Analysis of transcribed human endogenous retrovirus W env loci clarifies the origin of multiple sclerosis-associated retrovirus env sequences

<p>Abstract</p> <p>Background</p> <p>Multiple sclerosis-associated retrovirus (MSRV) RNA sequences have been detected in patients with multiple sclerosis (MS) and are related to the multi-copy human endogenous retrovirus family type W (HERV-W). Only one HERV-W locus (ERVWE1) codes for a complete HERV-W Env protein (Syncytin-1). Syncytin-1 and the putative MSRV Env protein have been involved in the pathogenesis of MS. The origin of MSRV and its precise relation to HERV-W were hitherto unknown.</p> <p>Results</p> <p>By mapping HERV-W <it>env </it>cDNA sequences (n = 332) from peripheral blood mononuclear cells of patients with MS and healthy controls onto individual genomic HERV-W <it>env </it>elements, we identified seven transcribed HERV-W <it>env </it>loci in these cells, including ERVWE1. Transcriptional activity of individual HERV-W <it>env </it>elements did not significantly differ between patients with MS and controls. Remarkably, almost 30% of HERV-W <it>env </it>cDNAs were recombined sequences that most likely arose <it>in vitro </it>between transcripts from different HERV-W <it>env </it>elements. Re-analysis of published MSRV <it>env </it>sequences revealed that all of them can be explained as originating from genomic HERV-W <it>env </it>loci or recombinations among them. In particular, a MSRV <it>env </it>clone previously used for the generation of monoclonal antibody 6A2B2, detecting an antigen in MS brain lesions, appears to be derived from a HERV-W <it>env </it>locus on chromosome Xq22.3. This locus harbors a long open reading frame for an N-terminally truncated HERV-W Env protein.</p> <p>Conclusion</p> <p>Our data clarify the origin of MSRV <it>env </it>sequences, have important implications for the status of MSRV, and open the possibility that a protein encoded by a HERV-W <it>env </it>element on chromosome Xq22.3 may be expressed in MS brain lesions.</p

The Rev/Rex homolog HERV-K cORF multimerizes via a C-terminal domain

AbstractExpression of human endogenous retrovirus K (HERV-K) is associated with germ-cell neoplasia. HERV-K encodes a protein of the Rev/Rex family, cORF, that supports cellular transformation and binds the promyelocytic leukemia zinc finger (PLZF) protein implicated in spermatogenesis. Rev/Rex function invariably depends on multimerization. Here we show that cORF likewise self-associates to form higher-order oligomers. Amino acids (aa) 47–87 in cORF are sufficient, aa 75–87 essential for self-association. Consistently, this domain is predicted to form a hydrophobic α-helix that may represent an oligomerization interface. The existence of a dimerization-competent cORF mutant lacking PLZF-binding activity (cORF47–87) suggests a way of dominant negative inhibition of the proposed tumor susceptibility factor cORF

Human endogenous retrovirus HERV-K(HML-2) encodes a stable signal peptide with biological properties distinct from Rec

<p>Abstract</p> <p>Background</p> <p>The human endogenous retrovirus HERV-K(HML-2) family is associated with testicular germ cell tumors (GCT). Various HML-2 proviruses encode viral proteins such as Env and Rec.</p> <p>Results</p> <p>We describe here that HML-2 Env gives rise to a 13 kDa signal peptide (SP) that harbors a different C-terminus compared to Rec. Subsequent to guiding Env to the endoplasmatic reticulum (ER), HML-2 SP is released into the cytosol. Biochemical analysis and confocal microscopy demonstrated that similar to Rec, SP efficiently translocates to the granular component of nucleoli. Unlike Rec, SP does not shuttle between nucleus and cytoplasm. SP is less stable than Rec as it is subjected to proteasomal degradation. Moreover, SP lacks export activity towards HML-2 genomic RNA, the main function of Rec in the original viral context, and SP does not interfere with Rec's RNA export activity.</p> <p>Conclusion</p> <p>SP is a previously unrecognized HML-2 protein that, besides targeting and translocation of Env into the ER lumen, may exert biological functions distinct from Rec. HML-2 SP represents another functional similarity with the closely related Mouse Mammary Tumor Virus that encodes an Env-derived SP named p14. Our findings furthermore support the emerging concept of bioactive SPs as a conserved retroviral strategy to modulate their host cell environment, evidenced here by a "retroviral fossil". While the specific role of HML-2 SP remains to be elucidated in the context of human biology, we speculate that it may be involved in immune evasion of GCT cells or tumorigenesis.</p

Evaluation of a New Automated, Standardized Generic Nucleic Acid Extraction Method (Total Nucleic Acid Isolation Kit) Used in Combination with Cytomegalovirus DNA Quantification by COBAS AMPLICOR CMV MONITOR

The new generic Total Nucleic Acid Isolation kit improved the detection limit of a cytomegalovirus (CMV) PCR from 400 to 200 copies/ml. Sensitivity and specificity in 30 CMV-positive and 100 negative samples were 100%. Analytical performance was excellent. The kit provides a reliable, standardized, and time-saving tool for DNA extraction

Physical and Functional Interactions of Human Endogenous Retrovirus Proteins Np9 and Rec with the Promyelocytic Leukemia Zinc Finger Protein▿

Only few of the human endogenous retrovirus (HERV) sequences in the human genome can produce proteins. We have previously reported that (i) patients with germ cell tumors often make antibodies against proteins encoded by HERV-K elements, (ii) expression of the HERV-K rec gene in transgenic mice can interfere with germ cell development and induce carcinoma in situ, and (iii) HERV-K np9 transcript is overproduced in many tumors including breast cancers. Here we document that both Np9 and Rec physically and functionally interact with the promyelocytic leukemia zinc finger (PLZF) tumor suppressor, a transcriptional repressor and chromatin remodeler implicated in cancer and the self-renewal of spermatogonial stem cells. Interaction is mediated via two different central and C-terminal domains of Np9 and Rec and the C-terminal zinc fingers of PLZF. One major target of PLZF is the c-myc proto-oncogene. Coexpression of Np9 and Rec with PLZF abrogates the transcriptional repression of the c-myc gene promoter by PLZF and results in c-Myc overproduction, altered expression of c-Myc-regulated genes, and corresponding effects on cell proliferation and survival. Thus, the human endogenous retrovirus proteins Np9 and Rec may act oncogenically by derepressing c-myc through the inhibition of PLZF

Human Endogenous Retrovirus Family HERV-K(HML-2) RNA Transcripts Are Selectively Packaged into Retroviral Particles Produced by the Human Germ Cell Tumor Line Tera-1 and Originate Mainly from a Provirus on Chromosome 22q11.21▿ †

The human germ cell tumor line Tera-1 produces retroviral particles which are encoded by the human endogenous retrovirus family HERV-K(HML-2). We show here, by quantitative reverse transcriptase PCR, that HML-2 gag and env RNA transcripts are selectively packaged into Tera-1 retroviral particles, whereas RNAs from cellular housekeeping genes and from other HERV families (HERV-H and HERV-W) are nonselectively copackaged. Assignment of cloned HML-2 gag and env cDNAs from Tera-1 retroviral particles to individual HML-2 loci in the human genome demonstrated that HML-2 RNA transcripts packaged into Tera-1 retroviral particles originate almost exclusively from an HML-2 provirus on chromosome 22q11.21. Based on relative cloning frequencies, this provirus was the most active among a total of eight transcribed HML-2 loci identified in Tera-1 cells. These data suggest that at least one HML-2 element, that is, the HML-2 provirus on 22q11.21, has retained the capacity for packaging RNA into HML-2-encoded retroviral particles. Given its elevated transcriptional activity and the presence of a full-length Gag open reading frame, the 22q11.21 HML-2 provirus may also significantly contribute to Gag protein and thus particle production in Tera-1 cells. Our findings provide important clues to the generation and biological properties of HML-2-encoded particles. In addition, copackaging of non-HML-2 HERV transcripts in HML-2-encoded particles should inform the debate about endogenous retroviral particles putatively encoded by non-HML-2 HERV families that have previously been described for other human diseases, such as multiple sclerosis

Np9 protein of human endogenous retrovirus K interacts with ligand of numb protein X

We have recently identified Np9 as a novel nuclear protein produced by the human endogenous retrovirus K and were able to document the exclusive presence of np9 transcript in tumors and transformed cells. With the aim of studying whether Np9 has a role in tumorigenesis, a systematic search for interacting proteins was performed. Here, we identify the RING-type E3 ubiquitin ligase LNX (ligand of Numb protein X) as an Np9-interacting partner. We furthermore show that the interaction involves N- and C-terminal domains of both proteins and can affect the subcellular localization of LNX. LNX has been reported to target the cell fate determinant and Notch antagonist Numb for proteasome-dependent degradation, thereby causing an increase in transactivational activity of Notch. We document that LNX-interacting Np9, like Numb, is unstable and degraded via the proteasome pathway and that ectopic Numb can stabilize recombinant Np9. Combined, these findings point to the possibility that Np9 affects tumorigenesis through the LNX/Numb/Notch pathway