Endogenous retrovirus-k (ERVK) encodes an alternate envelope protein

The present disclosure relates to an endogenous Retrovirus K protein (ERVK) with an alternative envelope protein titled CTXLP. Said CTXLP peptide is represented by the sequences set forth in SEQ ID NO: 1. Additionally, antibodies that specifically recognize the epitope(s) set forth in SEQ ID NO:1 are and methods of use thereof and kits comprising the peptide set forth in SEQ ID NO:1 are also included in the present disclosure.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created May 21, 2021, is named 51012-032001_Sequence_Listing_5_21_21_ST25 and is 130,650 bytes in size.

FIELD

The present disclosure relates generally an endogenous Retrovirus-K (ERVK) alternate envelope protein.

BACKGROUND

Conotoxins are neurotoxic peptides found in theConusgenus of marine snails used to immobilize prey22. Conusspecies are distinct in their ability to produce hundreds of different toxic peptides23. Conotoxins are disulfide-rich and are usually 10-30 amino acids in length22. Conotoxins act as antagonists to specific voltage and ligand-gated ion channels22. In humans, symptoms of conotoxin exposure include poor coordination, blurred vision, speech difficulties, and nausea23. Conotoxins have also been associated with episodes of delirium and psychosis24.

The O-superfamily of conotoxins exhibits an ICK fold. Members of the O-superfamily include μ-conotoxins, which inhibit voltage-gated sodium channels, and d-conotoxins, which delay sodium channel inactivation25. K-Conotoxins are inhibitors of voltage-gated potassium channels; ω-conotoxins inhibit N-type voltage-gated calcium channels (VGCCs)25. N-type VGCCs are located in presynaptic nerve terminals and are involved in neurotransmitter release26. ω-Conotoxin's selectivity for N-type VGCCs has allowed for their development as therapeutic agents. The ω-conotoxin MVIIA has been developed into a drug for relief of chronic and inflammatory pain27.

Genes encoding an ω-conotoxin-like protein (CTXLP) have also been identified in certain viruses. Nuclear polyhedrosis viruses (NPV) have been shown to secrete a small conotoxin-like peptide28. NPVs are insect pathogens belonging to the family baculoviridae28. Although NPV-CTXLP's function has not been elucidated, its structure was found to have a nearly identical structure to the conserved ω-conotoxin's cysteine motif28.

SUMMARY

In one aspect there is described an isolated polypeptide that comprises or consists of: an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in SEQ ID NO:1 (CSDYGINCSHSYGCCSRSCIALFC).

In one example the isolated polypeptide comprises or consists of an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:1.

In one example the isolated polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:1.

In one aspect there is described an isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in SEQ ID NO:1.

In one example the isolated nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 90% identity with the nucleotide acid sequence encoding the polypeptide of SEQ ID NO: 1.

In one example the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 95% identity with the nucleotide acid sequence encoding the polypeptide of SEQ ID NO: 1.

In one aspect there is described a vector comprising the nucleic acid molecule according to any one of claims4to6.

In one aspect there is described a mammalian cell comprising the nucleic acid molecule of any one of claims4to6.

In one example said mammalian cell is a human cell or non-human primate cell.

In one aspect there is described a host cell comprising the nucleic acid molecule of any one of claims4to6.

In one example said host cell is a mammalian cell, an insect cell (such asDrosophila melanogaster), a bacteria cell, or a fungal cell.

In one aspect there is described a method for producing the peptide comprising: culturing a mammalian cell, or a host cell in a culture medium; and isolating the peptide from the mammalian cell, or host cell, or culture medium thereof.

In one aspect there is described an antibody that specifically recognizes the peptide of any one claims1to3.

In one example said antibody is a monoclonal antibody or a polyclonal antibody.

In one aspect there is described a method for treating or preventing conditions or disorders associated with CTXLP in a subject, comprising: administering to a subject in need thereof a therapeutically effective amount of active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity and/or CTXLP associated pathology.

In one aspect there is described a method for treating or preventing conditions or disorders associated with ERVK in a subject, comprising: administering to a subject in need thereof a therapeutically effective amount of an active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits CTXLP activity and/or CTXLP associated pathology.

In one example said condition or disorder is an infectious disease.

In one example said infection disease is HSV infection, HIV infection, EBV infection, HTLV infection,Toxoplasma Gondiiinfection, HSV infection, or prion disease.

In one example said condition or disorder is a neurological disease.

In one example said neurological disease is amyotrophic lateral sclerosis (ALS), bipolar disorder, Kennedy's disease, multiple sclerosis, or schizophrenia.

In one example said condition or disorder is a cancer.

In one example said associated pathology is a change in CNS function of said subject, a developmental disorder, a stroke, Alzheimer's disease, spinal cord injury, cerebral ischemia, Huntington's disease, Parkinson's disease, a peripheral neuropathy, or epilepsy ocular disease.

In one example said active agent a small molecule, an antibody, a nucleic acid, an aptamer, or a peptide.

In one example said active agent comprises a Michael acceptor electrophile (MAE).

In one example said active agent comprises gambogic acid.

In one example said active agent comprises celastrol.

In one example said active agent is a small molecule or antibody reversing CTXLP blockade on oligodendrocyte precursor cell maturation and oligodendrocyte myelination, such as clemastine fumarate.

In one example further comprising administering a human anti-Nogo-A antibody.

In one example said active agent is a small molecule enhancer of CaV2.2 and its calcium channel associated transcription regulator (CaV2.2 CCAT) expression or activity, such as EGTA, or glutamate.

In one aspect there is described a use of a therapeutically effective amount of active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity and/or CTXLP associated pathology for treating or preventing conditions or disorders associated with CTXLP in a subject.

In one aspect there is described a use of a therapeutically effective amount of active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity and/or CTXLP associated pathology in the manufacture of a medicament for treating or preventing conditions or disorders associated with CTXLP in a subject.

In one aspect there is described a use of a therapeutically effective amount of an active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits CTXLP activity and/or CTXLP associated pathology for treating or preventing conditions or disorders associated with ERVK in a subject.

In one aspect there is described a use of a therapeutically effective amount of an active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits CTXLP activity and/or CTXLP associated pathology in the manufacture of a medicament for treating or preventing conditions or disorders associated with ERVK in a subject.

In one example said condition or disorder is an infectious disease.

In one example said infection disease is HSV infection, HIV infection, EBV infection, HTLV infection,Toxoplasma Gondiiinfection, HSV infection, or prion disease.

In one example said condition or disorder is a neurological disease.

In one example said neurological disease is amyotrophic lateral sclerosis (ALS), bipolar disorder, Kennedy's disease, multiple sclerosis, or schizophrenia.

In one example said condition or disorder is a cancer.

In one example said associated pathology is a change in CNS function of said subject, a developmental disorder, a stroke, Alzheimer's disease, spinal cord injury, cerebral ischemia, Huntington's disease, Parkinson's disease, a peripheral neuropathy, or epilepsy ocular disease

In one example said active agent a small molecule, an antibody, a nucleic acid, an aptamer, or a peptide.

In one example said active agent comprises a Michael acceptor electrophile (MAE).

In one example said active agent comprises gambogic acid.

In one example said active agent comprises celastrol.

In one example said active agent is a small molecule or antibody reversing CTXLP blockade on oligodendrocyte precursor cell maturation and oligodendrocyte myelination, such as clemastine fumarate.

In one example further comprising the use of a human anti-Nogo-A antibody.

In one example said active agent is a small molecule enhancer of CaV2.2 and its calcium channel associated transcription regulator (CaV2.2 CCAT) expression or activity, such as EGTA, or glutamate.

In one aspect there is described a method for transcriptional activation, comprising contacting a DNA molecule comprising a gene with a peptide of any one of claims1to3.

In one aspect there is described a diagnostic reagent for use in the detection of CTXLP protein in a subject, comprising an antibody.

In one aspect there is described a diagnostic reagent for use in the detection of CTXLP mRNA in a subject, comprising an isolated nucleic acid according to any one of claims4to6.

In one aspect there is described a diagnostic reagent for use in the detection CTXLP activity in a subject, comprising a peptide of any one of claims1to3.

In one aspect there is described a method for treating or preventing conditions or disorders associated with CTXLP in a subject, comprising: measuring an amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA; and administering to a subject in need thereof a therapeutically effective amount of an active agent optionally in a physiological carrier or a pharmaceutically acceptable salt thereof when the amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA, is high, optionally compared to a control, wherein the active agent blocks or inhibits the CTXLP activity and/or CTXLP associated pathology.

In one aspect there is described a method for treating or preventing conditions or disorders associated with ERVK in a subject, comprising: measuring an amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA; and administering to a subject in need thereof a therapeutically effective amount of an active agent optionally in a physiological carrier or a pharmaceutically acceptable salt thereof when the amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA, is high, optionally compared to a control, wherein the active agent blocks or inhibits the CTXLP activity and/or CTXLP associated pathology.

In one example said condition or disorder is an infectious disease.

In one example said infection disease is HSV infection, HIV infection, EBV infection, HTLV infection,Toxoplasma Gondiiinfection, HSV infection, or prion disease.

In one example said condition or disorder is a neurological disease.

In one example said neurological disease is amyotrophic lateral sclerosis, bipolar disorder, Kennedy's disease, multiple sclerosis, or schizophrenia.

In one example said condition or disorder is a cancer.

In one example said associated pathology is a change in CNS function of said subject, a developmental disorder, a stroke, Alzheimer's disease, spinal cord injury, cerebral ischemia, Huntington's disease, Parkinson's disease, a peripheral neuropathy, or epilepsy ocular disease

In one example said active agent a small molecule, an antibody, a nucleic acid, an aptamer, or a peptide.

In one example said active agent comprises a Michael acceptor electrophile (MAE).

In one example said active agent comprises gambogic acid.

In one example said active agent comprises celastrol.

In one example said active agent is a small molecule or antibody reversing CTXLP blockade on oligodendrocyte precursor cell maturation and oligodendrocyte myelination, such as clemastine fumarate.

In one example, further comprising administering a human anti-Nogo-A antibody.

In one example said active agent is a small molecule enhancer of CaV2.2 and its calcium channel associated transcription regulator (CaV2.2 CCAT) expression or activity, such as EGTA, or glutamate.

In one example the amount of CTXLP polypeptide is determined using an antibody.

In one aspect there is described a kit comprising: (a) a container comprising a pharmaceutical composition containing the peptide, and/or a nucleic acid, and/or a vector, a mammalian cell, a host cell, and/or an antibody, in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; and (c) optionally, instructions for use.

In one example further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.

In one aspect there is described a method identifying CTXLP inhibitors, comprising: contacting a mammalian cell or a host cell, such as an insect cell (such asDrosophila melanogaster), a bacteria cell, or a fungal cell, with a test compound or test composition, and measuring an amount of CTXLP protein, CTXLP-mRNA, CTXLP-regulated gene, or CTXLP-associate biomarker.

In one aspect there is described a method identifying CTXLP inhibitors, comprising: contacting a organoid, with a test compound or test composition, and measuring an amount of CTXLP protein, CTXLP-mRNA, CTXLP-regulated gene, or CTXLP-associate biomarker

DETAILED DESCRIPTION

In one aspect, there is described herein the identification of a region in the ERVK provirus DNA which encodes a conotoxin-like polypeptide, and which may have significance in ERVK pathogenesis. In a specific example, the polypeptide is CTXLP (CSDYGINCSHSYGCCSRSCIALFC) (SEQ ID NO: 1).

In one example, there is described an isolated polypeptide that comprises or consists of: an amino acid sequence having at least about 70% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 75% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 80% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 99% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 100% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 70% identity to about 100% identify with the amino acid sequence set forth in SEQ ID NO:1.

In one example, there is described an isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence having at least about 70% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 75% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 80% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 99% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 100% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 70% to about 100% identity with the amino acid sequence set forth in SEQ ID NO:1.

The term “isolated”, as used herein, refers to altered or removed from the natural state. For example, a polypeptide or nucleic acid naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

Unless otherwise specified, a “nucleotide sequence encoding a polypeptide” (and the like) includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a polypeptide protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

In some examples, there is described a vector comprising the nucleic acid molecule described above and herein.

The term “vector” or “expression vector” as used herein refers to a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. In on example, the vector is a pcDNA3.1 vector.

“Similarity”, for example between two peptides, may be determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to a sequence of a second polypeptide. Variants are defined to include peptide sequences different from the original sequence, for example, different from the original sequence in less than 40% of residues per segment of interest, different from the original sequence in less than 25% of residues per segment of interest, different by less than 10% of residues per segment of interest, or different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence.

The term “sequence identity” of a polypeptide or polynucleotide as used herein refers to a degree of sameness in an amino acid residue or a base in a specific region of two sequences that are aligned to best match each other for comparison. The sequence identity is a value obtained via alignment and comparison of the two sequences in the specific region for comparison, in which a partial sequence in the specific region for comparison may be added or deleted with respect to a reference sequence. The sequence identity represented in a percentage may be calculated by, for example, comparing two sequences that are aligned to best match each other in the specific region for comparison, determining matched sites with the same amino acid or base in the two sequences to obtain the number of the matched sites, dividing the number of the matched sites in the two sequences by a total number of sites in the compared specific regions (i.e., a size of the compared region), and multiplying a result of the division by 100 to obtain a sequence identity as a percentage. The sequence identity as a percentage may be determined using a known sequence comparison program, for example, BLASTP or BLASTN (NCBI), CLC Main Workbench (CLC bio), or MegAlign™ (DNASTAR Inc).

A polypeptide of may be synthesized by conventional techniques. For example, the peptides may be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods. Automated synthesis may be used.

In some example, a polypeptide may be produced by culturing a cell comprising a nucleic acid which encoded the polypeptide, and isolating the polypeptide from the host cell or culture medium thereof.

The peptides of the invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes orXenopusegg extracts to a standard translation reaction.

In some examples, the polypeptides described herein may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.

A “cell” or “host cell” refers to an individual cell or cell culture that can be or has been a recipient of any recombinant vector(s), isolated polynucleotide, or polypeptide. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell which comprises a recombinant vector of the invention is a recombinant host cell.

In one example, the host cell is a cell obtained or derived from a subject.

In one example the cell host is a human cell.

In one example, the cell is SVGA (astrocytes), RenCell CX (neuroprogenitor cells), or NCCIT (teratocarcinoma).

In some examples, there is described an antibody that specifically binds to a polypeptide as described herein. In one example, the polypeptide comprises or consists of the sequence of SEQ ID NO: 1.

The term “antibody” or “antibodies” is used herein refers to both polyclonal and monoclonal antibodies. In addition to intact or “full” immunoglobulin molecules, also included in the term “antibodies” are fragments (e.g., CDRs, Fv, Fab and Fc fragments) or polymers of those immunoglobulin molecules and humanized versions of immunoglobulin molecules, as long as they exhibit any of the desired properties according to the description.

Antibodies of the description may also be generated using well-known methods.

In some examples, a polypeptide may be used for generating an antibody of the description may be partially or fully purified from a natural source, or may be produced using recombinant DNA techniques.

In some examples, the antibodies may be purchased commercially.

In some examples, the generation of two or more different sets of monoclonal or polyclonal antibodies may maximize or increase the likelihood of obtaining an antibody with the specificity and affinity required for its intended use.

The antibodies produced may tested for their desired activity by known methods, in accordance with the purpose for which the antibodies are to be used (e.g., Immunoblooting, ELISA, immunohistochemistry, immunotherapy, etc).

For example, antibodies may be tested in ELISA assays or, Western blots, immunohistochemical staining of formalin-fixed or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested according to known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e.; the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity.

Monoclonal antibodies may be prepared using hybridoma methods. In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods.

In some example, the antibodies are humanized antibodies. Methods for humanizing non-human antibodies are well known in the art.

In some examples, antibodies may be labeled with probes suitable for detection by various imaging methods. Methods for detection of probes include, but are not limited to, fluorescence, light, confocal and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography and positron emission tomography.

Examples of probes may include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium and other lanthanides, paramagnetic iron, fluorine-18 and other positron-emitting radionuclides. Antibodies may be directly or indirectly labeled with said probes. Attachment of probes to the antibodies includes covalent attachment of the probe, incorporation of the probe into the antibody, and the covalent attachment of a chelating compound for binding of probe, amongst others well recognized in the art.

In one example, there is described a method for treating or preventing conditions or disorders associated with CTXLP in a subject, comprising: administering to a subject in need thereof a therapeutically effective amount of an active agent or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity.

In one example, there is described a method for treating or preventing conditions or disorders associated with ERVK in a subject, comprising: administering to a subject in need thereof a therapeutically effective amount of an active agent or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity.

In one example, the active agent is a CTXLP inhibitors.

In one example, a CTXLP inhibitors inhibits or reduces the activity of CTXLP polypeptide.

In one example, a CTXLP inhibitors inhibits or reduces the level or amount of CTXLP polypeptide.

In one example, a CTXLP inhibitors inhibits or reduces the level or amount of of CTXLP mRNA.

In some example, a CTXLP inhibitor may be, without being limiting thereto, a small molecule, an antibody, a nucleic acid, an aptamer, a peptide.

The term “small molecule” as used herein refers to a molecule of less than about 1,000 daltons, in particular organic or inorganic compounds.

In one example, the small molecule may be a small molecule inhibitor of HIV Tat. In one example, the small molecule inhibitor of HIV Tat is a Michael acceptor electrophile (MAE). In one example, the MAE is curcumin, rosmarinic acid, gambogic acid, celastrol (15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), cyclopentenone prostaglandins (CyPG), such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), N-acetylcysteine amide (NACA), or D-penicillamine (also called Cuprimine). In one example, the small molecule inhibitor of HIV Tat is a sulfhydryl compound with chelating properties. In one example, the sulfhydryl compound with chelating properties is N-(2-Mercapto-propionyl)-glycin (MPG), 2,3-Dimercapto-propanol (DMP), 2,3-Dimercapto-propane-sulfonic acid (DMPS), Nitric oxide (NO), or sulphated polysaccharides. In one example the small molecule inhibitor of HIV Tat is a Thioredoxin reductase 1 (TRR1) inhibitor. In one example, the Thioredoxin reductase 1 (TRR1) inhibitor is B5 (curcumin analog).

In one example, the CTXLP inhibitor is a nucleic acid molecule interfering specifically with CTXLP expression. In some example, the nucleic acid CTXLP inhibitor may be an antisense against CTXLP, a siRNA against CTXLP, a shRNA against CTXLP, or a ribozyme.

The term “RNAi” or “interfering RNA” refers an RNA, which is capable of down-regulating the expression of the targeted polypeptide, such as CTXLP. It encompasses small interfering RNA (siRNA), double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules. RNA interference, designates a phenomenon by which dsRNA specifically suppresses expression of a target gene at post-translational level. In normal conditions, RNA interference is initiated by double-stranded RNA molecules (dsRNA) of several thousand base pairs in length. In vivo, dsRNA introduced into a cell is cleaved into a mixture of short dsRNA molecules called siRNA. The enzyme that catalyzes the cleavage, Dicer, is an endo-RNase that contains RNase III domains

siRNA are usually designed against a region 50-100 nucleotides downstream the translation initiator codon, whereas 5′UTR (untranslated region) and 3′UTR are usually avoided. The chosen siRNA target sequence should be subjected to a BLAST search against EST database to ensure that the only desired gene is targeted. Various products are commercially available to aid in the preparation and use of siRNA. In a preferred embodiment, the RNAi molecule is a siRNA of at least about 15-50 nucleotides in length, preferably about 20-30 base nucleotides.

RNAi can comprise naturally occurring RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end of the molecule or to one or more internal nucleotides of the RNAi, including modifications that make the RNAi resistant to nuclease digestion.

RNAi may be administered in free (naked) form or by the use of delivery systems that enhance stability and/or targeting, e.g., liposomes, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors. They may also be administered in the form of their precursors or encoding DNAs.

Antisense nucleic acid can also be used to down-regulate the expression of CTXLP. The antisense nucleic acid can be complementary to all or part of a sense nucleic acid encoding CTXLP e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence, and is thought to interfere with the translation of the target mRNA.

An antisense nucleic acid can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. Particularly, antisense RNA can be chemically synthesized, produced by in vitro transcription from linear (e.g. PCR products) or circular templates (e.g., viral or non-viral vectors), or produced by in vivo transcription from viral or non-viral vectors.

Antisense nucleic acid may be modified to have enhanced stability, nuclease resistance, target specificity and improved pharmacological properties. For example, antisense nucleic acid may include modified nucleotides designed to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides.

Ribozyme molecules can also be used to block the expression of CTXLP. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. Ribozyme molecules specific for CTXLP can be designed, produced, and administered by methods commonly known to the art.

The term “aptamer” refers to a molecule of nucleic acid or a peptide able to bind specifically to CTXLP polypeptide.

The term “administering” as used herein includes all means of introducing the compounds and compositions described herein to the subject, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, and the like. The compounds and compositions described herein may be administered in unit dosage forms and/or formulations containing conventional nontoxic pharmaceutically-acceptable carriers, adjuvants, and vehicles.

Non limiting examples of oral administration include tablets, capsules, elixirs, syrups, and the like.

Non limiting examples of parenteral administration include intravenous, intraarterial, intraperitoneal, epidurial, intraurethral, intrasternal, intramuscular and subcutaneous, as well as any other art recognized route of parenteral administration.

Non limiting examples of means of parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques, as well as any other means of parenteral administration recognized in the art. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH in the range from about 3 to about 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art. Parenteral administration of a compound is illustratively performed in the form of saline solutions or with the compound incorporated into liposomes. In cases where the compound in itself is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be applied.

The dosage of each compound(s) depends on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage used.

In one example, examples of ERVK associated diseases may include but are not limited to infectious diseases, autoimmune diseases, neurological diseases, cancer, and other conditions such as idiopathic nephrotic syndrome.

In one example, examples of CTXLP associated diseases may include but are not limited to infectious diseases, autoimmune diseases, neurological diseases, cancer, and other conditions such as idiopathic nephrotic syndrome.

Other, including but not limited to, Idiopathic nephrotic syndrome.

In one example, there is described a method for transcriptional activation, comprising contacting a DNA molecule comprising a gene with a polypeptide as described herein.

In one example, there is described a diagnostic reagent for use in the detection of CTXLP polypeptide in a subject, comprising an antibody specific for CTXLP polypeptide.

In one example, there is described a diagnostic reagent for use in the detection of CTXLP mRNA in a subject, comprising an isolated nucleic acid specific for CTXLP.

In one example, there is described a diagnostic reagent for use in the detection CTXLP activity in a subject, comprising a polypeptide as described herein.

In one example, there is described a method for treating or preventing conditions or disorders associated with CTXLP in a subject, comprising: measuring an amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA; and administering to a subject in need thereof a therapeutically effective amount of an active agent or a pharmaceutically acceptable salt thereof when the amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA, is high, optionally compared to a control, wherein the active agent blocks or inhibits the CTXLP activity.

In one example, there is described a method for treating or preventing conditions or disorders associated with ERVK in a subject, comprising: measuring an amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA; and administering to a subject in need thereof a therapeutically effective amount of an active agent or a pharmaceutically acceptable salt thereof when the amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA, is high, optionally compared to a control, wherein the active agent blocks or inhibits the CTXLP activity.

Method are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

In one example, there is described a kit comprising: (a) a container comprising a pharmaceutical composition containing a polypeptide as described herein, and/or a nucleic acid as described herein, and/or an expression vector, and/or a host cell, and/or an antibody as described herein, in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; and (c) optionally, instructions for use.

In one example, the kit further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.

To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.

EXAMPLES

Endogenous retroviruses (ERVs) are host genetic elements originating from prior infection of host germ-line cells that are subsequently inherited through the germline. ERVs represent approximately 8% of human genomic DNA. ERVs can benefit their host, or in other contexts are proposed to be involved in pathogenesis and disease. Notably, our interest in ERVK CTXLP lies in its association to motor neuron conditions such as Amyotrophic Lateral Sclerosis (ALS), as well in cancers.

ERVK CTXLP Bioinformatics: Endogenous retrovirus-K (ERVK) conotoxin-like protein (CTXLP) is produced following a ribosomal frameshifting event and is subject to post-translational modifications (PTMs). PTMs and alternative start sites allow for a variety of CTXLP isoforms which may drive distinct pathogenic mechanisms. The prevalence and polymorphic variability of ERVK CTXLP-encoding insertions suggests that CTXLP is a pervasive and conserved ERVK protein. The molecular characterization of CTXLP revealed a conotoxin domain which predicts that it acts as antagonist to specific voltage-gated calcium channels. CTXLP also contains a cysteine motif that aligned to multiple cone snail, spider and viral toxins, which are known to function as antagonists to voltage-gated ion channels. This intrinsic capacity to interfere with calcium channels through these motifs suggests a putative mechanism by which ERVK can act in the pathogenesis of motor neuron diseases such as ALS.

CTXLP biological characterization: CTXLP protein isoform expression in NCCIT and SVGA cells was elucidated by Western blots which indicated presumed isoform sizes of 32 kDa, 51 kDa, and 90/110 kDa. In NCCIT cells, endogenous CTXLP is ubiquitously expressed in the nucleus, and also identified in the cytoplasm and cell membrane, based on cell fractionation and confocal experiments. In contrast, in SVGA cells basal CTXLP levels are limited, but highly inducible by pro-inflammatory stimuli. In addition, CTXP expression is almost exclusively in the chromatin fraction and demonstrates a prominence in the nucleus upon confocal imaging. The notable exception is that after pro-inflammatory activation for 24 hours CTXLP puncta appear in the cytoplasm and on cellular membranes reminiscent of pathogenic protein aggregates. Moreover, the localization pattern in response to pro-inflammatory activators resulting in a prominence in the nucleus ability to bind chromatin suggests that CTXLP may be involved in viral transcription. A primary candidate as a viral transcription factor is the 32 kDa CTXLP isoform, as small cysteine-rich proteins have previously been identified as transcriptional activators, as per HIV-1 Tat (15 kDa) and HTLV Tax (40 kDa) role as viral transcription co-activators.

CTXLP Expression in disease states: ERVK CTXLP localized to the motor cortex in spinal cord sections from autopsy samples of patients with ALS, but not neuro-normal controls. Concomitantly, CTXLP expression was substantially enhanced in diseased ALS tissues, aligning with oligodendrocytes, Nogo-A expression and demyelinated lesions. In addition, cancer cell lines and tissue expressed greater levels of CTXLP relative to normal controls. Together, these findings provide significant evidence for the activity of CTXLP in ALS and certain cancers.

Pathological consequences of CTXLP expression: ERVK CTXLP has the capacity to enhance NF-κB p65 and p50 proteins that play a critical role in ALS pathogenesis. In addition, CTXLP administration or transfection induced significant levels of capase-3. The induction of caspase-3 activation and apoptosis by CTXLP was inhibited by excess extracellular calcium pointing to a calcium channel mediated activation of toxicity. Remarkably, despite the initial die off of cells, cells remaining in the cultures appeared to demonstrate appreciable cellular proliferation relative to control suggesting the induction of a carcinogenic process. CTXLP also had a notable effect on the depletion of CaV2.2 voltage-gated calcium channel-associated transcriptional regulator (CaV2.2 CCAT) from the nucleus.

ERVK CTXLP can be targeted by small molecule therapeutics: A drug screen revealed that celastrol and gambogic acid have the capacity to inhibit endogenous CTXLP expression in NCCIT cancer cell line. Moreover, gambogic acid was able to reduce inducible CTXLP expression the presence of TNFα and ameliorate the concomitant expression of pathogenic marker Nogo-A. This strongly suggests that therapeutic targeting of CTXLP in human disease could be an agent in the efforts to ameliorate the devastation of ALS.

Development of cell and animal models to investigate CTXLP pathogenesis: Human tissue and animal models for the study of CTXLP in ALS and cancer are needed. We are actively working to further develop our human tissue culture models. In addition, together with Dr. Alberto Civetta, we are in the process of developing a model inDrosophiliaat the University of Winnipeg. Importantly, we will continue to pursue mammalian models with our collaborators which offer an opportunity to explore multiple features of pathogenesis as we continue to elucidate the processes involved in CTXLP pathogenesis.

ERVK CTXLP is a novel pathological target for the development of therapeutics for inflammatory, neurological and oncogenic diseases.

ABBREVIATIONS

Abbreviations used in text.

ALS Amyotrophic lateral sclerosis

BLAST Basic local alignment search tool

CC Cervical spinal cord

cDNA Complimentary deoxyribonucleic acid

ChIP Chromatin immunoprecipitation

CNS Central nervous system

DNA Deoxyribonucleic acid

Env Envelope

GA Gambogic acid

HAART Highly active antiretroviral therapy

HCV Hepatitis C virus

HIV Human Immunodeficiency virus

HTLV Human T-lymphotrophic virus

HML Human Mouse mammary tumour virus-like

ICK Inhibitor cysteine knot

IP Immunoprecipitation

IRES Internal ribosomal entry site

ISRE Interferon response element

LATS Large tumor suppressor kinase

LC Lumbar spinal cord

LIGHT Homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes

LTR Long terminal repeat

MAE Michael acceptor electrophile

MMTV Mouse mammary tumour virus

mRNA Messenger ribonucleic acid

MS Multiple sclerosis

MUSCLE MUltiple Sequence Comparison by Log-Expectation

NCCIT National Cancer Center Institute Tokyo, teratocarcinoma cell line

NCBI National Centre for Biotechnology Information

NLS Nuclear localization signal

NPV Nuclear polyhedrosis virus

OPC Oligodendrocyte precursor cell

ORF Open reading frame

PCA Principle component analysis

PLP Proteolipid protein

PRF Programmed ribosomal frameshift

Q-PCR Quantitative polymerase chain reaction

RA Rheumatoid arthritis

RNA Ribonucleic acid

RT Reverse transcriptase

SRA Sequence Read Archive

SU Surface unit

SVGA SV40 T antigen glial astrocytes

Tat Trans-activator of transcription

Tax Transactivator from the X-gene region

TM Transmembrane

TM EV Theiler's Murine Encephalomyelitis Virus

TRAF-2/6 TNF receptor associated factor

VEGF Vascular endothelial growth factors

VGCC Voltage gated calcium channel

WCE Whole cell extract

Endogenous Retroviruses

Retroviruses are single-stranded RNA viruses that replicate through reverse transcription1. Retroviruses use the enzyme reverse transcriptase to convert their genomic RNA to DNA, and then use a viral integrase to insert itself into a host genome2. Retroviruses are categorized as being either exogenous or endogenous3. Examples of exogenous retroviruses include Human-Immunodeficiency virus (HIV) and Human T-lymphotropic virus (HTLV). Alternatively, endogenous retroviruses (ERVs) are genetic elements originating from prior infection of host germ-line cells, allowing them to be inherited through Mendelian genetics3. ERVs represent approximately 8% of human genomic DNA4. ERVs can benefit their hosts, or in other contexts are proposed to be involved in pathogenesis and disease6.

Endogenous Retrovirus-K (ERVK) is the most recently endogenated retrovirus in the human genome1. ERVK is a group of similar viruses that are categorized into 10 clades (sub-groups). ERVK (HML-2 clade) first entered the human genome approximately 28 million years ago, occurring before the divergence of hominids and old-world monkeys7. More recent insertions of ERVK occurred up to 200,000 years ago, and are specific to the human lineage. This has resulted in several human-specific ERVK insertions8. Approximately 1000 ERVK loci have been identified in the human genome9. Although the majority of ERVK insertions have been silenced through mutations and negative selection, there are an estimated 24 fixed loci capable of producing viral proteins3,6. ERVs are also found to be highly polymorphic between individuals and different ethnic groups7. ERVK expression has been detected in several tissues throughout the body at varying levels between individuals3,10.

The ERVK genome consists of the essential retroviral genes gag-pro-pol-env, along with its own accessory genes1(FIG.1). The group specific antigen (gag) gene encodes structural proteins including the viral capsid8,11. The protease (pro) gene encodes a protease which cleaves newly synthesized viral proteins1. The polymerase (pol) gene encodes for proteins including reverse transcriptase and integrase2,11. The envelope (env) gene encodes the glycoproteins of the viral envelope11. The ERVK genome is flanked by long terminal repeats (LTRs), which were assistive in retroviral DNA insertion into the host12. Once inserted into the host genome, the virus is considered a provirus. LTRs contain elements of enhancers and promoters, including transcription factor binding-sites and interferon-stimulated response elements that regulate both retroviral and host gene expression6,11.

ERVK can be organized into two types based on their genome. Type 1 proviruses contain a 292 base-pair deletion near the 5′ end of env not found in type 2 proviruses13. The presence or absence of this deletion affects the accessory proteins the provirus produces13,14.

ERVK Envelope Protein

The ERVK envelope (Env) protein is initially translated as a large, inactive polyprotein15,16. The polyproteins dimerizes or trimerizes and are then cleaved by the cellular protease furin, forming a surface unit (SU) and transmembrane (TM) subunit15. Like other retroviral envelope proteins, the assembled Env trimer is heavily glycosylated and is expressed on the viral capsid membrane, as well as infected host cell membranes, allowing for incorporation of the virus into host cells15,17.

Cysteine Knot Proteins

Cysteine knots are protein structural motifs found throughout animals, fungi and plants18. Cysteine knot proteins are known for their stability, attributed to their 3 disulfide bonds; two of the disulfide bonds and their peptide backbone form a ring that the third bond goes through, thus forming a “knot” structure18. Cysteine knot proteins are categorized as cyclic cysteine knots, growth factor cysteine knots, or inhibitor cysteine knots (ICK). Cyclic cysteine knots are found in plants and often have defense functions as bactericides and insecticides18. Growth factor cysteine knots are found in extracellular signaling molecules and are involved in various functions including cell-cell communication and embryonic development19. Examples include the vascular endothelial growth factors (VEGFs), and nerve growth factor19. ICK proteins are found in fungi, plants and animals and act as antagonists to a variety of receptors and ion channels18.

ICK proteins include a vast array of peptides found in various living organisms. The ICK structure consists of six conserved (connected as Cys1-CysIV, CysII-CysV, and CysIII-CysVI) cysteine residues and an otherwise variable peptide backbone18(FIG.3). Within the animal kingdom, ICK peptides are found in the venoms of spiders, scorpions, and marine snails, and function either as pore-blockers or gate-modifiers of ion channels18. Mammalian ICK peptides have also been identified, including agouti-signalling protein (ASIP) and agouti-related peptide (AGRP)20.

Animal ICKs are proposed to be a result of divergent evolution21. Functional constraints during evolution have resulted in spider, snail, and scorpion ICKs maintaining a similar gene structure, protein fold, and target receptor, which are all evidence for a common ancestor21. Alternatively, plant and fungi ICK do not have these similarities to animal ICKs, suggesting they are a product of convergent evolution21. In certain baculoviruses, a cysteine-rich ORF has been detected, that potentially translates into an ICK fold21. The putative ICK motif resembles the animal ICKs, suggesting that viruses may have obtained this genetic sequence by a gene transfer event after infecting an ICK-carrying host21.

Conotoxins are neurotoxic peptides found in theConusgenus of marine snails used to immobilize prey22. Conusspecies are distinct in their ability to produce hundreds of different toxic peptides23. Conotoxins are disulfide-rich and are usually 10-30 amino acids in length22. Conotoxins act as antagonists to specific voltage and ligand-gated ion channels22. In humans, symptoms of conotoxin exposure include poor coordination, blurred vision, speech difficulties, and nausea23. Conotoxins have also been associated with episodes of delirium and psychosis24.

The O-superfamily of conotoxins exhibits an ICK fold. Members of the O-superfamily include μ-conotoxins, which inhibit voltage-gated sodium channels, and δ-conotoxins, which delay sodium channel inactivation25. K-Conotoxins are inhibitors of voltage-gated potassium channels; ω-conotoxins inhibit N-type voltage-gated calcium channels (VGCCs)25. N-type VGCCs are located in presynaptic nerve terminals and are involved in neurotransmitter release26. ω-Conotoxin's selectivity for N-type VGCCs has allowed for their development as therapeutic agents. The ω-conotoxin MVIIA has been developed into a drug for relief of chronic and inflammatory pain27.

Genes encoding an ω-conotoxin-like protein (CTXLP) have also been identified in certain viruses. Nuclear polyhedrosis viruses (NPV) have been shown to secrete a small conotoxin-like peptide28. NPVs are insect pathogens belonging to the family baculoviridae28. Although NPV-CTXLP's function has not been elucidated, its cysteine bridges were found to have a nearly identical structure to the conserved ω-conotoxin's cysteine motif28. We have discovered a novel CTXLP ORF in the envelope gene of ERVK. The full pathogenic potential of ERVK CTXLP domain remains unknown.

Identification of a Conotoxin-Like Domain in the ERVK Genome

Splicing and Conserved Domains in the ERVK Genome (Start Codon-Biased Analysis)

NetGene2 splice site prediction yielded a large number of predicted splice junctions (105-119 per ERVK sequence). However, after exhaustive analysis, none of these splice junctions resulted in the creation of domains that could be identified using the Conserved Domains Database. However, the predicted splicing patterns resulted in the identification of between 27 and 46 newly created ORFs per ERVK sequence.

After finding no conserved domains in the initial analysis, the requirement for a start codon (ATG, CTG, TTG, GTG or ATT) at the beginning of each ORF was removed, because a start codon could be introduced through splicing and thus was not strictly necessary. The removal of this requirement resulted in slightly different ORFs, which can be seen inFIG.2. Analysis of these ORFs identified a previously undescribed region that would generate a peptide with significant homology to known proteins. This ORF occurred in both type 1 and type 2 genomes (that is, it was not affected by the 292-base pair deletion). DNA with the potential to encode a peptide containing a domain with homology to the O-conotoxin superfamily was identified in a region of env, but in a different reading frame, from nucleotide 7863 to nucleotide 7934 in the 5′-3′ direction. This ORF did not contain the typical methionine codon that is often used as a start codon for translation.

The putative conotoxin-like domain contained six characteristic cysteine residues and one characteristic glycine residue, indicating that it is most similar to the ω-conotoxin family. Another group of viruses, Nuclear Polyhedrosis Viruses, which are insect-infecting Baculoviruses, produce a similar conotoxin-like protein (NPV CTXLP). The putative ERVK CTXLP showed the greatest similarity to these viral proteins.FIG.3shows the sequences of several ω-conotoxins produced by 3 cone snail species, as well as the sequence of a Nuclear Polyhedrosis Virus conotoxin-like domain. Although these sequences differ from each other in notable ways, 7 conserved residues (6 cysteines and 1 glycine) are found in all of them. These residues are also observed in the ERVK CTXLP domain.

The ERVK CTXLP sequence showed the greatest homology to NPV CTXLP sequences (E-value=1.09×10−5).FIG.4shows the sequence logo for ERVK CTXLP and 10 NPV sequences, in which several more amino-acid residues (in addition to the 7 described above) are conserved.

FIG.5summarizes theConusand NPV sequences with greatest similarity and centrality to ERVK CTXLP.FIG.6shows the logo of the knotin domain of CTXLP and its amino acid composition.

Three-Dimensional Modeling of the ERVK Conotoxin-Like Protein

Conotoxins adopt a knot-like conformation, called a knottin domain, which is important for their action. Omega-conotoxin and NPV CTXLP knottins include 3 disulfide bonds. Tertiary structure prediction of the ERVK-113 CTXLP protein using Knotter 1D3D software resulted in the conclusion that it too could form these characteristic features. The predicted 3-dimensional structure of the ERVK-113 CTXLP domain is shown inFIG.7.

This predicted structure was then superimposed on the predicted structure of an NPV CTXLP domain to examine the similarity between the two. This structure alignment (FIG.8) is based on sequence alignment and was prepared using UCSF Chimera software31.

The root mean square deviation between 24 atom pairs in this alignment is 0.426 angstroms. However, it can be difficult to see how similar the predicted structure of these two protein domains are from this image (FIG.8). As such, the structures were reduced to their respective peptide backbones. The resultant image can be seen inFIG.9.

Conotoxin-Like Proteins are Not Encoded by Other Retroviruses

After identifying that these two distantly related groups of viruses (ERVK and NPVs) both contain conotoxin-like protein coding capacity, we also searched for conotoxin-like domains within translations of all three reading frames of the env region of several other retroviral genomes (HIV-1, HTLV-1, MMTV, ERVW, ERVH). No conotoxin-like domains were identified in any of these retroviruses from our analysis.

Alignments of ERVK CTXLP and Other Cysteine-Rich Proteins

The ERVK CTXLP ORF is 39 amino acids long, with the cysteine-rich motif accounting for 30/39 amino acids (CSDYGINCSHSYGCCSRSCIALFCSVSKLC). The CTXLP cysteine-rich sequence was aligned to inhibitor cysteine knot (ICK) proteins and other cysteine-rich proteins using Geneious software (Version R8)30. A sequence logo was generated from the alignment to assess amino acid conservation between CTXLP and known cysteine-rich proteins (FIG.10). Geneious alignment software was used to compare ERVK CTXLP's cysteine-rich amino acid sequence to other cysteine-rich proteins (Table 1) to further understand CTXLP's structure and potential function.

TABLE 1Proteins and peptides from various organisms and theirrespective accession numbers compared to CTXLP cysteine-rich motif found using Geneious software.Protein/PeptideOrganismAccession #Guanxitoxin-2SpiderP84837.1Guanxitoxin-1DSpiderP84836.1Hainantoxin-ISpiderD2Y1X6.1Hainantoxin-IIISpiderD2Y1X9.1Hainantoxin-IVSpider1NIY_AHainantoxin-VSpiderP60975.1Hanatoxin-1SpiderP56852.1Hanatoxin-2SpiderP56853.1SgtxSpider1LA4_AGrammotoxinSpiderP60590.2Huwentoxin-ISpiderP56676.2Huwentoxin-XSpiderP68424.2agouti-related peptideHumanO00253.1agouti-signalling proteinHuman1Y7K_AVEGF-AHumanP15692.2VEGF-BHumanP49765.2VEGF-CHumanCAA63907.1VEGF-DHumanBAA24264.1VEGF-EHumanABA00650.1VEGF-FSnake1WQ8_APlacental Growth FactorHumanAAH07789.1TatHIV-1CCD30501.1TatHIV-2AAA76845.1TaxHTLV-1BAD95659.1TaxHTLV-2AFC76143.1TaxHTLV-3Q0R5R1.1EnvelopeHTLV-4CAA29690.1EnvelopeJaagsiekte SheepAAK38688.1Retrovirus

Several spiders are known to utilize ICK peptides in their toxins32. The putative ERVK CTXLP cysteine motif was aligned to 12 spider toxins from various species of spiders.FIG.11shows ERVK CTXLP peptide had significant similarity to the Hainantoxin-I and Hainantoxin-IV ICK motifs, with a pairwise identity above 25% suggesting conserved protein function (NCBI).

The sequence logo generated showed that the cysteine knot (C-C-CC-C-C) motif is conserved in ERVK CTXLP and the spider toxins examined. Although there was significant sequence diversity in other amino acids, each sequence contained the essential 6 cysteine residues for an ICK. Five of the 12 spider toxins also contained a glycine residue in an identical position to ERVK CTXLP's characteristic glycine. The overall cysteine spacing of the CTXLP motif was unique when compared with spider toxins, suggesting that despite forming an ICK fold, the overall protein conformations are likely divergent. This could explain receptor binding specificity of each toxin species. Spider toxins and CTXLP were then examined for conserved motifs (Table 2). Overall, there was little conservation outside of the ICK motif, with the most significant conservation found in Hainantoxin-I, a voltage gated sodium channel inhibitor.

When ERVK CTXLP peptide was aligned to the human ICK proteins agouti-signalling protein (ASIP) and agouti-related peptide (AGRP), significant similarity was found in the conserved cysteine domain (both with a pairwise identity of 21.9% with CTXLP), suggesting structural similarity and possible functional overlap (FIG.12).

Seven of ten cysteine residues found in the agouti family peptides aligned with ERVK CTXLP. Agouti proteins use 8 cysteines to form an ICK structure33, whereas CTXLP only has 7 cysteines and is likely to take on a simpler ICK fold. Agouti-like proteins are the only known ICK domain containing protein in humans; however, these findings suggest that CTXLP may also be a human-derived ICK protein.

ERVK CTXLP was also aligned to 7 VEGF proteins, which utilize a growth factor cysteine knot. Although there was some alignment between the cysteine residues and some similar motifs (ex. GCC) identified, the large gaps in spacing and the different spacing of cysteine residues in the VEGF proteins suggests that there is no significant similarity to ERVK CTXLP (all with identity≤7.7%) (FIG.13). The dissimilarity suggests that CTXLP does not take on a growth factor cysteine knot structure.

When ERVK CTXLP was aligned to the retroviral accessory protein Tat from HIV-1 and HIV-2, some degree of similarity was detected (identity 19.4% and 16.1%, respectively) (FIG.14). Tat (HIV-1 and HIV-2) contains a conserved C-C-CC-C-C motif that has a much tighter spacing than ERVK CTXLP. In contrast to HIV-1 Tat and CTXLP, HIV-2 Tat did not contain a central “CC” pair of cysteine residues. ERVK CTXLP was also aligned with Tax proteins from HTLV-1, HTLV-2, and HTLV-3 along with envelope from HTLV-4 and envelope from Jaagsiekte retrovirus, and no homology was detected (data not shown). The conservation of a cysteine motif and central “CC” cysteine pair in HIV-1 Tat and CTXLP is a potential basis for conserved structure and function of these viral proteins.

Aligning ERVK CTXLP to several cysteine-rich peptides provided insight into the potential function of the CTXLP protein domain. ERVK CTXLP showed the greatest similarity to ICK peptides. The cysteine knot motif was conserved in all of the spider toxins examined, and Hainaintoxin-I showed the greatest similarity to CTXLP with an identity of 26.5%, suggesting similarity in function (NCBI). All other amino acid residues were highly variable, suggesting that the conservation of the cysteine residues and the tertiary structure are more important for peptide function rather than the primary amino acid sequence. The spider toxins function as antagonists to voltage gated ion channels, suggesting CTXLP may have a similar function18. Hainantoxin-I is a voltage-gated sodium channel inhibitor34; thus, CTXLP may function as a voltage-gated sodium channel inhibitor. Although the ERVK CTXLP had significant similarity to Hainantoxin-I, ERVK CTXLP still had the greatest similarity (25.9-33.3%) to the cone snail ω-conotoxins, suggesting CTXLP functions as a VGCC inhibitor. Previous studies have also shown that ω-conotoxin's amino acid residues threonine 11, tyrosine 13, lysine 2, lysine 4, and arginine 22 are important for calcium channel receptor binding35. CTXLP has some similar conserved residues including a tyrosine in position 12 and an arginine in position 17. CTXLP may alternatively utilize different amino acid residues to bind to cognate VGCC targets.

ERVK CTXLP also showed significant identity to the human agouti-family proteins, specifically ASIP and AGRP peptides. ASIP and AGRP are mammalian ICK peptides that both function as antagonists to melanocortin receptors 1, 3 and 4 (MC1R, MC3R, and MC4R)36. ASIP is produced in the skin to promote pigment production, while AGRP is involved in metabolism36. The agouti-family of peptides contains a unique ICK pattern33. CTXLP is only capable of forming 3 of the 4 cysteine bridges identified in agouti, suggesting that CTXLP takes on the basic ICK fold. The similarity between ERVK CTXLP to the agouti family of peptides provides further support for CTXLP's structure as an ICK peptide, along with first evidence for the presence of viral ICK peptides in humans.

Vascular endothelial growth factors (VEGFs) contain a growth factor cysteine knot motif, and are signalling molecules involved in angiogenesis19. ERVK CTXLP did not show significant similarity (≤7.7%) to the VEGF proteins. A dissimilar cysteine motif with a different spacing of cysteine residues and a significant difference in overall protein size (12-47 kDa for VEGF versus 32 and 51 kDa for CTXLP), suggests that ERVK CTXLP does not function in a growth factor or cytokine manner19. CTXLP's similarity to the ICK peptides and dissimilarity to VEGF suggests that ERVK CTXLP likely functions as a receptor antagonist via an ICK motif.

Cysteine-rich peptides have also been identified in exogenous retroviruses. ERVK CTXLP has some sequence similarity with the Tat accessory protein of HIV-1 and HIV-2. Although Tat is not an ICK peptide and has a slightly different cysteine spacing pattern to ERVK CTXLP, a similar cysteine rich motif was identified in both proteins (19.6%). The cysteine-rich motif of Tat endows this protein with neurotoxic properties37. Tat expression in the brains of HIV-1 infected patients has been associated with neuronal apoptosis via caspase activation and calcium accumulation38. The structural similarities between Tat and ERVK CTXLP may suggest that they both use similar mechanisms for pathogenicity. HIV-2 is known to be a less pathogenic than HIV-139. A partial explanation to this decreased pathogenicity may lie in the structural differences between their respective Tat proteins. HIV-2 Tat has a deletion of one cysteine, losing the “CC” motif. Interestingly, all ERVK CTXLP domains examined contained a “CC” motif. The mechanisms surrounding HIV Tat neurotoxicity are diverse and manifold38,40,41, suggesting that substantial research may be required to address potential ERVK CTXLP cellular toxicity in the CNS. The cysteine motif in HIV Tat has also been associated with increased HIV transactivation, by translocating to the nucleus and interacting with transcriptional machinery38. HIV Tat can also transactivate ERVK42. Thus, the multiple functions of HIV-1 and HIV-2 Tat suggest that CTXLP's conserved cysteine motif may also contribute to neurotoxicity and retroviral transcription. Other retroviral proteins examined (HTLV Tax) did not show any homology to ERVK CTXLP, suggesting that this pathogenic mechanism is not conserved among all retroviruses.

Identification of CTXLP-Encoding ERVK Loci in the Human Genome

Nomenclature for each ERVK loci is based on their common names, as well as their chromosome location. Geneious was used to align both ERVK rec exon 1 and the predicted CTXLP DNA sequence with 95 ERVK HML-2 insertions identified in the human genome. After the ERVK insertions were aligned, many insertions (33) were excluded from further analysis due to the absence of an intact env. The ERVK insertions with an intact env were then aligned to both the rec gene and CTXLP cysteine motif nucleotide sequences.FIG.15shows an example of the rec exon 1 and CTXLP nucleotide alignments against intact ERVK envelope genes. The CTXLP sequence was found at bp 1413 of env, just at the 3′ end of the coding region for the envelope SU protein. This location is the cleavage site of the envelope polyprotein, where there is a junction between the SU and TM proteins. The aligned nucleotide sequences were then translated into an amino acid sequence and examined for intact Rec and CTXLP coding sequences. Table 3 shows that there are 25 ERVK insertions (out of the 62 examined) capable of producing a CTXLP protein, with 5 proviruses also capable of producing Rec.

Of the 95 ERVK DNA sequences, 33 were excluded due to an incomplete env sequence. The remaining 62 sequences were then translated and examined for an intact CTXLP in the appropriate reading frame. The resulting CTXLP peptide sequences were aligned and a sequence logo and consensus sequence were generated to assess amino acid conservation and detect polymorphisms (FIG.16).

In total, 25 ERVK insertions containing the CTXLP cysteine motif were analysed for overall conservation of the peptide sequence and to identify specific variants.FIG.16shows that although each CTXLP peptide has a nearly identical amino acid sequence (identity 95.6%), there are distinct CTXLP polymorphisms identified.

Ten distinct CTXLP polymorphisms were detected. The most prevalent polymorphism is found in ERVK-3, ERVK-104, ERVK-10, ERVK-9, ERVK-14, ERVK-14(b), ERVK-8, ERVK-103, ERVK-25, ERVK-7, ERVK-21, ERVK-16p11.2, and ERVK-113 (Allele 1). The second most prevalent substitutes glycine for serine, relative to the consensus, at two alignment positions (Ser16Gly, Ser27Gly) and is found in ERVK-5 and ERVK-20 (Allele 2). ERVK-HML-2_2q21.2 differs only at the latter Serine (Ser27Gly). ERVK-18 differs only at the former Serine (Ser16Gly). ERVK-51 has the former variation as well as a valine in position 20 (Ser16Gly, Ile20Val). ERVK-1 has phenylalanine at position 22 (Leu22Phe). ERVK-4 contains arginine at position 5 (Gly5Arg). ERVK-HML-2_12q13.2 contained an asparagine at position 16 (Ser16Asn). ERVK-23 shows three polymorphisms at positions 5, 17, and 27 (Gly5Arg, Arg17Lys, Val26Glu). The prevalence and polymorphic variability of ERVK CTXLP-encoding insertions suggests that CTXLP is a pervasive and conserved ERVK protein.

Out of the identified CTXLP encoding proviruses, 20 of 25 ERVK insertions were human-specific43. ERVK-18, ERVK-5, ERVK-69, ERVK-20, ERVK-HML-2_16p21, and ERVK-51 are found in other primates including orangutan, chimpanzee, and rhesus monkey, demonstrating that ERVK CTXLP is an evolutionarily conserved protein, which either entered the genome of a common primate ancestor or through cross-species infection with a specific CTXLP-encoding ERVK virus16(SeeFIG.59below in “primate models” section). The evolutionary age and clade of ERVK retroviruses that encode CTXLP suggests that CTXLP is unique to primates43,44, and that the human genome has an enrichment of this type of ERVK proviruses.

A re-analysis of CTXLP variants in the human genome using a different methodology resulted in similar conclusion regarding the polymorphic nature of CTXLP+ ERVK genomes (FIG.17).

ERVK CTXLP Domain and Disease Associations

CTXLP was identified in both type 1 and type 2 ERVK (FIG.1). The prevalence of CTXLP in both types of ERVK suggests that CTXLP originated early on in ERVK evolution, being present before the divergence of ERVK A env genomes45. Select CTXLP+ loci had known disease associations (Table 3). Out of the 25 CTXLP-encoding ERVK insertions examined, no insertions were associated with ALS—most likely due to a lack of research on this topic. Two of the 25 insertions (ERVK-18 and ERVK-10) were associated with the psychiatric condition schizophrenia. Two out of 3 insertions associated with MS contained CTXLP-encoding insertions. ERVK expression has previously been associated with neurological disease6, suggesting that CTXLP may be one mechanism for neurotoxicity. Surprisingly, 9 CTXLP-encoding insertions were associated with cancer. ERVK env expression has previously been identified in several cancers46. As we demonstrate below (see “CTXLP and disease” section,FIGS.25,36,43and44), there is a notable association between CTXLP and cancer, suggesting that currently identified (Table 3) and other CTXLP-encoding loci with no known disease association may serve as future biomarkers for cancer. The different polymorphisms of CTXLP identified did not associate with any specific disease conditions. Any effects these polymorphisms have on CTXLP function remains unknown.

Identification of ERVK CTXLP—An Alternate Form of the ERVK Envelope Protein

Predicted Full ERVK CTXLP Protein Sequence

The results of both the Pfam and NCBI-CDD databases indicated that the predicted CTXLP amino acid sequence (used to produce the CTXLP plasmids described below) shares similarities with both ERVK Rec, an oncogenic alternate splice product of the env gene, and ERVK Env. These results support the prediction that CTXLP is partially composed of the SU unit of the Env glycoprotein. The NCBI-CDD database also indicated the presence of a surface glycoprotein signal peptide domain. Lastly, the C-terminal portion of the CTXLP sequence was found to share similarities with the O-conotoxin superfamily, which ω-conotoxins are a part of. Lastly, the DUF4408 domain corresponds to a domain of unknown function which is primarily found in plants. Together, these results suggest that CTXLP is composed of the ERVK Env SU unit with a C-terminal ω-conotoxin domain (FIG.18).

Programmed Frameshifting and Internal Ribosomal Entry Site

RNAfold Analysis of RNA Structures in the ERVK Env Transcript

Our biomedical experiments suggested that there were CTXLP isoforms of different sizes, therefore we examined whether the conventional and alternative methionine start sites could be used to make both long and short CTXLP proteins. The first 350 bp of the ERVK Env-encoding RNA was inserted into RNAfold software to predict RNA secondary structure (iltrna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).FIG.19Bsuggests that the CTLXP proteins can originate from the conventional Env methionine start or potentially use IRES-like RNA hairpins to initiate translation at a downstream methionine.

This alternative mechanism for CTXLP expression is the use of internal ribosomal entry site (IRES), using an alternative translational start site (FIG.19B). The 51 and 32 kDa isoforms of CTXLP likely form using the start of the env ORF (nucleotide position 1) or an IRES in the env reading frame, starting from one of its many methionine start codons (specifically amino acid position 200). Translating env from alternative start codons would result in different sized isoforms of CTXLP. Certain viruses, including HIV, use IRES to allow for mRNA translation to begin in the middle of the transcript48,49. The mRNA forms a hairpin structure that allows the binding of the 40S RNA subunit followed by protein translation50. Using RNAfold software to predict mRNA secondary structure upstream of CTXLP, an RNA fold structure similar to HIV IRES was predicted (nucleotide 84 to 187, and 213 to 318 in env transcript), that could take advantage of alternate methionine start codon.

ERVK env nucleotide sequences were also inserted into RNAfold starting from 150 base pairs upstream of the CTXLP ORF to predict RNA secondary structure. RNA secondary structure was examined for evidence of -1 programmed ribosomal frameshifting motifs, including a slippery site with the X-XXY-YYZ form, a 5-10 nucleotide spacer and a downstream pseudoknot or hairpin structure (FIG.19C). The 1-350 bp region of the ERVK env gene was also examined for RNA secondary structure motifs, as it contains numerous alternative methionine start sites. RNA secondary structure was then examined for potential internal ribosomal entry site (IRES) binding sites, which take the form of complex RNA hairpins48.The likelihood that these ERVK RNA secondary structures represent an IRES was determined using IRES prediction software called IRESite {http://iresite.org/IRESite_web.php), and reported as a similarity with known cellular and viral IRES 2D structures.

PRF can occur when three elements are combined: i) a slippery site containing an X-XXY-YYZ motif which after frameshifting by -1 results in XXX-YYY reading, ii) a 5 to 10 nucleotide spacer sequence, and iii) a downstream hairpin-type pseudoknot. ERVK CTXLP-encoding insertions contained an appropriate U-UUA-AAU slippery site to allow for −1 frameshifting to UUU-AAA. After the slippery site there was a 5 nucleotide spacer sequence before the CTXLP ORF. All sequences examined showed a strong probability of forming a hairpin-type pseudoknot within the RNA sequence encoding the CTXLP cysteine-rich motif (FIG.19C). Paradoxically, if the envelope protein translates past the CTXLP ORF start and frameshifts by −4, this introduces a conserved KRQK nuclear localization sequence (NLS) into the hypothetical protein (FIG.20). Another transcription factor that contains a KRQK NLS is NE-κB p5051. There are no other known NLS in either the ERVK envelope protein or the predicted ERVK CTXLP proteins.

If CTXLP encoding originates from the conventional start site in the env transcript, followed by a −1 PRF then it may produce a 51 kDa CTXLP protein. Alternatively, if CTXLP encoding originates from an IRES site using an alternate methionine in the env transcript (FIG.20), followed by a −1 PRF then it may produce a 32 kDa CTXLP protein. The insertions that showed the highest probability of forming a potential IRES binding site upstream of the CTXLP ORF were ERVK-113 and ERVK-4. These sequences analysed by IRESite software predict that the IRES-like RNA hairpins most resemble those found in HIV, Theiler's Murine Encephalomyelitis Virus (TMEV), and Hepatitis C virus (HCV).

We had previously hypothesized that ERVK CTXLP be produced via alternative splicing of the Rec transcript. Although alternative splicing is a common mechanism in retroviruses, only 4 of 25 CTXLP-encoding insertions also had intact Rec protein. The prevalence of CTXLP-encoding insertions in the absence of Rec suggests that alternative splicing is not the mechanism of CTXLP formation. Upstream and downstream of the CTXLP ORF are several stop codons. One mechanism that retroviruses use to compensate for stop codons or a lack of methionine starts is called programmed minus-one ribosomal frameshifting52. This involves the formation of an H-type pseudoknot in the RNA transcript at the site of frameshifting. The H-type pseudoknot would likely halt the ribosome from continuing translation, leading to the −1 PRF52. The slippery site UUU-AAA-U would re-establish ribosomal tRNA and mRNA base pairing and allow for the continuation of translation after frameshifting52. We predict that this mechanism could be used to extend the ORF of the ERVK SU protein by adding on a C-terminal CTXLP domain (FIGS.19&20). Although the predicted structural stability of the ERVK env RNA H-type pseudoknot (FIG.18) was stronger in some ERVK insertions than others, the presence of this RNA secondary structure was conserved in all sequences examined using RNAfold. To our knowledge, frameshifting under conditions of inflammation has not been previously described. Enhanced frameshifting due to inflammation may be a unique mechanism in retroviruses, particularly upon exposure to TNFα (FIGS.27and28).

Together, this data suggests that ERVK CTXLP is likely expressed as a cryptic peptide through frameshifted translation of the env transcript (FIGS.19&20). Alternative forms of viral env proteins (CTXLP proteins may be considered isoforms of env) may be translated under specific physiological conditions53,54. When ERVK env is translated in this proposed alternative ORF, the CTXLP peptide would be expressed within the translated env as a cryptic peptide. Cryptic peptides are proposed to have significantly different functions than their precursor protein55. Cryptic epitopes within modified HIV proteins have been shown to be immunogenic56,57.

Therefore, ERVK CTXLP is likely formed from a −4 PRF occurring slightly upstream of the furin cleavage site in env. An IRES sequence likely allows for a shorter ERVK CTXLP protein isoform to be produced, explaining the distinct isoforms of CTXLP identified.

Prediction of Post-Translational Modifications for CTXLP

ERVK CTXLP is predicted to have the following post-translational modifications, including, but not limited to phosphorylation, SUMOylation, glycosylation and lipid addition (FIGS.20&21, Tables 4-8) using ELM software47and other resources.

The N-linked glycosylation of ERVK CTXLP has been verified experimentally using PNGase treatment of CTXLP protein fractions, followed by western blot analysis for shifts in high molecular weight protein banding patterns (FIG.26, n=3). Phosphorylation is postulated to occur to ERVK CTXLP when bound to chromatin, as seen in the 32 kDa band shift inFIG.30.

Moreover, bioinformatic predictions also predicted protein cleavage and interaction sites within CTXLP (Table 8) using the PROSPER website (https://prosper.erc.monash.edu.au/home.html). Among the proteins predicted to cleave CTXLP were HIV protease, furin, NEC1, and NEC2. The predicted furin cleavage site is consistent with the location of the known furin cleavage site that typically cleaves the Env polyprotein into discrete SU and TM peptide chains that are then assembled into multimer proteins. However, it is unclear how an overlapping NRS N-linked glycosylation site would impact the ability of furin the cleave the site. As well, the predicted cleavage by HIV protease is interesting as ERVK interactions with HIV proteins have been previously reported42,58,59. Of note, cleavage predictions only take into account primary amino acid sequence only, and do not account for how viral protein tertiary structure and cellular factors come into play.

Lastly, the predicted protein interactions of cellular proteins and CTXLP were equally intriguing (Table 9). Among the predicted interactions were proteins involved in cell cycle regulation such as MAPK and LATS. As well, interactions were also predicted with proteins involved in innate immunity such as UPS-7/HAUSP, TRAF-2 and TRAF-6, which are upstream of NF-κB in inflammatory signalling.

Design of a Custom ERVK CTXLP Antibody

Pierce Custom antibody services has produced a CTXLP-specific polyclonal rabbit antibody, used in all the experiments described below. The predicted epitopes are listed inFIG.22. The rabbit protocol and immunization plan is stated inFIG.23.

Design of a Custom ERVK CTXLP Vector, and Complementary ERVK Env and ERVK SU Vector

GenScript custom plasmid services has produced an ERVK CTXLP-expressing vector within a pcDNA3.1 backbone, used in all the experiments described below. We also synthesized a matching ERVK SU vector as a complementary plasmid devoid of the CTXLP domain. The sequences used to produce the vectors are listed below:

First portion is ERVK Env SU, aa residues 1-465 (furin cleavage site) of ERVK 113 (19p12b) with normal frame. Second portion is ERVK CTXLP, aa residues 463-500 of ERVK 113 (19p12b) with +3 reading frame and no start codon bias. Env SU ends at KR before bolded font indicating where Env CTXLP starts (QK). Bolded portion of sequence represents the allele 1 portion of Env CTXLP.

NRS is an N-linked glycosylation site, RSKR is the furin cleavage site, KRQK is the nuclear localization sequence (NLS). NRS may mask furin site allowing for NLS function.

→aa residues 466-699 (furin cleavage site to end of aa sequence) of ERVK 113 (19p12b). Bolded portion of sequence represent the ISU domain of ERVK TM.

The ERVK CTXLP Domain is Found in Several Distinct Proteinaceous Forms

Western Blot Analysis of ERVK-Expressing NCCIT Cells

Whole cell extract of ERVK-expressing NCCIT cells and an immunoprecipitated (IP) fraction enriched for CTXLP were analyzed by Western blot (FIG.25). In the whole cell extract, major bands were identified at 90 and 110 kDa, suggesting this may be the predominant form of CTXLP found in the cell. Unmodified CTXLP would be expected to correspond to a 51 kDa band; thus, is it possible that heavier bands are due to PTM such as glycosylation, phosphorylation or sumoylation. It should be noted that higher molecular weight bands are also observed in other cell types (see below). It is likely that these heavier bands reflect PTM including phosphorylation (≈2 kDa) and glycosylation (≥2 kDa) (FIGS.21and26) or sumoylation. The possibility that the higher molecular weights reflect post-translational modification of the 32 and 51 kDa CTXLP isoforms through glycosylation, would be in accordance with the results of the bioinformatic analysis which indicate that ERVK Env and CTXLP proteins are heavily glycosylated (FIG.21, Table 6,FIG.26)15,60,61. Another possibility for observing larger than expected protein bands is detergent-resistant multimerization, as retroviral envelope proteins form trimers15. Light bands were also observed at 51 kDa and 32 kDa, the latter form possibly due to an alternative start site within or cleavage of CTXLP protein.

In the CTXLP-enriched fraction, the most heavily enriched band was detected at 51 kDa. In this fraction, CTXLP-reactive bands were also found at 110 and 142 kDa, which are also possible results of protein PTM or multimerization. Lighter bands were found at 26 and 29 kDa, again these bands suggest CTXLP cleavage or alternative methionine start site products.

ERVK CTXLP is Inducible Through the Action of Pro-Inflammatory Signalling

Astroctye Expression of ERVK CTXLP in the Presence of Pro-Inflammatory Agents

NCCIT, used as control cells, were cultured along side-SVGA cells. In addition to higher molecular weight bands (not shown) NCCIT cells demonstrated a 51 kDa band and 32 kDa band. Unlike NCCIT cells, in the astrocytic cell line SVGA ERVK CTXLP protein is spontaneously expressed at low levels, with a minor 32 kDa protein being apparent (FIG.25). However, CTXLP expression in SVGA cells was upregulated upon treatment with pro-inflammatory cytokines tumor neurosis factor (TNFα) and LIGHT (lymphotoxin-like inducible member of the TNF superfamily protein) (FIG.27). In contrast to CTXLP expression, Env protein expression was not induced by pro-inflammatory stimulus.

As with NCCIT cells, larger 90/110 kDa CTXLP reactive bands were also observed upon TNFα or LIGHT treated SVGA andReNcell neurons (FIGS.28,30,56and58). As detailed above, these larger bands may represent post-translational modification (PTM) of CTXLP isoforms.

The Localization of ERVK CTXLP Expression is Cell Type and Inflammation Dependent

Ubiquitous Expression of ERVK CTXLP Expression in NCCIT Cells

The localization of CTXLP protein expression was examined through Western blot analysis of cellular fractions and confocal imaging. In NCCIT cells, endogenous CTXLP protein appeared ubiquitously expressed and localizes to the cytoplasm, nucleus and chromatin enriched fractions. CTXLP protein was also found in both the soluble and insoluble NCCIT whole cell lysates (FIGS.29Aand B). The latter further suggests that there is interaction of CTXLP with cell membranes. The ubiquitous nature of CTXLP was supported by confocal imaging (FIG.27C).

CTLXP expression was also associated with indicators of autophagy in NCCIT cells (FIG.29B). Autophagy occurs when dysfunctional proteins and damaged organelles accumulate within a cell, and has been associated with cell dysfunction in neurodegenerative disease62. NCCIT cells exhibited insoluble caspase-3, which is an indicator for autophagy63. There was also evidence of LC3B cleavage (data not shown) which occurs during autophagy.

Nuclear Localization of ERVK CTXLP Expression in SVGA Cells

In contrast to NCCIT cells, in SVGA cells endogenous CTXLP protein localization occurred predominantly in the chromatin cellular fraction (FIG.29A) and was poorly detected by confocal imaging (FIG.29C). However, CTXLP protein levels were strongly elevated in the nucleus upon treatment of 0.1 and 1 ng/mL TNFα when compared to cells alone (FIG.30). This increase in CTXLP expression upon treatment of astroctyes with low levels of TNFα or LIGHT, suggests that chronic, low level inflammation may augment CTXLP levels physiologically. Moreover, with pro-inflammatory cytokine exposure, CTXLP puncta formed within the nucleus, but staining was excluded from the nucleoli (FIG.31). Higher resolution images of CTXLP expressing astrocytes showed that CTXLP puncta also formed in the cytoplasm and on the cell surface, suggesting that potential isoforms of CTXLP may have location-specific functions (FIG.31B).

Enhanced CTXLP expression was also associated with enhanced cytoplasmic RT expression (FIG.30), suggesting regulation of global ERVK gene expression may be linked. Pro-inflammatory cytokines have been shown to induce ERVK expression64-67. During inflammatory events, ERVK Env has been shown to be either neuroprotective68and neuropathological69. Indeed, chronic exposure to TNFα may facilitate frameshifting in ERVK env translation leading to CTXLP being produced as a cryptic protein. TNFα expression and its subsequent downstream signalling cascade products may result in enhanced IRES-dependant translation70, promoting the formation of the smaller CTXLP isoform. Nonetheless, the observation that ERVK CTXLP did not co-localize with ERVK Env protein which localized (FIG.31), suggesting that these proteins may have different localization sequences and/or patterns. Thus, the relationship between CTXLP and Env in ERVK gene regulation remain unclear.

Overexpression of the CTXLP cysteine-rich domain in SVGA cells also resulted in increased CTXLP 32 kDa and 90/110 kDa protein bands (FIG.32), suggesting that this protein domain is sufficient to enhance its own expression.

Consistent with our observation that CTXLP is enriched in the chromatin fraction (FIG.29), DNABIND prediction (dnabind.szialab.org/)71 predicts that CTXLP binds DNA. Prediction parameters were as follows: false positive rate of 6%, expected sensitivity of 58.7%, expected Matthews correlation coefficient of 0.55, score threshold is set to 1.577 (threshold probability of 0.8288). Prediction results of the CTXLP sequence were a score of 1.771 and a probability of DNA binding of 0.8546.

An analysis is DNA interactions revealed that CTXLP bound the interferon response elements (ISREs) within the ERVK viral promoter (5′ LTR) (FIG.33). This binding is enhanced in the presence of inflammatory cytokine stimuli, and is distinct in select cell types (FIG.33). This suggests that CTXLP, and specifically the 32 kDa form of CTXLP (FIG.24), may bind DNA and regulate gene expression. Indeed, preliminary data suggest that CTXLP can alter both ERVK expression (FIG.32) and the transcription of NF-κB transcripts (see below). Further, CTXLP may alter the gene expression patterns of numerous viral and cellular genes containing an ISRE elements in their promoters72,73. Thus, CTXLP may regulate ERVK gene expression, as well as other genes containing ISREs.

In summary, CTXLP protein isoform expression in NCCIT and SVGA cells was elucidated by Western blots which indicated presumed isoform sizes of 32 kDa, 51 kDa, and 90/110 kDa. In NCCIT cells, endogenous CTXLP is ubiquitously expressed being present in the nucleus and also identified in the cytoplasm and cell membrane, based on cell fractionation and confocal experiments. In contrast, in SVGA cells basal CTXLP levels are limited, but highly inducible by pro-inflammatory stimuli. In addition, CTXP expression in almost exclusively in the chromatin fraction and demonstrates a prominence in the nucleus upon confocal imaging. The notable exception is that after pro-inflammatory activation for 24 hours CTXLP puncta appear in the cytoplasm and on cellular membranes reminiscent of pathogenic protein aggregates. Moreover, the localization pattern in response to pro-inflammatory activators resulting in a prominence in the nucleus (FIGS.29-31), ability to bind chromatin (FIGS.29and33) and absence from the nucleoli (FIG.31) suggests that CTXLP may be involved in viral transcription. A primary candidate as a viral transcription factor is the 32 kDa CTXLP isoform, as small cysteine-rich proteins have previously been identified as transcriptional activators74,75, as per HIV-1 Tat (15 kDa) and HTLV Tax (40 kDa) role as viral transcription co-activators76,77. Additionally, low basal CTXLP staining in non-diseased cells suggests that it might have a role in normal physiology and gene regulation processes.

CTXLP Expression in Disease States

CTXLP is Expressed In Vivo in Humans

RNAseq Analysis of CTXLP+ Transcripts in Disease States

To evaluate the significance of CTXLP in disease, we evaluated the expression of CTXLP encoding ERVK loci in publicly available RNA-Seq datasets in the Sequence Read

TABLE 10RNA-Seq datasets in the Sequence Read Archive (SRA) used for the analysis of ERVK expression.Characteristics of RNA-Seq StudiesSelec-StudyConditionTissueInstrumentStrategySourcetionLayoutRead LengthSRP090259schizophroniadorsolateral prefrontalAB SOLiD 4RNA-SeqtranscriptomiccDNAsingle50bpcortexSystemSRP074904bipolar disorderputamen or candidateIllumina HiSeq 2000RNA-SeqtranscriptomiccDNAsingle99bpnucleusSRP110016multiple sclerosisoptic chiasmIllumina HiSeq 3000RNA-SeqtranscriptomiccDNAsingle50bpSRP102685rheumatoid arthritissynoviumIllumina HiSeq 2000RNA-SeqtranscriptomiccDNApaired101/99bpSRP068424HIV+/CD4+ T-cellsIllumina HiSeq 2500RNA-Seqtranscriptomicrandompaired100/100bpHCV+PCR
Archive (SRA) (Table 10). This analysis is summarized in Table 11 andFIGS.34-37. These loci were identified by searching the SRA by disease affiliation and then evaluating each potential study based on samples size, tissue and sequencing quality. Preference was given to studies with large sample sizes, autologous controls, ex vivo disease-relevant tissue, and high sequencing quality. Paired-end reads were preferred to single-end. We focused on studies with fewer measures selecting for particular RNA sub-populations, which could have depleted ERVK RNA from the input.

The studies examine included breast cancer, prostate cancer, Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Rheumatoid Arthritis (RA), schizophrenia, bipolar disorder and HIV+/HCV+ infection. ERVK expression by HML group is summarized in Table 11. We found that the overall expression of ERVK in these disease states was low, and all HML groups were expressed. ERVK expression was highest in blood and cancerous tissue. In addition, we found loci with significantly different expression between patients and controls, but these were different for each study. Breast cancer, prostate cancer, and Multiple Sclerosis datasets contained expression patterns which could potentially distinguish patients from controls. These patterns were driven by differences in expression of CTXLP− loci and loci with inactivating Env mutations.

TABLE 11ERVK expression by HML group in RNAseq datasetsstudyhmlMinimumMedianMeanSDMaximumSRP090259HML100.01250010.11796620.321337911.465101HML1000.03891120.18014320.35993585.855237HML200.02144371.038610428.96567952800.179981HML300.00855360.07723900.19345355.249185HML400.02748620.08636980.18119183.655131HML500.00000000.08595800.23501174.212269HML600.00000000.11337780.30164794.628222HML700.01284470.06023450.14120953.037872HML800.01931170.291558311.70069471289.465670K14C00.00000000.05425100.16676013.168318LTR22B1#LTR/ERVK00.00000000.11615270.36747008.431398LTR22B2#LTR/ERVK00.00000000.12411510.32843462.537287LTR22C2#LTR/ERVK00.00000000.08262170.23263122.358517LTR22E#LTR/ERVK00.00000000.14239270.36075233.823776LTR3B_#LTR/ERVK00.00000000.08551710.22277433.975364SRP074904HML100.00000000.10619610.30185003.824492HML1000.00000000.17892090.46873007.527670HML200.00723850.16916231.8123759197.784008HML300.00000000.09179080.335519412.499732HML400.00000000.12520370.28513075.880441HML500.00000000.09090870.35537319.824726HML600.00000000.10702340.29502233.474829HML700.00000000.07868690.20058972.337186HML800.00000001.084554256.40084415752.139071K14C00.00000000.05661770.18891852.725355LTR22B1#LTR/ERVK00.00000000.09291500.26387504.531172LTR22B2#LTR/ERVK00.00000000.16434430.55388006.122761LTR22C2#LTR/ERVK00.00000000.06970170.22673123.086240LTR22E#LTR/ERVK00.00000000.13071250.39803594.137923LTR3B_#LTR/ERVK00.00000000.10793250.424786811.348338SRP110016HML100.00000000.09399170.40840616.709148HML1000.02464430.19779490.61067228.991545HML200.07481450.19238790.701392525.584209HML300.02074530.08501210.405298414.993567HML400.06763250.20559830.583387117.361348HML500.00000000.06275470.23043484.292697HML600.00000000.11766310.36524454.943406HML700.01654610.05717580.15347952.665017HML800.02376770.07799740.558615831.923344K14C00.00000000.03190940.11706431.377055LTR22B1#LTR/ERVK00.00000000.07442160.25812932.729679LTR22B2#LTR/ERVK00.00000000.14623190.69899674.712645LTR22C2#LTR/ERVK00.00000000.03411520.12448661.373369LTR22E#LTR/ERVK00.00000000.04832350.17737401.849842LTR3B_#LTR/ERVK00.00000000.04787080.16858032.643292SRP102685HML100.00000000.12687670.33537424.052137HML1000.03042810.19509710.34552982.487003HML200.02298320.19389481.8896213219.947046HML300.00000000.12233290.381517110.135958HML400.02378790.17313270.35624145.434816HML500.00000000.10890930.30432747.381214HML600.00000000.15853090.39217064.257929HML700.00000000.09122980.28785457.654548HML800.00811542.090130398.51469058515.001781K14C00.00000000.07065360.23918373.356916LTR22B1#LTR/ERVK00.00000000.12347180.31681245.835195LTR22B2#LTR/ERVK00.00000000.19373940.71348055.204694LTR22C2#LTR/ERVK00.00000000.09027750.23361342.321173LTR22E#LTR/ERVK00.00000000.17644750.48526934.627528LTR3B_#LTR/ERVK00.00000000.12587780.35180044.700366SRP068424HML100.00000000.15461030.912832532.738476HML1000.00000000.19100840.51564098.690614HML200.00000000.24080152.7415319195.191293HML300.00000000.10167560.721286132.342361HML400.00000000.30649751.565190656.983694HML500.00000000.17370370.966825122.605106HML600.00000000.15132850.738777015.850478HML700.00000000.06474190.24888505.277685HML800.00000000.20231576.0970185705.290157K14C00.00000000.07945240.45410368.772424LTR22B1#LTR/ERVK00.00000000.13250970.644764913.163549LTR22B2#LTR/ERVK00.00000000.06938380.27072592.985159LTR22C2#LTR/ERVK00.00000000.04401000.18204652.502976LTR22E#LTR/ERVK00.00000000.09898930.32363254.761848LTR3B_#LTR/ERVK00.00000000.10854890.50311988.617390SRP064478HML100.00000000.06018780.19127653.648491HML1000.00462140.10859390.28463233.347339HML200.01141720.10689181.092057560.595378HML300.00000000.06630530.285564811.555712HML400.01224220.08186990.20046832.824911HML500.00000000.05447990.16957443.303590HML600.00000000.06875350.18992001.844958HML700.00000000.04536270.12147061.274142HML800.00000001.625206286.08804099238.633200K14C00.00000000.03564870.12480002.091446LTR22B1#LTR/ERVK00.00000000.06030750.17066183.324758LTR22B2#LTR/ERVK00.00000000.08903970.32380182.795000LTR22C2#LTR/ERVK00.00000000.05320440.14801411.725137LTR22E#LTR/ERVK00.00000000.08901930.25721002.633204LTR3B_#LTR/ERVK00.00000000.05481180.16285872.074693SRP058722HML100.00000000.10999290.537408619.771974HML1000.00000000.31207554.0788949209.074420HML200.00000000.30833417.0141530716.132793HML300.00000000.08192580.572117144.512233HML400.00556(20.16318020.566941115.160832HML500.00000000.07405190.29915518.570903HML600.00000000.15085480.757216935.954051HML700.00000000.07215020.26531967.406958HML800.00000000.13476035.66204041058.007060K14C00.00000000.09424041.301332377.419996LTR22B1#LTR/ERVK00.00000000.13814691.336836154.714245LTR22B2#LTR/ERVK00.00000000.12026720.60004576.857656LTR22C2#LTR/ERVK00.00000000.05242240.34729649.886468LTR22E#LTR/ERVK00.00000000.11791830.598601119.318676LTR3B_#LTR/ERVK00.00000000.08714520.472209617.621088ERP000550HML100.00000000.09136130.672110825.754908HML1000.00000000.52653473.7211529104.449407HML200.00000000.38297479.4737435494.573463HML300.00000000.07580830.611352924.861316HML400.00000000.12263140.522354739.400311HML500.00000000.05450700.357373415.142432HML600.00000000.16651881.197963346.109402HML700.00000000.06532120.521710511.243053HML800.00000000.07922742.3560620344.064632K14C00.00000000.02083340.09933721.730934LTR22B1#LTR/ERVK00.00000000.04966530.21144733.282185LTR22B2#LTR/ERVK00.00000000.12430720.67552996.645951LTR22C2#LTR/ERVK00.00000000.05429080.30509226.011783LTR22E#LTR/ERVK00.00000000.03863060.13914342.082330LTR3B_#LTR/ERVK00.00000000.06490270.40066988.956652

The lack of differential total RNA expression in controls versus the ALS cohort (FIG.37), which is intriguing given data from protein immunostaining showing obvious differences between clinical groups (see below,FIGS.38-42). PCA analysis reveals that expression of select ERVK CTXLP+ loci cluster in controls versus the ALS cohort (FIG.37), suggesting that specific CTXLP loci may drive the expression of CTXLP protein in ALS.

ERVK CTXLP Expression is Enhanced in CNS Tissues from Patients with Amyotrophic Lateral Sclerosis (ALS)

ALS pathology involves degeneration of upper (brain) and lower (spinal cord) motor neurons, leading to muscle weakness and paralysis (reviewed in78-80). Brain and spinal cord inflammation is a hallmark of ALS (reviewed in81,82). The majority of ALS cases are sporadic, and the cause of this disease remains unknown. Here, we focus on the connection between neuropathology associated with ALS and ERVK CTXLP, such as proteinopathy83,84, aberrant calcium signalling85, demyelination86, and oligodendrocyte dysfunction87.

To show that CTXLP protein is not only expressed in in vitro cell cultures, but also in ex vivo (autopsy) human tissues, spinal cord and brain tissues from neuro-normal controls and patients with ALS were assayed for CTXLP by western blot (FIG.38) and confocal microscopy (FIGS.39-42). Western blot analysis of motor cortex specimens from neuronormal controls and patients with ALS reveals significantly enhanced CTXLP expression in ALS (FIG.38A, p<0.05). CTXLP was concomitantly expressed with inflammation and tissue injury marker CX3CL1 (FIG.38B)88. Analysis of cervical spinal cord tissues also demonstrates elevated CTXLP and CX3CL1 expression in ALS as compared to controls, alongside a modest decrease in levels of voltage-gated calcium channel CaV2.2 in ALS (FIG.38, C-F). Together, these results point to tissue injury and inflammation in CTXLP+ tissues from patients with ALS.

In addition, confocal microscopy of cervical spinal cord (FIG.39A) and motor cortex specimens (FIG.39B) from neuro-normal controls and patients with ALS reveals substantially enhanced CTXLP expression in ALS. In the motor cortex, CTXLP+ cells were neurons (based on MAP2 neuronal marker). This is consistent with previous observations of ERVK proteins present in the motor cortex of patients with ALS67,89,90. Notably, basal CTXLP expression was mostly nuclear in neuronormal tissues, whereas CTXLP exhibited a pattern of cytoplasmic aggregation in motor cortex tissues from patients with ALS (FIG.39B). Enhanced MAP2 staining in the axon hillock of CTXLP+ pyramidal neurons may be an indicator of virus activity, as seen during rabies infection91,92. This pattern of CTXLP expression coincided with a notable decrease in CaV2.2 expression in ALS as compared to controls. Based on staining pattern, this decrease may represent a loss of CaV2.2 expressing pyramidal neurons, as well as smaller CaV2.2+ cells93. Remarkably, CTXLP patterning in the cervical spinal cord exhibited a ring pattern surrounding MAP2+ neurons (MAP2 marks neuronal axons in grey,FIG.39A, far right panel).

Our evidence further indicates that CTXLP can alter oligodendrocyte behavior. In the CNS, highly specialized cells called oligodendrocytes protect neuronal axons by wrapping them in an extensive plasma membrane compacted to produce the myelin sheath94. Oligodendrocyte precursor cells (OPCs) are a pool of immature oligodendrocytes, which express characteristic markers such TCF4, Olig1 and Olig295,96. Upon differentiation into mature oligodendrocytes, they begin to express myelin proteins such as PLP, MOG and MAG95. Oligodendrocytes must myelinate early post-differentiation and myelination occurs within a short timeframe (12-18 hours), where their extended processes ensheath 50-60 axonal segments simultaneously97. Some CNS regions (spinal cord, brainstem and visual cortex) exhibit early myelination during human development, whereas other regions undergo myelination into adulthood (prefrontal cortex and association fibers). Pools of OPCs can remain in tissues and are capable of migration and later differentiation into mature oligodendrocytes, often in response to brain injury98. However, in many disease states, an attempt at remyelination is most often unsuccessful98. A prevailing theory surrounding defects in remyelination is that despite increased numbers of OPCs in injured tissue, these precursor cells become stalled in an immature state and fail to properly differentiate into mature oligodendrocytes96,98. Alterations in OPC markers, such as enhanced TCF4 and Olig1 occurs in tissue lesions from patients with MS99,100.

Our observations show that CTXLP expression occurs in either lateral and/or anterior cortical spinal tracts in ALS (FIG.40). Strong CTXLP+ staining coincides with demyelinating lesions, as shown by solochrome cyanine staining of adjacent tissues (FIG.40). Increased TCF4 (oligodendrocyte precursor marker) is also evident in association with CTXLP expression in tissue from patients with ALS (FIG.40).

FIG.41depicts increased numbers of TCF4+ and Olig1+ cells expressing CTXLP in cervical spinal cord tissue from patients with ALS, as compared to controls. This indicates that CTXLP expression in the spinal cord of patients with ALS does indeed occur in oligodendrocytes (FIGS.39-41), specifically in cells expressing OPC markers.

Neurite outgrowth inhibitor (Nogo-A) is a key regulator of oligodendrocyte precursor cell (OPC) differentiation; when OPCs express Nogo-A they are unable to progress towards a mature oligodendrocyte phenotype, which is capable of myelination101,102. Thus, enhanced expression of Nogo-A in OPCs in the context of inflammation and disease states prevents axonal regeneration by restricting OPC maturation103-105. As an example, demyelinated MS lesions show an increased abundance of Nogo-A+ OPCs, yet the inability of OPCs to mature is proposed as the mechanism driving a non-permissive environment leading to remyelination failure103,106,107. In mature oligodendrocytes, Nogo-A expression prevents axonal sprouting and is expressed in these cells until the initiation of active myelination.

Nogo-A is implicated in a variety of neurological conditions, such as spinal cord injury, peripheral neuropathies, stroke, temporal lobe epilepsy, Alzheimer's disease, ALS, MS and schizophrenia101,108-110. Nogo-A has been identified as a prognostic marker and therapeutic target in ALS due to its substantial expression in muscle tissue from patients with motor neuron disease111,112. Mechanistically, Nogo-A expression destabilizes neuromuscular junctions113-116. Indeed, clinical trials using human anti-Nogo-A antibodies have been performed (ATI 355 from Novartis Pharma and Ozanezumab and GSK1223249 from GlaxoSmithKline)101,117,118. These therapies were designed to target Nogo-A expression in the periphery (intravenous infusions), but may fail to block Nogo-A expression in the CNS (FIG.42), thus explaining the negative results in Phase II clinical ALS trials with Ozanezumab119,120. Of note, genetic polymorphism reticulon 4 receptor (RTN4R) gene encoding the Nogo-A receptor (NgR1), is associated with sporadic ALS121.

FIG.42demonstrates that CTXLP expression in the spinal cord of patients with ALS is associated with elevated Nogo-A expression, specifically in OPCs and other cell types. This specifically occurs in areas of myelin depletion (seeFIG.41). It has been demonstrated in human spinal cord, that select myelin protein rings (PLP, MOG, but not MAG) are detectable by immunohistochemistry even 3 years after injury in degenerating fibre tracts exhibiting the absence of intact axons122. Nogo-A expression also persists in degenerating spinal tissue and may create a non-permissive environment for axon regeneration122. Furthermore, it has been shown that Nogo-A favours a pro-inflammatory context123, one that would promote ERVK expression via modulation of NF-κB and pro-inflammatory cytokine secretion67.

ERVK CTXLP Expression is Enhanced in Cancer Cells

To further evaluate the potential pathogenic activity of CTXLP, we examined CTXLP levels in cancer to follow-up on our observation that NCCIT human embryonic carcinoma line spontaneously expressed CTXLP. The localization pattern that included the cytoplasm also suggested that this represented a stage in course of aberrant CTXLP expression. Thus, we assayed prototypic teratocarcinoma (NCCIT) and breast cancer cells (T47D) for CTXLP expression as compared to the karyotypically normal, non-cancerous, cell lines astrocytic SVGA cells (FIG.43). Cancer cells clearly show higher levels of ERVK CTXLP as compared to non-cancerous cells. A cancer screen also reveals several cancers exhibiting enhanced CTXLP levels, including T cell lymphoma, Acute T-cell leukemia, epithelioid carcinoma, Burkitt's lymphoma, neuroepithelioma, prostate, breast, ovary, testis and skin cancers (FIG.44).

In summary, ERVK CTXLP localized to the motor cortex and spinal cord sections from autopsy samples of patients with ALS, but not neuronormal controls. Concomitantly, CTXLP expression was substantially enhanced in diseased ALS tissues aligning with oligodendrocytes, Nogo-A expression and demyelinated lesions. In addition, cancer cell lines and tissue expressed greater levels of CTXLP relative to normal controls. Together, these findings provide significant evidence for the activity of CTXLP in ALS and certain cancers.

Pathological Consequences of CTXLP Expression

Real Time PCR analysis of Transfected 293T Cells

To investigate how cells may react to the expression of CTXLP and SU, RT-PCR analysis was used to measure the expression of the pro-inflammatory NF-κB p65 subunit and the anti-viral response protein IRF7. This analysis showed that both CTXLP and SU triggered a marked increase in the mRNA expression of NF-κB p65. Conversely, neither protein was able to trigger an upregulation of IRF7 (FIG.45). The upregulation of NF-κB p65 may be beneficial to the ERVK provirus as it is able to 1) act as a direct transcriptional activator of the ERVK LTR, and 2) trigger inflammatory conditions that are conducive to ERVK activation64,67.

Confocal Microscopy Analysis of Transfected 293T Cells

SU and CTXLP transfected 293T cells were also stained to determine whether the presence of either of these proteins was sufficient to trigger the expression of NE-κB p65. It was observed that CTXLP was able to trigger NF-κB p65 protein expression, whereas SU was unable (FIG.46). This was in stark contrast to the RT-PCR results inFIG.45, where both ERVK SU and CTXLP induced NF-κB p65 transcription. As expected, there was a marked increase in SU expression in CTXLP-expressing cells, as the epitope for the SU antibody binds to either SU or CTXLP as both proteins contain the SU amino acid sequence.

To further confirm whether the effect of NF-κB induction by CTXLP is a general phenomenon occurring the multiple cell types, astrocytic SVGA cells were also transfected as described above and evaluated for NF-κB protein expression. Interestingly, both NF-κB p65 and p50 proteins were induced by CTXLP, but not ERVK SU overexpression (n=4). This finding is notable, considering we have shown that ERVK transcription is mediated by IRF1, p50 and p65 transcription factors, and impacts ERVK expression in ALS67. It is also intriguing considering TRAF proteins were predicted to be interacting partners of CTXLP, and may alter NF-κB signalling124,125.

A surprizing feature of several voltage-gated calcium channels (VGCCs) is the ability of their C-terminal fragments to translocate to the nucleus and impact gene expression. Termed calcium channel-associated transcription regulator (CCAT) by Gomez-Ospina et al. in 2006126, these novel gene products encoded within the VGCC sequences. In most cases, an antibody targeting a C-terminal CACNA1 epitope will identify an approximately 75 kDa CCAT fragment with a cellular distribution within the nucleus (or nuclear fractions), unlike the intact channel protein localized to the cytoplasm and membrane126,127. VGCC CCAT proteins can be regulated by cell signalling events. For example, cellular signals that promote CaV1.2 CCAT nuclear localization include treatment of neurons with 2.5 mM EGTA (a chelator which reduces free extracellular calcium), whereas 65 mM KCI treatment (mimicking tonic activity of VGCC) decreased nuclear CCAT levels126. Several signals which drive high intracellular calcium levels in neurons, including 100 μM glutamate, depolarization and NMDA signalling, lead to decreased nuclear CCAT levels126.

We have previously demonstrated an inverse correlation between CTXLP and voltage-gated calcium channel CaV2.2 expression in ALS brain and spinal cord tissues (FIGS.38and39). To further extend this observation, we performed in vitro experiments of CTXLP and ERVK SU exposure by overlaying immunoprecipitation products on human astrocytes (FIG.47). Treatment of SVGA cells with CTXLP, but not ERVK SU, resulted in the depletion of Cav2.2 CCAT (75 kDa), as measured by western blot analysis and confocal microscopy. A decreased in the full size CaV2.2 channel (220 kDa) was also observed (n=2, data not shown). Thus, CTXLP in the CNS may lead to an overall decrease in CaV2.2 channel and CaV2.2 CCAT expression, thus explaining the observed decreased expression of CaV2.2 in ALS (FIGS.38and39). The regulatory role of CaV2.2 on cellular transcription is currently unknown.

ERVK CTXLP is Toxic Via Mechanisms that Differ from ERVK Env-Mediated Toxicity

Treatment of SVGA Astrocyte Cells with ERVK Env Proteins Isolated Via Immunoprecipitation

To determine the neurotoxicity of CTXLP, SVGA astrocyte cells were treated with CTXLP proteins isolated from NCCIT cells via immunoprecipitation (IP). This simulates conditions wherein CTXLP would enter the cell from the outside and possibly exert its effects by binding to cell surface receptors (such as calcium channels). There was considerable variation in the neurotoxicity assays, which may be due in part to the fact that cells were dosed by volume of CTXLP. There was no reliable way to measure the concentration of the protein in the IP product, as protein concentration was well below sensitivity of our in-house BCA assay (20 μg/ml). However, a much higher number of cells treated with CTXLP expressed caspase-3 (apoptosis marker used in the toxicity assays) than control cells demonstrating that CTXLP was toxic to astrocytes, even at unmeasurably low concentrations (FIG.48).

A separate toxicity assay was performed by treating astrocytes with SU and CTXLP (respectively) in the presence and absence of calcium. Theoretically, if CTXLP does in fact contain an ω-conotoxin domain, by flooding cells with calcium and thus saturating calcium channels, it's ability to exert toxic effects on cells via calcium channel binding should be blocked. This is what was seen in CTXLP, but not SU, toxicity assays. In the presence of calcium, the levels of caspase expression in CTXLP-treated cells were similar to controls conditions. They were also less than those of cells treated with CTXLP in the absence of calcium. Further, cells treated with SU in the presence and absence of calcium expressed similar levels of caspase-3 in comparison to CTXLP alone (FIGS.49and50).

Cells in this same toxicity assay were also analyzed at later time points. It was observed that CTXLP and SU-treated cells appeared to be able to continue to replicate despite high levels of caspase expression. After 8 days, cells in both conditions increased in cell density despite high levels of caspase-3 expression and without the addition of media. These observations suggest that these cells may have been transformed oncogenically129-131. Conversely, control cells were not viable after 8 days in culture (FIG.51). It is interesting to note that some viruses, such as the influenza virus, require caspase expression to replicate efficiently132. Moreover, HIV Tat, the viral transactivator, induces caspase activation as part of its neurotoxic mechanism38.

It is notable that the trend of enhanced caspase-3 positivity is seen in both treated (FIGS.48-51) and transfected cells (FIG.52), suggesting that exposure to CTXLP and/or cellular production of CTXLP in vivo may be toxic to cells.

ERVK CTXLP Expression Drives Morphological Changes in Cells

ERVK CTXLP-treated SVGAs and 293T cells transfected with ERVK CTXLP-encoding plasmids were imaged to observe the cellular morphology. Though many of the cells looked normal in appearance, it was observed that an increased number of the cells produced long filipodia (FIG.53). As well, the formation of syncytia (multinucleated cells) was also observed amongst cells exposed to CTXLP. These features are characteristic of retrovirus-infected cells and are known to promote virus transfer between cells133.

This data indicates that ERVK CTXLP expression has the capacity to enhance NF-κB p65/p50 and CaV2.2 proteins that play a critical role in ALS pathogenesis. In addition, CTXLP administration or transfection induced significant levels of capase-3. The induction of caspase-3 activation and apoptosis by CTXLP was inhibited by excess extracellular calcium pointing to a calcium channel mediated activation of toxicity. Remarkably, despite the initial die off of cells, cells remaining in the cultures appeared to demonstrate appreciable cellular proliferation relative to control suggesting the induction of a carcinogenic process.

ERVK CTXLP can be Targeted by Small Molecule Therapeutics

Taken together this data strongly indicates that targeting CTXLP would have significant therapeutic value in ALS. To this end, we have began investigating A small molecule inhibitors to capable of counteracting the pathological effects associated with CTXLP expression is of therapeutic value. A drugs screen in ERVK CTXLP-expressing NCCIT cells was performed to evaluate potential efficacity against CTXLP (FIG.54). Michael acceptor electrophile (MAE) compounds are known to inhibit HIV Tat-dependent transcription by interfering with thiols in its cysteine-rich domain134,135. As CTXLP and HIV Tat share commonality in their cysteine-rich domains (FIG.14), we evaluated a series of MAE compounds including curcumin, rosmarinic acid, gambogic acid and celastrol. Two MAE drugs, celastrol (Cel) and gambogic acid (GA), were identified as suppressing CTXLP expression in NCCIT cells in the low micromolar range (FIG.54).

Derived as an active compound from the Thunder God vine (Tripterygium wilfordii Hook F), celastrol (pubchem.ncbi.nlm.nih.ciov/compound/celastrol) is a plant-derived triterpene with antioxidant, anti-viral and anti-inflammatory activity134,136,137. Celastrol is currently used as a therapeutic agent for rheumatoid arthritis (RA) and lupus in China136,138.Celastrol has also been shown to impact pathological outcomes and symptoms in animal models of RA139, as well as inflammatory markers in activated fibroblast-like synoviocytes from patients with rheumatoid arthritis140. Celastrol has been shown to limit beta-amyloid pathology and neuronal degeneration in Alzheimer's disease models141. Its anti-cancer properties are also under investigation142.

Gambogic acid (pubchem.ncbi.nlm.nih.ciov/compound/16072310) is an active compound from the Gamboge tree (Garcinia hanburyi), with antioxidant, anti-viral and anti-inflammatory properties)134,143-145. It has been shown to be neuroprotective146, and inhibit spinal cord injury and inflammation in a rat model147. Both celastrol and gambogic acid can prevent mutant huntingtin protein aggregation and its neuronal toxicity148. The anti-cancer properties of gambogic acid are also under investigation144,145.

Improvements on drug efficacy, toxicity and tissue-targeting are possible by using MAE-derivatives, related compounds (Table 12), soluble analogues and nanosystem delivery to the brain149,150. Here we provide proof-of-concept that CTXLP is druggable using small molecules (FIGS.54-58); further drug development may improve upon the anti-CTXLP effects of celastrol and gambogic acid.

Gambogic Acid Remedies the Levels of CTXLP-Associated Pathological Markers CaV2.2 CCAT and Nogo-A

FIG.56highlights than not only CTXLP is reduced in the presence of gambogic acid, but that the drug treatment also restores CaV2.2 CCAT expression (known to be suppressed by CTXLP,FIG.47). Furthermore, we demonstrate show that gambogic acid is a potent inhibitor of Nogo-A (which inhibits remyelination) expression in cancerous NCCIT cells (FIG.56). As the decrease in Nogo-A expression directly correlates with a drop in CTXLP expression, this therapeutic strategy may also reduce pathogenic Nogo-A expression in ALS (FIG.42). Nogo-A inhibitors have been previously identified, such as green tea polyphenols and other natural product extracts151. Proteolytic turnover of Nogo-A is a physiological mechanism to reduce Nogo-A levels152. Gambogic acid may reduce Nogo-A expression by having an effect of CTXLP-driven pathology or a more direct effect on specific protein turnover153,154.

Development of Cell and Animal Models to Investigate CTXLP Pathogenesis

Identification of CTXLP-Encoding ERVK Loci in Primate Genomes and Their Human Homologues

Our close relatives also encode ERVK, but some ERVK loci are unique to humans. Examination of the ERVK content of three non-human primate genomes,Pan troglodytes(Common chimpanzee),Gorilla gorilla gorilla(Western lowland gorilla), andCercocebus atys(Sooty Mangabey) shows that CTXLP is not limited to humans.

The most recent genomic assembly for each primate species was searched for CTXLP in the same manner as the human genome (Table 13). panTro5 and gorGor5 were retrieved from UCSC, and Caty_1.0 was retrieved from NCBI. Chimpanzee ERVK were identified using UCSC table panTro5.nestedRepeats, but no such table exists for the Gorilla or Sooty Mangabey. Gorilla and Mangabey ERVK were identified directly from RepeatMasker output. To reduce the number of small ERV fragments to be BLASTed and to increase the accuracy of orthology predictions by including flanking genomic regions, the loci annotated in RepeatMasker were extended by 1000 bp to either side and then any less than 10 bp apart were merged.

The expected relationship between the four species is displayed by the number of orthologs recognized. Humans are most closely related to Chimpanzees, then to Gorillas, and most distantly to the Sooty Mangabey. We can see in the second table that the number of CTXLP and Env positive ERVK loci varies minimally between species.

Only two human CTXLP+ loci are present in all four species. There is also 1 mangabey locus present in all four. No loci were CTXLP+ and present in all four species.FIG.59depicts MUSCLE alignments of tBLASTx results from loci in human, chimpanzee, gorilla, and mangabey where an orthologue in at least one species encodes a Toxin_18+ ORF. The first four alignments are split in two, where the top half contains sequences belonging to orthologous sets aligned horizontally, and the bottom half contains the remaining sequences from each species. The fifth alignment is tBLASTx results for the cd-hit cluster representative sequence of the largest cluster of Toxin_18+ ORFs from each species. Only tBLASTx results for loci which encoded a representative sequence of a cluster containing more than one member were included. This alignment was generated by MUSCLE, then curated so that the Cys residues of chimpanzee_108932 aligned better with the other sequences.FIG.60suggests that there is more conservation between orthologues in different species than between paralogues from the same species. This pattern is much more apparent for CTXLP than for Env reading frame, where CTXLP appear to be under diversifying selection as indicated by increased dissimilarity between the CTXLP reading frame as compared to the Env reading frame of given sequences. differences are smaller if present at all. Taken together, this suggests that non-human primates are viable models for CTXLP research.

Murine Model of ERVK CTXLP

Avindra Nath's group has successfully developed a murine model which supports the neurotoxic potential of the ERVK envelope gene towards motor neurons69. ERVK env gene transgenic mice exhibit progressive motor dysfunction and hallmark pathology associated with ALS such decreased motor cortex volume and injury to pyramidal neurons and anterior horn cells in the spinal cord. This murine model is a solid platform for ERVK research, yet it remains unclear whether pathology and clinical outcomes were driven by canonical retroviral envelope proteins or CTXLP. This is because the insert used to generate the transgenic mice has the capacity to encode CTXLP (FIGS.57and58). However, these mice do represent a putative model of CTXLP-driven neuropathology and neurodegeneration.

Drosophila(fruit flies) are a widely-used model organism, often used to study the cellular effects of pathogenic human viruses155. Moreover, TDP-43 null and TDP-43 mutant flies develop measurable motor deficits156-158, making this model an exceptional tool to evaluate the impact of ERVK on ALS-like neuropathology and clinical outcomes. In collaboration with Dr. Alberto Civetta (University of Winnipeg), have designed an animal model system in which the ERVK proteins are transgenically expressed inDrosophila melanogaster.

ERVK Env, SU, TM and CTXLP open reading frames have been cloned (by GenScript, USA) into a pUAST vector (DrosophilaGenomic Resource Center, #1000), allowing for Gal4 control of transgene expression patterns (see section above on design of custom CTXLP, SU and Env vectors). Generation of an ERVK protein transgenic flies is outsourced to BestGene Inc. (Chino Hills, CA). Flies will be crossed with neuronal (ELAV156), glia (repol159) or astrocyte (aIrm160)-restricted Gal4 fly strains (BloomingtonDrosophilaStock Center 8760, 7415 & 67032, respectively) to generate flies selectively expressing cell-type specific ERVK proteins. For each experimental group, lifespan analysis and locomotor impairment (# of walks/focal) will be monitored as previously described156,161,162ENREF 80. To perform pathological examinations, flies will be cold-sacrificed, heads removed, and tissue either flash frozen for western blot or fixed for immunohistochemical analyses. Biological readouts will be correlated with survival and motor-impairment metrics, as to assess how pathological events track with clinical outcomes. In a second series of experiments, fly models exhibiting clinical impairment will be used to assess the efficacy of a panel of CTXLP inhibitors, such as celastrol and gambogic acid (FIGS.54-58). Inhibitors will be dissolved in DMSO and spiked into standard fly food just before solidification, at therapeutic concentrations of drug. Impact of drug administration on neuropathology and clinical outcomes will be evaluated.

Human Tissue Culture Models of CTXLP Expression

CTXLP was detectable in all human cell lines assayed (SVGA, ReNcell CX, NCCIT, T47D, cancer cell line panel), with varying degrees of expression. Based on the data shown above (FIGS.54-58), ERVK CTXLP-expressing NCCIT teratocarcinoma cells are a viable model for drug screening applications. Additionally, we have developed a transient vector (pcDNA3.1,FIGS.46and52) and a drug (cumate)-inducible lentiviral system (SBI SparQ QM812B-1163) allowing for stable overexpression of ERVK CTXLP, SU and Env. By using the feeder-independent pluripotent stem cell line WA09 (WiCell, mTeSR™ 1/Matrigel™ Platform) we can establish cerebral organoids, as previously described164. The above described protocols will form the foundation for generating ERVK CTXLP-expressing cerebral organoids, a human, druggable, three-dimensional brain model. Lentivirus transduction and flow cytometry selection will be used to generate WA09 stem cells containing the previously described cumate-inducible CTXLP vector. To establish a model of ERVK CTXLP-mediated pathology in intact cerebral tissue, these genetically modified WA09 cells will be grown to maximally sized cerebral organoids in the absence of cumate. Both wild-type and CTXLP-inducible cerebral organoids will be treated with varying doses of cumate, to allow for dose-response and time course experiments. Other potential human systems to study CTXLP in the future includes patient-derived human induced pluripotent stem cells systems165-167.

In summary, Human tissue and animal models for the study of CTXLP in ALS and cancer are needed. We are actively working to further develop our human tissue culture models. In addition, together with Dr. Alberto Civetta, we are in the process of developing a model inDrosophiliaat the University of Winnipeg. Importantly, we will continue to pursue mammalian models with our collaborators which offer an opportunity to explore multiple features of pathogenesis as we continue to elucidate the processes involved in CTXLP pathogenesis.

Discussion

Endogenous retroviruses (ERVs) are host genetic elements, representing approximately 8% of human genomic DNA. ERV activation can benefit their host, or in other contexts are proposed to be involved in pathogenesis and disease6. Our interest in ERVK and the CTXLP protein lies in its association to motor neuron conditions such as Amyotrophic Lateral Sclerosis (ALS), as well in cancers.

ERVK is known to be up-regulated in the neurons of many individuals with ALS67,89,90,168. The motor impairment in ALS is linked to calcium channel dysfunction, which is considered a viable therapeutic target. Thus, we were particularly intrigued by the CTXLP region of the ERVK genome when we discovered that it encoded a conotoxin-like peptide indicative of a pathogenic mechanism in ERVK associated disease. O-superfamily conotoxins are known to inhibit voltage gated calcium ion channels169. These channels are predominantly expressed at the presynaptic terminal of neural synapses170. When an impulse reaches this region, they allow calcium ions into the neuron, thereby increasing calcium ion concentration. This increase in concentration leads to fusion of synaptic vesicles containing neurotransmitters with the presynaptic membrane. The neurotransmitters are then released into the synaptic cleft where they bind to receptors on the postsynaptic terminal and stimulate downstream signaling. Inhibition of these channels with conotoxins can lead to tremors and an inhibition of motor function23,24. Our findings indicate that ERVK CTXLP may likewise be able to inhibit VGCCs and their calcium channel-associated transcriptional regulator (CCAT) and elicit similar responses resulting in impaired motor function.

It may be that ERVK CTXLP was previously implicated in ALS pathology. Notably, a 1997 study found that sera from 5 out of 6 ALS patients was able to reduce calcium ion currents when applied to mouse dorsal root ganglia171. The sera from a variety of disease control groups did not exhibit any effect on calcium ion currents. The authors concluded that “serum factors” from ALS patients can be passively transferred to affect calcium ion channel activity. It is possible that ERVK CTXLP may be the mediator of this effect. If this was the case, the protein would either have to be produced in non-brain cells or tissues. ERVK reverse transcriptase has been detected in the serum of many ALS patients172,173. If this enzyme originates from ERVK, it would demonstrate that ERVK proteins enter the serum during ALS. Thus, it would be possible that CTXLP could be present in the serum as well. In addition, a compromised blood-brain barrier observed in ALS and other neurology inflammatory events would allow CTXLP to cross into the serum174.

Conotoxins are able to specifically inhibit ion channels of certain types of neurons169, which may correlate with the loss of motor function and neurocognitive decline that is observed in ALS175. Other neurotoxin models have been proposed as etiological agents of ALS. The most prominent example is the suspected link between beta-N-methylamino-L-alanine (BMAA), a neurotoxin produced by a group of terrestrial cyanobacterial symbionts in cycad plants, and ALS (or an ALS-like syndrome)176. However, large scale spatial clustering of individuals with ALS has been inconsistent with the range of BMAA-producing cyanobacteria and other suspected environmental risk factors (although small, regional concordances have been identified)176. That is, no yet-proposed neurotoxin etiology has been able to explain the vast majority of cases of ALS. Therefore, although an environmental neurotoxin model for ALS makes sense at a physiological level, a genetic-based model (with environmental/epigenetic influence) seems more likely at an epidemiological level. A genetically-encoded neurotoxin etiology of ALS, such as ERVK CTXLP, would be consistent with both of these approaches. This model would not rely on the requirement to identify unique genes in individuals with ALS, as the different phenotypes (having ALS or not having it) could be caused by differential expression of the same genetic material. Additionally, two active ERVK loci unique to ALS patients have been identified90. It is possible that the CTXLP proteins of these loci are more functional than other ERVK sequences. Another alternative possibility is that insertional polymorphisms or single nucleotide polymorphisms result in differential ERVK CTXLP production or function. Thus, it is possible that sequence variation in ALS patients leads to differentially functional ERVK CTXLP proteins.

Additionally, there are several seemingly disparate features of conotoxin toxicity and ALS pathophysiology that would have to be resolved in order for such a link to be possible. For instance, calcium ion concentrations in the neurons of many ALS patients are elevated175,177. Since omega-conotoxins inhibit calcium influx into neurons169, elevated calcium levels are the opposite of what would normally be expected in an ERVK CTXLP etiology of ALS175. However, it may be possible that these two features are not inconsistent, as many factors (aside from VGCCs) control neuronal calcium ion concentrations170. For instance, calcium-binding proteins such as calbindin-D28K and parvalbumin are absent in motor neurons lost early in ALS170,177. These proteins were present in significantly higher concentrations in healthy motor neurons, and in those affected later in the course of the disease170. Impaired mitochondrial calcium buffering has also been observed in ALS neurons170,177.

Apart from pathology associated with VGCC disruption, we have also shown that CTXLP expression is correlated with elevated Nogo-A in the spinal cord of patients with ALS. Nogo-A is a key regulator of oligodendrocyte precursor cell (OPC) differentiation, ultimately negatively impacting remyelination and tissue repair. Demyelinated spinal cord lesions show an increased abundance of Nogo-A+ OPCs, yet the inability of OPCs to mature is proposed as the mechanism driving a non-permissive environment leading to remyelination failure103-107. Additionally, Nogo-A favours a pro-inflammatory context123, one that would promote ERVK expression via modulation of NF-κB and pro-inflammatory cytokine secretion67.

Nogo-A is implicated in a variety of neurological conditions, such as spinal cord injury, peripheral neuropathies, stroke, temporal lobe epilepsy, Alzheimer's disease, ALS, MS and schizophrenia101,108-110. Nogo-A has been identified as a prognostic marker and therapeutic target in ALS due to its substantial expression in muscle tissue from patients with motor neuron disease111,112. Mechanistically, Nogo-A expression destabilizes neuromuscular junctions113-116. Indeed, clinical trials using human anti-Nogo-A antibodies have been performed (ATI 355 from Novartis Pharma and Ozanezumab and GSK1223249 from GlaxoSmithKline)101,117,118. These therapies were designed to target Nogo-A expression in the periphery (intravenous infusions), but may fail to block Nogo-A expression in the CNS, thus explaining the negative results in Phase II clinical ALS trials with Ozanezumab119,120. Yet, anti-Nogo-A and remyelination-based therapies may be of value in the treatment of CTXLP+ disease states.

As ERVK CTXLP is present in the tissues of ALS patients, it may be used as a biomarker for the disease. This is significant given that ALS is often difficult to diagnose in its initial stages175. Furthermore, if it is found to be an etiological agent of disease, ERVK CTXLP levels could be useful in assessing disease progression or prognosis. Perhaps most importantly, therapeutics could be designed to target it in order to reduce motor function deficits and increase longevity. For instance, a humanized monoclonal antibody could be designed against ERVK CTXLP for intravenous immunoglobulin (IVIG) therapy. Alternatively, small molecule inhibitors, such as MAEs celastrol and gambogic acid, could be used to target ERVK CTXLP DNA binding, gene transactivation effects, enhancement of NF-κB expression and modulation of pathogenic biomarkers. If ERVK CTXLP is found to play pathological roles in other diseases, for example spinal cord injury, multiple sclerosis, schizophrenia or cancers to name a few, this avenue of research could have implications on the diagnosis and treatment of these diseases as well.

ERVK expression is up-regulated in schizophrenia and bipolar disorders178(unpublished data). This may be worth investigating further if ERVK CTXLP production is confirmed, given the fact that omega-conotoxins can cause emotional distress and prolonged delirium with psychotic features24,179.

Additionally, HIV and HTLV infections are known to lead to up-regulation of ERVK expression180,181. Both of these infections are associated with poorly understood, reversible ALS-like syndromes in a small number of patients182-184. HIV-associated ALS can be treated effectively with highly active antiretroviral therapy (HAART)182,183. It is possible that ERVK CTXLP is a pathological contributor to exogenous retrovirus infections and these ALS-like diseases.

Many cancers are associated with ERVK expression185. Evidence that increased ERVK CTXLP expression occurs in cancers cells implicates this viral protein in oncogenesis and possibly metastasis. Specifically, the induction of NF-κB is likely a key feature of ERVK CTXLP activity which may facilitate cancer development and progression186,187.

Together, the results of this analysis provide a basis for further research into the ERVK genome and the relationship between ERVK and inflammatory disease. Given the possible correlations between ERVK CTXLP and disease pathology, this line of research deserves further study.

FIGS.63-65summarize possible implications of our discoveries.

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The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.