The NP9 protein encoded by the human endogenous retrovirus HERV-K(HML-2) negatively regulates gene activation of the Epstein-Barr virus nuclear antigen 2 (EBNA2).

Epstein-Barr virus (EBV) is a human tumour virus that efficiently growth-transforms primary human B-lymphocytes in vitro. The viral nuclear antigen 2 (EBNA2) is essential for immortalisation of B-cells and stimulates viral and cellular gene expression through interaction with DNA-bound transcription factors. Like its cellular homologue Notch, it associates with the DNA-bound repressor RBPJκ (CSL/CBF1) thereby converting RBPJκ into the active state. For instance, both EBNA2 and Notch activate the cellular HES1 promoter. In EBV-transformed lymphocytes, the RNA of the NP9 protein encoded by human endogenous retrovirus HERV-K(HML-2) Type 1 is strongly up-regulated. The NP9 protein is detectable both in EBV-positive Raji cells, a Burkitt's lymphoma cell line, and in IB4, an EBV-transformed human lymphoblastoid cell line. NP9 binds to LNX that forms a complex with the Notch regulator Numb. Therefore, the function of NP9 vis-à-vis Notch and EBNA2 was analysed. Here, we show that NP9 binds to EBNA2 and negatively affects the EBNA2-mediated activation of the viral C- and LMP2A promoters. In contrast, NP9 did neither interfere in the activation of the HES1 promoter by Notch nor the induction of the viral LMP1 promoter by EBNA2. In an electrophoretic mobility shift analysis, NP9 reduced the binding of EBNA2 to DNA-bound RBPJκ by about 50%. The down-regulation of EBNA2-activity by NP9 might represent a cellular defence mechanism against viral infection or could, alternatively, represent an adaptation of the virus to prevent excessive viral protein production that might otherwise be harmful for the infected cell.

An N-terminally truncated envelope protein encoded by a human endogenous retrovirus W locus on chromosome Xq22.3.

BACKGROUND: We previously showed that the envelope (env) sequence of a human endogenous retrovirus (HERV)-W locus on chromosome Xq22.3 is transcribed in human peripheral blood mononuclear cells. The env open reading frame (ORF) of this locus is interrupted by a premature stop at codon 39, but otherwise harbors a long ORF for an N-terminally truncated 475 amino acid Env protein, starting at an in-frame ATG at codon 68. We set out to characterize the protein encoded by that ORF. RESULTS: Transient expression of the 475 amino acid Xq22.3 HERV-W env ORF produced an N-terminally truncated HERV-W Env protein, as detected by the monoclonal anti-HERV-W Env antibodies 6A2B2 and 13H5A5. Remarkably, reversion of the stop at codon 39 in Xq22.3 HERV-W env reconstituted a full-length HERV-W Xq22.3 Env protein. Similar to the full-length HERV-W Env protein Syncytin-1, reconstituted full-length Xq22.3 HERV-W Env is glycosylated, forms oligomers, and is expressed at the cell surface. In contrast, Xq22.3 HERV-W Env is unglycosylated, does not form oligomers, and is located intracellularly, probably due to lack of a signal peptide. Finally, we reconfirm by immunohistochemistry that monoclonal antibody 6A2B2 detects an antigen expressed in placenta and multiple sclerosis brain lesions. CONCLUSIONS: A partially defective HERV-W env gene located on chromosome Xq22.3, which we propose to designate ERVWE2, has retained coding capacity and can produce ex vivo an N-terminally truncated Env protein, named N-Trenv. Detection of an antigen by 6A2B2 in placenta and multiple sclerosis lesions opens the possibility that N-Trenv could be expressed in vivo. More generally, our findings are compatible with the idea that defective HERV elements may be capable of producing incomplete HERV proteins that, speculatively, may exert functions in human physiology or pathology.

Interaction of human immunodeficiency virus gp120 with the voltage-gated potassium channel BEC1.

Retrovirus replication critically depends on a dynamic interplay between retroviral and host proteins. We report on the binding of the surface subunit (glycoprotein 120 (gp120)) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) to the cytoplasmic C-terminus of the voltage-gated potassium channel BEC1 (brain-specific ether-a-go-go-like channel 1), an interaction that can result in the repression of BEC's activity and the inhibition of HIV-1 particle-release. BEC1 protein was found to be expressed in T cells and macrophages, the major target cells of HIV-1. Thus, gp120/BEC1 interaction may be involved in HIV-1 life cycle and/or pathogenesis.

Human endogenous retrovirus protein Rec interacts with the testicular zinc-finger protein and androgen receptor.

More than 2000 human endogenous retrovirus (HERV) sequences are present in the human genome, yet only a few are intact and able to produce proteins. The normal functions of these, if any, are unknown, but some HERV proteins have been implicated in cancers, in particular germ-cell cancers. For instance, it has been documented that (i) patients with germ-cell tumours frequently produce antibodies against HERV proteins; (ii) transgenic mice expressing HERV-K (HML-2) rec are prone to testicular carcinoma in situ; and (iii) Rec can bind and suppress a guardian of germline stem-cell pluripotency, the promyelocytic leukaemia zinc-finger protein (PLZF). This study identified the PLZF-related testicular zinc-finger protein (TZFP) as a binding partner of HERV-K (HML-2) Rec. Interactions occurred via the N- and C-terminal domains of Rec and the C-terminal DNA-binding zinc-finger domain of TZFP (aa 375-450). Not much is known about the function of TZFP. The protein is expressed predominantly in the testis, where it functions as a transcriptional repressor that is active during specific stages of spermatogenesis. The most intensely studied function of TZFP is that of a co-repressor of the activated androgen receptor (AR). Here, it was shown that Rec can form a trimeric complex with TZFP and AR, and can relieve the TZFP-mediated repression of AR-induced transactivation. In addition, Rec was able to overcome the direct transcriptional repression by TZFP of the c-myc gene promoter in reporter assays. Thus, HERV-K (HML-2) Rec may function as an oncoprotein by de-repressing oncogenic transcription factors such as AR.

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

BACKGROUND: 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. RESULTS: By mapping HERV-W env cDNA sequences (n = 332) from peripheral blood mononuclear cells of patients with MS and healthy controls onto individual genomic HERV-W env elements, we identified seven transcribed HERV-W env loci in these cells, including ERVWE1. Transcriptional activity of individual HERV-W env elements did not significantly differ between patients with MS and controls. Remarkably, almost 30% of HERV-W env cDNAs were recombined sequences that most likely arose in vitro between transcripts from different HERV-W env elements. Re-analysis of published MSRV env sequences revealed that all of them can be explained as originating from genomic HERV-W env loci or recombinations among them. In particular, a MSRV env 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 env locus on chromosome Xq22.3. This locus harbors a long open reading frame for an N-terminally truncated HERV-W Env protein. CONCLUSION: Our data clarify the origin of MSRV env sequences, have important implications for the status of MSRV, and open the possibility that a protein encoded by a HERV-W env element on chromosome Xq22.3 may be expressed in MS brain lesions.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

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.

Lack of immune responses against multiple sclerosis-associated retrovirus/human endogenous retrovirus W in patients with multiple sclerosis.

The multiple sclerosis-associated retrovirus (MSRV), originally identified in cell cultures from patients with multiple sclerosis (MS), is closely related to the human endogenous retrovirus family type W (HERV-W). Different lines of evidence appear compatible with a potential role of MSRV/HERV-W in the pathogenesis of MS. The authors therefore analyzed humoral and cellular immune responses against MSRV/HERV-W antigens in patients with MS, patients with other inflammatory and noninflammatory neurological diseases, and healthy controls, using indirect immunofluorescence and enzyme-linked immunospot assays. Antibodies against the HERV-W envelope (Env) protein, Syncytin-1, were found in one of 50 patients with MS and none of 59 controls, whereas antibodies against MSRV matrix and capsid (Gag) or Env proteins were not detectable in any of the patients or controls. Similarly, in a screening of human leukocyte antigen (HLA)-B7+ patients with MS (n = 23) and controls (n = 29) for cytotoxic T-lymphocyte responses against 36 predicted HLA-B7-restricted MSRV/HERV-W Gag-, protease-, and reverse transcriptase-derived peptides, no such responses could be detected in any of the subjects studied. These data suggest that there are no appreciable humoral or cellular immune responses against MSRV/HERV-W in patients with MS. While this may be due to immunological tolerance of physiologically expressed HERV-W proteins, strategies other than measurement of immune responses will be required to further elucidate the relationship between MSRV/HERV-W and MS.

Estimation of human herpesvirus 8 prevalence in high-risk patients by analysis of humoral and cellular immunity.

BACKGROUND: Immunocompromized individuals, such as patients with end-stage renal disease, transplant recipients, and HIV-infected patients, are at increased risk of acquiring human herpesvirus (HHV)-8 associated infectious complications. The prevalence of HHV-8 infection generally is determined by detection of immunoglobulin G. However, because serological assays differ greatly, estimations on the actual HHV-8 prevalence vary considerably. METHODS: HHV-8-specific cellular and humoral immunity were analyzed in 128 controls, 73 patients on dialysis, 67 transplant recipients, and 69 HIV-infected patients with the use of flow cytometry and indirect immunofluorescence microscopy. RESULTS: A higher seroprevalence (from 13.7% to 44.9%) was confirmed for all groups of immunocompromised individuals as compared with healthy controls (3.9%). Among immunocompetent individuals, as little as 12.5% had HHV-8 reactive T-cell frequencies greater than the detection limit. In line with a higher seroprevalence in immunosuppressed patients, HHV-8-specific T cells were detectable in 30.1% of dialysis patients, 20.9% of transplant recipients, and 24.6% of HIV-infected individuals. When combining the individual presence of either HHV-8-specific antibodies or T cells or both, the prevalence of HHV-8 infection approached 15.6% in healthy individuals and 41.1%, 40.3%, and 55.1% in dialysis patients, transplant recipients, and HIV-infected individuals, respectively. CONCLUSIONS: The exclusive serological analysis considerably underestimates the prevalence of HHV-8 infection in all study groups. Thus, the combined quantitation of both humoral and cellular immunity may instead be a superior method to assign the individual HHV-8 status. Moreover, this study suggests that the relative contributions of humoral and cellular immunity in control of HHV-8 may be different depending on the type of immunodeficiency.

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.

Permanent loss of anti-HBc after reactivation of hepatitis B virus infection in an anti-HBs and anti-HBc-positive patient after allogeneic stem cell transplantation.

BACKGROUND: Reactivation of a hepatitis B virus (HBV) infection after transplantation is associated with a high morbidity and mortality. HBV infections generally result in anti-HBc persisting lifelong. CASE REPORT: A 44-year-old female presented 10 years after allogeneic stem cell transplantation with a chronic hepatitis B. The infection was reactivated from a resolved (anti-HBs and anti-HBc positive) HBV infection acquired some years prior to transplantation. Interestingly, she lost all antibodies to HBV including anti-HBc and is upto now anti-HBc negative. The sequence of the surface and the core gene did not reveal any escape mutations. Thus, the loss of anti-HBc might suggest an immunotolerance of the donor's immune system against HBcAg. CONCLUSION: This data illustrate that an HBV infection might be reactivated despite high anti-HBs levels prior to transplantation. Furthermore, this is the first patient in which a complete loss of anti-HBc could be documented. Moreover, since anti-HBc is often used as a screening marker for HBV it should be kept in mind that anti-HBc negative patients with high viremic HBV infection may occur.

Prevalence of herpes simplex virus (types 1 and 2), varicella-zoster virus, cytomegalovirus, and human herpesvirus 6 and 7 DNA in cerebrospinal fluid of Middle Eastern patients with encephalitis.

HSV-1 DNA was detected in 32 (30%) of 106 cerebrospinal fluid samples from patients with encephalitis. Cytomegalovirus, varicella-zoster virus, and human herpesvirus 6 (HHV-6) DNAs were each detected in three patients (3%); herpes simplex virus type 2 (HSV-2) and HHV-7 PCRs were negative. HSV detection was associated with seizure (P = 0.02), especially focal seizure (P = 0.0002), and pathological computed tomography (P = 0.02) with focal lesions (P = 0.0004).

Differences in CMV-specific T-cell levels and long-term susceptibility to CMV infection after kidney, heart and lung transplantation.

Patients after kidney, heart and lung transplantation differ in their immunosuppressive drug regimens and in susceptibility to infectious complications with cytomegalovirus (CMV). In this study, CMV-specific T-cell responses were characterized in long-term transplant recipients and associated with the frequency of infectious complications. CMV-reactive CD4 T cells from 50 healthy controls, 68 renal, 14 heart and 24 lung transplant recipients were flow cytometrically quantified by the induction of cytokines after specific stimulation. Moreover, the immunosuppressive effect of calcineurin inhibitors on specific T-cell reactivity was quantified in vitro and compared with responses in vivo. Median CMV-specific T-cell frequencies in long-term renal (1.48%; range 0.06-17.26%) and heart transplant recipients (0.90%; 0.13-12.49%) did not differ from controls (1.82%; 0.26-21.00%). In contrast, CMV-specific T-cell levels were significantly lower in lung transplant recipients (0.50%; <0.05-4.98%) and showed a significant correlation with the frequency of infectious episodes (r =-0.57, p = 0.005). The differences within the groups were associated with increasing dosages of immunosuppressive drugs, as exemplified for calcineurin inhibitors that dose dependently reduced specific T-cell reactivity in vitro. In conclusion, monitoring CMV-specific CD4 T cells may serve as a measure for long-term disease susceptibility and may contribute to an improved management of CMV complications after lung transplantation.

Human endogenous retrovirus rec interferes with germ cell development in mice and may cause carcinoma in situ, the predecessor lesion of germ cell tumors.

Germ cell tumors (GCTs) are among the most common malignancies in young men. We have previously documented that patients with GCT frequently produce serum antibodies directed against proteins encoded by human endogenous retrovirus (HERV) type K sequences. Transcripts originating from the env gene of HERV-K, including the rec-relative of human immunodeficiency virus rev, are highly expressed in GCTs. We report here that mice that inducibly express HERV-K rec show a disturbed germ cell development and may exhibit, by 19 months of age, changes reminiscent of carcinoma in situ, the predecessor lesion of classic seminoma in humans. This provides the first direct evidence that the expression of a human endogenous retroviral gene previously established as a marker in human germ cell tumors may contribute to organ-specific tumorigenesis in a transgenic mouse model.

Detection of herpes simplex virus, cytomegalovirus and Epstein-Barr virus DNA in atherosclerotic plaques and in unaffected bypass grafts.

BACKGROUND: Herpes virus infections are suspected to be involved in the pathogenesis of atherosclerosis. OBJECTIVE AND METHOD: Viral DNA of herpes simplex virus (HSV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) was analyzed by real-time PCR on 48 biopsies from atherosclerotic plaques extracted by end-arterectomy (46 coronary arteries, 2 carotid arteries), and in tissue from non-atherosclerosis vessels from the same patient as controls (23 internal mammary arteries, 43 saphenous veins). RESULTS: HSV-1 DNA was detected significantly more frequently in plaques (35%) than in control veins (9%, P = 0.006). However, the frequency of HSV-1 DNA detection in the internal mammary artery grafts was as high as in plaques (22%, P = 0.28). CMV and EBV DNA were exclusively found in plaques but not in controls, with 10% for CMV (P = 0.06 versus veins, P = 0.17 versus graft arteries) and 2% for EBV (P = 1.0), respectively. HSV-2 was neither detected in plaques nor in controls. Herpes viral DNA was significantly associated only with arterial hypertension but not with other classical risk factors (P = 0.02), in accordance with the hypothesis that herpes viral infection may alter the vessel wall. CONCLUSION: We conclude that herpes viral infections may have a role in atherosclerosis and that the presence of herpes viral DNA in the grafts used for bypass surgery might constitute a potential risk for atherosclerosis or restenosis.

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.

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.

HERV-K(HML-2) GAG/ENV antibodies as indicator for therapy effect in patients with germ cell tumors.

Germ cell tumors (GCT) are strictly associated with the expression of HERV-K(HML-2) proviruses, and the majority of GCT patients produce antibodies to structural proteins of these proviruses. The objective of our study was to determine the significance of the serological response to HERV-K(HML-2) Gag and Env proteins for diagnosis, management of GCT patients and estimation of the therapy success. The data document a strong association of HERV-K(HML-2) antibodies and the clinical manifestation of the disease and therapy success. HERV-K(HML-2) antibodies seem to have an important diagnostic value as well as indicator of chemotherapy success.

Is the cytomegalovirus serologic status always accurate? A comparative analysis of humoral and cellular immunity.

BACKGROUND: The precise knowledge of cytomegalovirus (CMV) status is important in a variety of clinical settings such as transplantation or blood transfusion. There are, however, various situations where an immunoglobulin-based diagnostic approach has limitations. METHODS: The CMV status of 388 individuals was determined by the analysis of both CMV-specific immunoglobulins and cellular immunity using a standard enzyme-linked immunosorbent assay and a rapid-flow cytometric approach, respectively. RESULTS: In 386 of 388 individuals, both assays yielded concordant results in assigning the CMV status (294 positive and 92 negative). In 2 of 94 (2.1%) CMV immunoglobulin (Ig) G-negative individuals, CMV-specific CD4 T cells were detectable. Repeated testing of the index and follow-up samples yielded one positive and one marginally positive IgG titer. CONCLUSIONS: Both assays yielded highly concordant results. Thus, the T-cell-based approach may represent a reliable alternative for defining CMV status in any clinical situation where serologic testing is limited by borderline titers or by the presence of passively administered antibodies.

Risk of occupational human herpesvirus 8 infection for health care workers.

Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated herpesvirus, is highly prevalent in certain risk groups (human immunodeficiency virus-infected patients, transplant recipients, and patients on hemodialysis). Heath care workers caring for these patients were found to be more frequently infected with HHV-8 than staff caring for other patients (P < 0.01).