# Patent

## Title
LTR region of MSRV-1 and the proteins it encodes, and probes and methods for detecting MSRV-1 retrovirus

## Abstract

The invention relates to: a nucleotide fragment of an MSRV-1 LTR-RU5 region, comprising a nucleotide sequence encoding the expression of a protein, wherein the protein comprises a peptide sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4; complementary nucleotide fragments; probes and primers that hybridize to the fragment; proteins encoded by the fragment; antibodies directed against the proteins encoded by the fragment; and processes for detecting the presence of MSRV-1 using a probe or an antibody of the invention.

## Background
This is a Continuation of Application No. 09/890,340, now abandoned, which is a U.S. National Stage Application of PCT/IB00/00159, filed Feb. 15, 2000. The entire disclosure of the prior applications is hereby incorporated by reference herein in its entirety.


Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) the cause of which remains as yet unknown.
Many studies have supported the hypothesis of a viral aetiology of the disease, but none of the known viruses tested has proved to be the causal agent sought: a review of the viruses sought for several years in MS has been compiled by E. Norrby and R. T. Johnson.
Recently, a retrovirus different from the known human retroviruses has been isolated in patients suffering from MS. The authors were also able to show that this retrovirus could be transmitted in vitro, that patients suffering from MS produced antibodies capable of recognizing proteins associated with the infection of leptomeningeal cells by this retrovirus, and that the expression of the latter could be strongly stimulated by the immediate-early genes of some herpesviruses.
All these results point to the role in MS of at least one unknown retrovirus or of a virus having reverse transcriptase activity which is detectable according to the method published by H. Perron and qualified as “LM7-like RT” activity.
The Applicant's studies have enabled two continuous cell lines infected with natural isolates originating from two different patients suffering from MS to be obtained by a culture method as described in U.S. Pat. No. 5,650,318, the content of which is incorporated in the present description by reference. These two lines, derived from human choroid plexus cells, designated PLI-2 and LM7PC, were deposited with the ECACC on 22nd July 1992 and 8th January 1993, respectively, under numbers 92072201 and 93010817, in accordance with the provisions of the Budapest Treaty. Moreover, the viral isolates possessing LM7-like RT activity were also deposited with the ECACC under the overall designation of “strains”. The “strain” or isolate harboured by the PLI-2 line, designated POL-2, was deposited with the ECACC on 22nd July 1992 under No. V92072202. The “strain” or isolate harboured by the LM7PC line, designated MS7PG, was deposited with the ECACC on 8th January 1993 under No. V93010816.
Starting from the cultures and isolates mentioned above, characterized by biological and morphological criteria, the next step was to endeavour to characterize the nucleic acid material associated with the viral particles produced in these cultures.
The portions of the genome which have already been characterized have been used to develop tests for molecular detection of the viral genome and immunoserological tests, using the amino acid sequences encoded by the nucleotide sequences of the viral genome, in order to detect the immune response directed against epitopes associated with the infection and/or viral expression.
The viral system discovered by the Applicant is related to a complex retroviral system. In effect, the sequences to be found encapsidated in the extracellular viral particles produced by the different cultures of cells of patients suffering from MS show clearly that there is coencapsidation of retroviral genomes which are related but different from the “wild-type” retroviral genome which produces the infective viral particles. This phenomenon has been observed between replicative retroviruses and endogenous retroviruses belonging to the same family, or even heterologous retroviruses. The notion of endogenous retroviruses is very important. In the case of MSRV-1, it has been observed that endogenous retroviral sequences comprising sequences homologous to the MSRV-1 genome exist in normal human DNA. The existence of endogenous retroviral elements (ERV) related to MSRV-1 by all or part of their genome explains the fact that the expression of the MSRV-1 retrovirus in human cells is able to interact with closely related endogenous sequences.
Defective clones which only express proteins may be involved in the pathology.
The Applicant has made an unexpected discovery, according to which RU5 region of a retroviral LTR that is defined in the present invention, encodes the expression of at least one protein. This is unusual for LTRs, in particular in the RU5 region.
The present invention first relates to a nucleotide fragment of a LTR-RU5 region comprising a nucleotide sequence which encodes the expression of a protein, wherein said protein comprises at least six, preferably at least eight, and more preferably at least twelve, amino acids of a peptide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and a complementary nucleotide fragment.
Advantageously, a nucleotide fragment of the invention, or the complementary nucleotide fragment thereof, comprises a nucleotide sequence which encodes the expression of a protein, wherein said protein comprises a peptide sequence selected from SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and complementary nucleotide fragment. Preferably said protein consists of SEQ ID NO:3 or comprises or consists of SEQ ID NO:2 and SEQ ID NO:4.
The invention also relates to the following matter:

    
    
        a nucleic acid probe for the detection of MSRV-1 retrovirus, which comprises 10 to 1000 monomers and specifically hybridizes with the nucleotide fragment of the invention, in high stringency conditions;
        a primer for the amplification by polymerization of a nucleic acid retroviral sequence of MSRV-1 virus, which comprises 10 to 30 monomers and hybridizes with the nucleotide fragment of the invention, in high stringency conditions
        a protein encoded by a nucleotide fragment of the invention preferably, said protein comprises a peptide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, or consists of a peptide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:2 and SEQ ID NO:4;
        a polypeptide comprising at least six, preferably at least eight, and more preferably at least twelve, amino acids of SEQ ID NO:4;
        a polyclonal or monoclonal antibody directed against a protein of the invention or a polypeptide of the invention;
        a process for detecting, in a biological sample, the presence of MSRV-1 retrovirus comprising:
    
    


contacting a probe of the invention with said biological sample, determining whether the probe binds to a nucleic acid in said biological sample, wherein binding indicates the presence of MSRV-1 virus; said process may comprise an amplification step wherein said nucleic acid is amplified with a primer of the invention;

    
    
        a process for detecting, in a biological sample, the presence of MSRV-1 retrovirus comprising:
    
    


contacting an antibody of the invention with said biological sample,
determining whether the antibody binds to a protein of the invention, in said biological sample, wherein binding indicates the presence of MSRV-1 virus;

    
    
        a process for detecting, in a biological sample, the presence of MSRV-1 retrovirus comprising detecting the antigenic or biological properties of a protein of the invention or a fragment thereof; advantageously, said protein fragment is a polypeptide of the invention.
    
    


Before describing the invention in detail, different terms used in the description and the claims are now defined:

    
    
        the term “MSRV” used in the present description denotes any pathogenic and/or infective agent associated with MS, in particular a viral species, the attenuated strains of the said viral species or the defective-interfering particles or particles containing coencapsidated genomes, or alternatively genomes recombined with a portion of the MSRV-1 genome, derived from this species. Viruses, and especially viruses containing RNA, are known to have a variability resulting, in particular, from relatively high rates of spontaneous mutation;
        human virus is understood to mean a virus capable of infecting, or of being harboured by human beings,
        according to the invention, a nucleotide fragment or an oligonucleotide or polynucleotide is an arrangement of monomers, or a biopolymer, characterized by the informational sequence of the natural nucleic acids, which is capable of hybridizing with any other nucleotide fragment under predetermined conditions, it being possible for the arrangement to contain monomers of different chemical structures and to be obtained from a molecule of natural nucleic acid and/or by genetic recombination and/or by chemical synthesis; a nucleotide fragment may be identical to a genomic fragment of the MSRV-1 virus discussed in the present invention;
        thus, a monomer can be a natural nucleotide of nucleic acid whose constituent elements are a sugar, a phosphate group and a nitrogenous base; in RNA the sugar is ribose, in DNA the sugar is 2-deoxyribose; depending on whether the nucleic acid is DNA or RNA, the nitrogenous base is chosen from adenine, guanine, uracil, cytosine and thymine; or the nucleotide can be modified in at least one of the three constituent elements; as an example, the modification can occur in the bases, generating modified bases such as inosine, 5-methyldeoxycytidine, deoxyuridine, 5-(dimethylamino)deoxyuridine, 2,6-diaminopurine, 5-bromodeoxyuridine and any other modified base promoting hybridization; in the sugar, the modification can consist of the replacement of at least one deoxyribose by a polyamide, and in the phosphate group, the modification can consist of its replacement by esters chosen, in particular, from diphosphate, alkyl- and arylphosphonate and phosphorothioate esters;
        <<informational sequence) is understood to mean any ordered succession of monomers whose chemical nature and order in a reference direction constitute or otherwise an item of functional information of the same quality as that of the natural nucleic acids;
        hybridization is understood to mean the process during which, under suitable working conditions, two nucleotide fragments having sufficiently complementary sequences pair to form a complex structure, in particular double or triple, preferably in the form of a helix in particular in high stringency conditions (see Maniatis et al., Molecular Cloning, Cold Spring Harbor, 1982); in general, depending on the length of the probes used, these conditions are the following: the temperature for the hybridization reaction is between approximately 20° C. and 65° C., and especially between 35° C. and 65° C., in a saline solution at a concentration of approximately 0.8 to 1 M;
        a probe comprises a nucleotide fragment synthesized chemically or obtained by digestion or enzymatic cleavage of a longer nucleotide fragment, comprising at least six monomers, advantageously from 10 to 100 monomers and preferably 10 to 30 monomers, and possessing a specificity of hybridization under high stringency conditions; preferably, a probe possessing fewer than 10 monomers is not used alone, but is used in the presence of other probes of equally short size or otherwise; under certain special conditions, it may be useful to use probes of size greater than 100 monomers; a probe may be used, in particular, for diagnostic purposes, such molecules being, for example, capture and/or detection probes;
        the capture probe may be immobilized on a solid support by any suitable means, that is to say directly or indirectly, for example by covalent bonding or passive adsorption
        the detection probe may be labelled by means of a label chosen, in particular, from radioactive isotopes, enzymes chosen, in particular, from peroxidase and alkaline phosphatase and those capable of hydrolysing a chromogenic, fluorogenic or luminescent substrate., chromophoric chemical compounds, chromogenic, fluorogenic or luminescent compounds, nucleotide base analogues and biotin;
        the probes used for diagnostic purposes of the invention may be employed in all known hybridization techniques, and in particular the techniques termed <<DOT-BLOT>>, <<SOUTHERN BLOT>>, <<NORTHERN BLOT>>, which is a technique identical to the <<SOUTHERN BLOT>> technique but which uses RNA as target, and the SANDWICH technique; advantageously, the SANDWICH technique is used in the present invention, comprising a specific capture probe and/or a specific detection probe, on the understanding that the capture probe and the detection probe must possess an at least partially different nucleotide sequence,
        a primer is a probe comprising at least six monomers, and advantageously from 10 to 30 monomers, possessing a specificity of hybridization under high stringency conditions for the initiation of an enzymatic polymerization, for example in an amplification technique such as PCR (polymerase chain reaction), in an elongation process such as sequencing, in a method of reverse transcription or the like;
    
    


In view of the fact that a virus possessing reverse transcriptase enzymatic activity may be genetically characterized equally well in RNA and in DNA form, both the viral DNA and RNA will be referred to for characterizing the sequences relating to a virus possessing such reverse transcriptase activity, termed MSRV-1 according to the present description.
Detection of a substance or agent is understood below to mean both an identification and a quantification, or a separation or isolation, of the said substance or said agent.



A better understanding of the invention will be gained on reading the detailed description which follows, prepared with reference to the attached figures, in which:



FIG. 1 shows the 5′ nucleotide sequence of the RU5 region obtained from clone LB15 nucleotides 1 to 623 of SEQ ID NO:14) and the 5′ gag region obtained from clones CL2 (nucleotides 624 to 1680 of SEQ ID NO:14) and CL17 nucleotides 1681 to 2052 of SEQ lID NO:14). The amino acid (AA) translation is shown under the gag nucleotide sequence (SEQ ID NOS:15-17, wherein AA 1-420 are found in SEQ ID NO:15, AA 422-469 are found in SEQ ID NO:16, and AA 471-486 are found in SEQ ID NO:17). The polyadenylation site in R is boxed and the polyadenylation downstream signal in U5 is underlined. (II) indicates a frameshift and (.) represents a stop codon. Amino acid positions in bold correspond to the major homology region (MHR) in the sequence encoding the capsid. Underlined amino acid positions correspond to the conserved positions in the sequence encoding the nucleocapsid.
FIG. 2 shows the 5′ env region obtained from clone C15 (nucleotides 1 to 1479 of SEQ ID NO:18) and the 3′ env and LTR sequences obtained from clone CL6 (nucleotides 1480 to 2030 of) (SEQ ID NO:18). The amino acid (AA) translation is shown under the env nucleotide sequence (SEQ ID NOS:19 and 20, wherein AA 1-38 are found in SEQ ID NO:19 and AA 39-542 are found in SEQ ID NO:20). Horizontal arrows indicate the env region and the U3, and R substructures. In the env region, the signal peptide and the putative immunosuppressive peptide are underlined, the N-linked glycosylation sites are boxed, and the two putative cleavage sites are indicated by vertical arrows. In the U3R region; the CAAT regulatory element, the polyA signal, and the TATA box are underlined, and the cap site is indicated.
FIG. 3 is a graph of the promoter activity of U3R clones obtained from MS plasma RNA (CL6), normal placenta RNA (PH74, accession number AF072506) and human cell DNA (5M6). U3R sequences were cloned into the pCAT3 Enhancerr reporter vector. CAT activity, evaluated after 48 hours of incubation, represents the promoter efficiency of the corresponding sequences. The values presented correspond to the mean of 3 independent experiments.
EXAMPLE 1:
Determination of MSRV-LTR regions
A RT-PCR amplification was performed with antisense 3′ primers located in the gag region and a 5′ sense primer defined from the R sequence previously obtained in the 3′ end (clone 6, described below). The clone LB15 (SEQ ID NO:1), encompassing R (87 bp), U5 (456 bp), PBS, and 5′ gag regions, was thus obtained from concentrated culture particles. A polyadenylation site is compatible with the “CA” dinucleotide motif located at the junction of R and U5 regions and a putative poly(A) downstream signal is located at a distance of 24 nucleotides downstream of the poly(A) signal, with the consensus sequence “YGTGTTYY” (SEQ ID NO:5). The putative primer-binding site (PBS), identified downstream of the U5 region, has proved to be related to the 3′ end of the avian tRNATrp complementary sequence.
It is also noteworthy that 2 rather short in-frame orfs (SEQ ID NO:2 and SEQ ID NO:3 were found in the RU5 region. The 3′ LTR U3R region was identified in the CL6 clone (FIG. 2) obtained by amplification on DNase-treated RNA extracted from MS plasma. By comparison with the 5′ LTR sequences obtained by 5′ extension on MSRV RNA, a 299 bp-long U3 region and a 84 bp-long R region in CL6 LTR region, located downstream the env orf and ending with the polyA-tail has been identified. The typical regulatory element CAAT box was observed in two locations in the putative U3 region, but other putative binding sites for several transcription factors were also detected with Transcription Factor database (Heinemeyer T, Wingender, E, Reuter, I, Hermjakob, H, Kel, A E, Kel, O V, Ignatieva, E V, Ananko, E A, Podkolodnaya, O A, Kolpakov, F A, Podkolodny, N L, and Kolchanov, N A. (1998). Databases on transcriptional regulation: TRANSFAC, TRRD, and COMPEL. Nucleic Acids Res. 26, 364-370).
A TATA box (TATAAA) was observed in the U3 region as described for numerous retroviruses. Finally, a poly(A) signal (AATAAA) was found 83 bp downstream of the TATA box.
In order to evaluate the promotor activity of LTR clones from different origins, CAT assays with sub-cloned U3R regions from LTR clones obtained from MS plasma RNA (CL6) and from related HERV-W copies in non-MS DNA (5M6) and in non-MS placenta RNA (PH74) were performed. As shown in FIG. 3, a potent activity was associated with the LTR sequence in CL6 clone from MS plasma, whereas a weak and moderate promotor activity was found with 5M6 and PH74 clones respectively. Similar results were obtained with different cell types.
EXAMPLE 2
Material and Methods Used in Example 1
Total RNA was extracted from MS plasma or purified particles by standard acidified guanidium thiocyanate procedure or by the <<viral RNA extraction kit>> (Boehringer Mannheim). After a DNase I treatment, RNA was reverse transcribed (ExpandTM RT, Boehringer Mannheim) with either random hexanucleotide primers, specific MSRV primer or anchored oligodT primer, and was amplified by nested or semi-nested PCR (Long Expand PCR kit—Boerhinger Mannheim). For each assay, a no-RT control was performed 5′ and 3′ extension from the MSRV pol region was performed to obtain clones with primers indicated below.









LB15:



Sense primer







(SEQ ID NO:8)









5′GCAACAGCAACCCCCTTTGGGT 3′



 



Antisense primers







(SEQ ID NO:9)









5′CTTGGAGGGTGCATAACCAGGGAAT 3′



 







(SEQ ID NO:10)









5′CTATGTCCTTTTGGACTGCTGTTTGGGT 3′



 



CL6:



Sense primers







(SEQ ID NO:11)









5′GCCATCAAGCCACCCAAGAACTCTTAACTT 3′



 







(SEQ ID NO:12)









5′CCAATAGCCAGACCATTATATACACTAATT 3′



 



Antisense primer







(SEQ ID NO:13)









5′GACTCGCTGCAGATCGATTTTTTTTTTTTTTTTT 3′






PCR fragments were cloned into a pCR2.1 of the TA cloningTM kit (Invitrogen) and were sequenced in the both directions using the ‘Prism ready reaction kit dye deoxyterminator cycle sequencing kit’ (Applied Biosystems), with the Applied Biosystem 377 and 373A automated DNA sequencers.
U3R regions from CL6 (as shown in WO-99/02666 in the name of Applicant), PH74 (as shown in WO-99/02696 in the name of Applicant) and 5M6 (as shown in WO-99/02666 in the name of Applicant) clones (respectively obtained from MS plasma RNA, placenta RNA and Human DNA), were cloned into pCAT3 (Promega-Biotech, Madison, Wis., USA) for CAT assays. Transfection experiments were performed on Hela, PG4, BeWo or Jurkat cells using the Superfect Transfection Kit (Qiagen GmbH, Germany) with 2 μg of purified recombinant plasmid. After 48 h of incubation, the cells were harvested in order to evaluate CAT activity by the use of the CAT Enzyme Assay System (Promega-Biotech). For this purpose the Liquid Scintillation Counting (LSC) protocol was followed as recommended by the manufacturer. A positive control consisted of cells transfected by 2 μg of pCAT3 Contol′ (Promega-Biotech).

## Claims

1. An isolated nucleotide fragment comprising a nucleotide sequence that encodes a protein having a sequence comprising SEQ ID NO: 2, wherein the encoding nucleotide sequence is located in the RU5 region of MSRV-1.

2. The nucleotide fragment of claim 1, wherein said protein has a sequence comprising SEQ lID NO: 2.

3. The isolated nucleotide fragment of claim 1, wherein said protein has a sequence comprising SEQ ID NO: 4.

4. The isolated nucleotide fragment of claim 1, wherein said protein has a sequence consisting of SEQ ID NO: 2 or SEQ ID NO: 4.