Christine Leib-Mösch

Comprehensive analysis of human endogenous retrovirus transcriptional activity in human tissues with a retrovirus-specific microarray.

ABSTRACT Retrovirus-like sequences account for 8 to 9% of the human genome. Among these sequences, about 8,000 pol-containing proviral elements have been identified to date. As part of our ongoing search for active and possibly disease-relevant human endogenous retroviruses (HERVs), we have recently developed an oligonucleotide-based microarray. The assay allows for both the detection and the identification of most known retroviral reverse transcriptase (RT)-related nucleic acids in biological samples. In the present study, we have investigated the transcriptional activity of representative members of 20 HERV families in 19 different normal human tissues. Qualitative evaluation of chip hybridization signals and quantitative analysis by real-time RT-PCR revealed distinct HERV activity in the human tissues under investigation, suggesting that HERV elements are active in human cells in a tissue-specific manner. Most active members of HERV families were found in mRNA prepared from skin, thyroid gland, placenta, and tissues of reproductive organs. In contrast, only few active HERVs were detectable in muscle cells. Human tissues that lack HERV transcription could not be found, confirming that human endogenous retroviruses are permanent components of the human transcriptome. Distinct activity patterns may reflect the characteristics of the regulatory machinery in these cells, e.g., cell type-dependent occurrence of transcriptional regulatory factors.

Development of a LightCycler PCR Assay for Detection and Quantification of Aspergillus fumigatus DNA in Clinical Samples from Neutropenic Patients

ABSTRACT The increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the diagnostic tools for this disease. We established a LightCycler-based real-time PCR assay to detect and quantify rapidly, specifically, and sensitively Aspergillus fumigatus DNA in both bronchoalveolar lavage (BAL) and blood samples from high-risk patients. The primers and hybridization probes were derived from an A. fumigatus-specific sequence of the mitochondrial cytochrome b gene. The assay is linear in the range between 13.2 fg and 1.3 ng of A. fumigatus DNA, corresponding to 3 to 300,000 CFU per ml of BAL fluid or blood. No cross-amplification was observed with human DNA or with the DNA of fungal or bacterial pathogens. For clinical evaluation we investigated 10 BAL samples from nine neutropenic patients with malignant hematological diseases and 12 blood samples from seven neutropenic patients with malignant hematological diseases. Additionally, we tested one blood sample and one BAL sample from each of two neutropenic patients. In order to characterize the validity of the novel PCR assay, only samples that had shown positive results by a previously described sensitive and specific nested PCR assay were tested. Twelve of 12 BAL samples and 6 of 14 blood samples gave positive results by the LightCycler PCR assay. Eight of 14 blood samples gave negative results by the novel method. The LightCycler PCR-mediated quantification of the fungal burden showed 15 to 269,018 CFU per ml of BAL sample and 298 to 104,114 CFU per ml of blood sample. Twenty of 20 BAL samples and 50 of 50 blood samples from subjects without evidence of invasive pulmonary aspergillosis (IPA) were PCR negative. Compared to a previously described nested PCR assay, these preliminary data for the novel real-time PCR assay indicate a less sensitive rate of detection of IPA in high-risk patients, but the assay may be valuable for quantification of the fungal burden in individual clinical samples.

Human Endogenous Retrovirus Expression Profiles in Samples from Brains of Patients with Schizophrenia and Bipolar Disorders

ABSTRACT The detection and identification of retroviral transcripts in brain samples, cerebrospinal fluid, and plasma of individuals with recent-onset schizophrenia and schizoaffective disorders suggest that activation or upregulation of distinct human endogenous retroviruses (HERVs) may play a role in the etiopathogenesis of neuropsychiatric diseases. To test this hypothesis, we performed a comprehensive microarray-based analysis of HERV transcriptional activity in human brains. We investigated 50 representative members of 20 HERV families in a total of 215 brain samples derived from individuals with schizophrenia or bipolar disorders and matched controls. A characteristic brain-specific retroviral activity profile was found that consists of members of the class I families HERV-E, HERV-F, and ERV9 and members of HERV-K taxa. In addition to these constitutively expressed HERVs, a number of differentially active HERV elements were identified in all brain samples independent of the disease pattern that may reflect differences in the genetic background of the tested individuals. Only a subgroup of the HML-2 family (HERV-K10) was significantly overrepresented in both bipolar-disorder- and schizophrenia-associated samples compared to healthy brains, suggesting a potential association with disease. Real-time PCR analysis of HERV env transcripts with coding capacity potentially involved in neuroinflammatory conditions revealed that env expression of HERV-W, HERV-FRD, and HML-2 remains unaffected regardless of the clinical picture. Our data suggest that HERV transcription in brains is weakly correlated with schizophrenia and related diseases but may be influenced by the individual genetic background, brain-infiltrating immune cells, or medical treatment.

Variable Transcriptional Activity of Endogenous Retroviruses in Human Breast Cancer

ABSTRACT Human endogenous retroviruses (HERVs) account for up to 9% of the human genome and include more than 800 elements related to betaretroviruses. While mouse mammary tumor virus (MMTV) is the accepted etiological agent of mammary tumors in mice, the role of retroviral elements in human breast cancer remains elusive. Here, we performed a comprehensive microarray-based analysis of overall retroviral transcriptional activities in 46 mammary gland tissue specimens representing pairs of nonmalignant and tumor samples from 23 patients. An analysis of nonmalignant tissue samples revealed a distinct, mammary gland-specific HERV expression profile that consists of 18 constitutively active HERV taxa. For corresponding tumor samples, a general trend toward lower levels of HERV transcription was observed, suggesting common regulatory mechanisms. In various subsets of patients, however, increased transcript levels of single class I HERV families (HERV-T, HERV-E, and HERV-F) and several class II families, including HML-6, were detected. An analysis of transcribed HML-6 sequences revealed either the activation of some or the increased activity of several proviral loci. No evidence for MMTV or human MMTV-like virus transcripts was found, indicating that transcriptionally active, MMTV analogous, exogenous viruses were not present in the breast cancer samples analyzed.

Human SSAV-related endogenous retroviral element: LTR-like sequence and chromosomal localization to 18q21

A new family of human endogenous retroviral sequences was recently discovered by way of its relationship to the simian sarcoma-associated virus (SSAV). One molecular clone, termed S71, contains sequences related to the genes coding for the group-specific antigens (gag) and polymerase (pol) proteins of SSAV. At the 3 end of this human retroviral element we have now found a 535-bp region which shows features characteristics of a retroviral long terminal repeat, including potential signal sequences essential for transcriptional control. By means of Southern blotting and in situ hybridization, the sequence was mapped to chromosome 18 band q21.

Isolation of an SSAv-related endogenous sequence from human DNA

We have found human DNA to contain a number of sequences related to simian sarcoma associated virus (SSAV). One of these sequences was isolated from a human genomic library. The molecular clone, termed S71, contains regions homologous to SSAV gag and pol fragments and SSAV LTR. Furthermore, hybridization experiments and DNA sequencing revealed distinct homologies to the reverse transcriptase coding region of several other retroviruses including baboon endogenous virus (BaEV) and murine leukemia viruses (MuLV) as well as retrovirus-like elements. Some sequence homology was also found with the C-type retrovirus-related multicopy human clone 4-1. S71 is present in only one copy per human genome equivalent and exhibits an EcoRI restriction fragment length polymorphism.

S71 is a phylogenetically distinct human endogenous retroviral element with structural and sequence homology to simian sarcoma virus (SSV)

Human endogenous retroviral element S71 had previously been shown to contain gag- and pol-related regions and a 3 LTR-like sequence. The nucleotide sequence of S71 was determined and compared with the corresponding regions of SSV and its helper virus SSAV. The 1.48-kb S71 gag region consists of matrix protein p15 (MA)-, capsid protein p30 (CA)-, and nucleocapsid protein p10 (NC)-related sections and the 1.82-kb pol region of tether, RNase H (RH), and endonuclease/integrase (IN) sections. The S71 nucleotide sequence contains a 167 amino acid open reading frame encompassing MA. The boundaries of the S71 element are delimited by direct repeats and the entire element is 5.4 kb long. Similarity between S71 and the v-sis-bearing, defective SSV provirus also covers overall structural organization, including the presence of presumably nonretroviral sequences. Both the gag and the pol regions of S71 contain sequences highly conserved in numerous retroviruses. Phylogenetic analysis with conserved CA, RH, and IN sequences showed that of all other (C-type) human retroviral elements available for comparison, S71 is most closely related to infectious primate and murine retroviruses. This suggests that S71 represents a phylogenetic subgroup of its own. In addition we identified short ranges of conserved amino acid sequences within C-type retroviral gag and pol genes sufficient for phylogenetic analysis. Use of these may facilitate large-scale phylogenetic evaluation of C-type retroviral elements and allow rapid classification of new elements.

Oncogenic retrovirus from spontaneous murine osteomas II. Molecular cloning and genomic characterization

An N-ecotropic murine leukemia virus (OA MuLV), originally isolated from spontaneous osteomas of strain 101 mice, was molecularly cloned. The virus induces osteomas, osteopetrosis, and malignant lymphomas in NMRI mice. The cloned virus was analyzed by heteroduplex analysis, restriction enzyme mapping, and oligonucleotide mapping. The data show a very close relationship to the endogenous Akv prototype virus with some differences in the gag and the env region. The nucleotide sequence of the U3 region of OA MuLV LTR revealed a structure within the presumable enhancer region very similar to the U3 sequences of the FBJ murine sarcoma virus and its associated helper virus. The significance of these specific structures for the oncogenicity of the virus and the development of the typical disease pattern is discussed.

Biological significance of human endogenous retroviral sequences

Endogenous retroviruses (ERVs) have been known for many years to exist in numerous natural and laboratory animal species. In humans it has been demonstrated that at least 1% of the genome consists of retrovirus-related sequences. Involvement of ERVs in the development of neoplastic and autoimmune diseases in the mouse model implicated a potentially pathogenic role of ERVs for humans, too. The research in this field led to a number of results strongly suggesting that human endogenous retroviral sequences (HERVs) are biologically active, on the RNA and even on the protein level. Particle formation, regulation or dysregulation of cellular gene expression, and synthesis of potentially pathogenic viral proteins indicate the broad spectrum of mechanisms by which HERVs may obtain biological significance.

Expression Profiles of Endogenous Retroviruses in Old World Monkeys

ABSTRACT Human endogenous retroviruses (HERVs) are a major component of the human genome and an active part of the transcriptome. Some HERVs play vital biological roles, while others potentially contribute to diseases. Many HERVs are relatively new in the primate genome, having entered or expanded after the lineages leading to the platyrrhines (New World monkeys) and catarrhines (Old World monkeys and apes) separated. Most HERVs are active in at least some tissues, though tissue specificity is common for most elements. We analyzed multiple tissues from several Old World monkeys using retroviral pol-based DNA microarrays and quantitative PCR methods to determine their ERV expression profiles. The results demonstrate that while many ERVs are active in nonhuman primates, overall the tissue expression specificity is unique to each species. Most striking is that while the majority of HERVs analyzed in this study are expressed in human brain, almost none are expressed in Old World monkey brains or are only weakly expressed.