Nikolaus Mueller-Lantzsch

Levels of virus-specific CD4 T cells correlate with cytomegalovirus control and predict virus-induced disease after renal transplantation.

Background. Immunosuppressive treatment in transplant patients frequently causes infectious complications with cytomegalovirus (CMV). The extent of CMV replication can be followed by a number of diagnostic methods. There is, however, no simple diagnostic tool to assess the quality of the cellular antiviral immune response of an individual patient. This would be of particular importance for therapy decisions, as patients with detectable virus load do not necessarily develop CMV-related disease. Using a rapid whole blood assay, the frequencies of CMV-reactive CD4 and CD8 T cells were followed after renal transplantation to characterize their relative contribution in the containment of CMV infection. Methods. T cells from transplant patients and healthy control persons were stimulated with CMV antigen in vitro. Based on specific cellular activation and induction of intracellular cytokines, the frequency of CMV-reactive CD4 and CD8 T cells was determined using flow cytometry. Viral load was quantified using the “hybrid-capture” assay. Results. The absence of CMV complications in long-term transplant recipients is reflected by stable virus-specific T-cell frequencies, which do not differ from healthy CMV-positive controls. In contrast, during the first months after transplantation, clinical symptoms are preceded by a decrease in CMV-reactive CD4 T-cell frequencies and an increase in CMV load. Conclusions. The individual immune response and CMV replication are critically balanced and can be characterized by assessing both viral load and antiviral T cells. Our experimental design allows the identification of patients with sufficient, insufficient, or absent T-cell activity and can serve as diagnostic tool to facilitate decisions on antiviral therapy.

An almost-intact human endogenous retrovirus K on human chromosome 7

I n contrast to the many defective endoge- nous retroviral sequences, the human endogenous retrovirus HERV-K(HML-2) (ref. 1) group shows conservation of intact retroviral genes (for review, see ref. 2). We have previously reported evidence that human chromosome 7 contains both intact HERV-K gag and env genes, indicating the possible existence of an HERV-K provirus with intact open reading frames (ORFs) for all retroviral genes3, 4.

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 protein cORF supports cell transformation and associates with the promyelocytic leukemia zinc finger protein.

Human endogenous retrovirus sequences (HERVs) reside in the genomes of primates and humans for several million years. The majority of HERVs is non-coding but a limited set is intact and can express proteins. We have recently identified an almost intact HERV-K(HML-2) provirus on chromosome 7 and have documented that most patients with germ cell tumors (GCTs) display antibodies directed against proteins of HERV-K(HML-2). To address whether these proteins merely represent tumor markers or contribute to neoplastic transformation, we examined the transforming potential of various HERV sequences and studied physical interactions between HERV and cellular proteins by yeast two-hybrid and biochemical assays. cORF, a protein encoded by the C-terminal open reading frame within the env gene, supports tumor growth in nude mice and associates with the promyelocytic leukemia zinc finger protein (PLZF). The interaction domains map between amino acid residues 21 and 87 of cORF, and between residues 245 and 543 of PLZF. PLZF is critical for spermatogenesis in mice. Abnormal spermatogenesis or maturation of gonocytes is thought to predispose humans to the development of germ cell tumors. Thus, cORF of human endogenous retroviruses may contribute to tumor development by interfering with processes during spermatogenesis that involve PLZF.

Characterization of DP103, a Novel DEAD Box Protein That Binds to the Epstein-Barr Virus Nuclear Proteins EBNA2 and EBNA3C

The Epstein-Barr virus-encoded nuclear antigens EBNA2 and EBNA3C both interact with the cellular transcription factor RBP-Jκ and modulate the expression of several shared target genes, suggesting a tight cooperation in latently infected cells. In a survey for additional cellular factors that bind to EBNA2 as well as EBNA3C, we have isolated and characterized DP103, a novel human member of the DEAD box family of putative ATP-dependent RNA helicases. The interaction with DP103 is mediated by amino acids (aa) 121–213 of EBNA2 and aa 534–778 of EBNA3C, regions that are not involved in binding of the viral proteins to RBP-Jκ. TheDP103-cDNA encodes a protein of 824 aa that harbors all of the common DEAD box motifs. Monoclonal antibodies raised against DP103 detect a protein of 103 kDa in mammalian cells that resides in high molecular weight complexes in vivo. We have detected an ATPase activity intrinsic to or closely associated with DP103. By subcellular fractionation, we find DP103 in both a soluble nuclear fraction as well as in the insoluble skeletal fraction. Whereas the protein and its mRNA are uniformly expressed in all tested cell lines, we observed differential expression of the mRNA in normal human tissues.

Evaluation of Use of Epstein-Barr Viral Load in Patients after Allogeneic Stem Cell Transplantation To Diagnose and Monitor Posttransplant Lymphoproliferative Disease

ABSTRACT Epstein-Barr virus (EBV)-induced posttransplant lymphoproliferative disease (PTLD) continues to be a serious complication following transplantation. The aim of the present study was to evaluate the EBV load as a parameter for the prediction and monitoring of PTLD. The EBV load was analyzed by a quantitative competitive PCR with 417 whole-blood samples of 59 patients after allogeneic stem cell transplantation (SCT). The EBV load was positive for all 9 patients with PTLD and for 17 patients without PTLD. The viral loads of patients with manifest PTLD differed from the loads of those without PTLD (median loads, 1.4 × 106 versus 4 × 104 copies/μg of DNA; P < 0.0001). A threshold value of 105 copies/μg of DNA showed the best diagnostic efficacy (sensitivity, 87%; specificity, 91%). However, in patients with less than three major risk factors for PTLD, the positive predictive value of this threshold was rather low. One week prior to the manifestation of PTLD, the EBV load was as low in patients who developed PTLD as in patients without disease (median, 2.2 × 104 copies/μg of DNA; P was not significant). EBV DNA tested positive first at 20 to 71 days prior to the clinical manifestation of PTLD and occurred with the same delay after transplantation regardless of disease (median delay, 52 versus 63 days; P was not significant). EBV DNA was detected earlier in patients with primary infections than in those with reactivations (33 versus 79 days; P = 0.01), but the peak levels were similar in the two groups. EBV primary infection or EBV reactivation is frequent in patients after allogeneic SCT but results in PTLD only in a subgroup of patients. Although evaluation of the EBV load has limitations, the EBV load represents a valuable parameter to guide therapy.

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.

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

ABSTRACT 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.

Epstein-Barr Virus Nuclear Antigen 1 Forms a Complex with the Nuclear Transporter Karyopherin α2

The Epstein-Barr virus (EBV) is implicated in the induction of several malignancies. The nuclear antigen 1 (EBNA1) is the only viral protein that is expressed consistently in all EBV-associated tumors. EBNA1 is involved in the replication and maintenance of the viral episome in the infected cell and exhibits oncogenic activity in transgenic mice. Here we report the identification of the nuclear transporter karyopherin α2 as a cellular partner of EBNA1 using the yeast “two-hybrid system.” Karyopherin α2 is also called importin α or Rch1. The binding to karyopherin α2 was mediated through a C-terminal region of EBNA1 encompassing the nuclear localization signal, whereas clones of EBNA1 devoid of the nuclear localization signal failed to bind to karyopherin α2. The interaction was biochemically confirmed by far-Western analysis using bacterially expressed karyopherin α2 and karyopherin α2-specific monoclonal antibodies. The nuclear transport of EBNA1 was impaired by expression of N-terminally truncated karyopherin α2. Zone velocity sedimentation in a sucrose gradient indicated that: (i) EBNA1 and Rch1 colocalize; and (ii) the association of karyopherin α2 with high molecular weight protein complexes might be impeded by the presence of EBNA1.

Np9 Protein of Human Endogenous Retrovirus K Interacts with Ligand of Numb Protein X

ABSTRACT 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.