pradel and G. Paranhos-Baccala have participatedqually in the molecular characterization presented in this report. We

hank Professor J. Pellat and Dr. O. Moreau (Centre Hospitalier Univer-itaire Grenoble, France), Dr. P. Giraud, Professors E. Broussolle and G.hazot (Hopital Neurologique P. Wertheimer, Lyon), and Drs. Boulliatnd Lemaitre (Centre Hospitalier, Bourg en Bresse), for providing usith MS samples. We also thank Carine Malcus-Vaucanson (bio-erieux, Lyon) for her assistance in bioethics, as well as F. Penin and. Deleage for their assistance on nucleic acids analysis. This workas supported by bioMerieux S. A.

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