Activation of a HERV-H LTR induces expression of an aberrant calbindin protein in human prostate carcinoma cells

The human genome contains around 500 000 LTR retrotransposons including endogenous retroviruses (HERVs). Most of these LTR elements are silenced by epigenetic conditions, but may be reactivated by environmental factors such as chemicals, radiation or exogenous viruses. We have analyzed a HERV-H element located upstream of the human calbindin gene (CALB-1) that leads to expression of a truncated calbindin protein by alternative splicing in a human prostate carcinoma cell line (PC3). PC3 cells are polyploid with one or two complete alleles of chromosome 8, and four to five pieces of the 8q-arm, all containing the CALB-1 locus. Analysis of these loci did not reveal substantial alterations on DNA sequence level, such as modification of splice sites, suggesting an epigenetic activation of the HERV-H LTR. Therefore, we compared the DNA methylation status of this LTR in PC3 cells and in three prostate carcinoma cell lines not expressing the truncated calbindin protein. We found that the HERV-H LTR is hypomethylated in two cell lines including PC3. Chromatin immunoprecipitation (ChIP) analysis, however, revealed that the chromatin is associated with active marks (acetylated histone H4 and tri-methylated lysine4 at histone H3) at this locus only in PC3 cells. This data suggests that reactivation of the HERV-H LTR requires at least two steps: demethylation of DNA and modification of histones. There is evidence that expression of the truncated calbindin prevents apoptosis and may thus contribute to the malignant phenotype of PC3 cells

Expression pattern analysis of transcribed HERV sequences is complicated by ex vivo recombination

<p>Abstract</p> <p>Background</p> <p>The human genome comprises numerous human endogenous retroviruses (HERVs) that formed millions of years ago in ancestral species. A number of loci of the HERV-K(HML-2) family are evolutionarily much younger. A recent study suggested an infectious HERV-K(HML-2) variant in humans and other primates. Isolating such a variant from human individuals would be a significant finding for human biology.</p> <p>Results</p> <p>When investigating expression patterns of specific HML-2 proviruses we encountered HERV-K(HML-2) cDNA sequences without proviral homologues in the human genome, named HERV-KX, that could very well support recently suggested infectious HML-2 variants. However, detailed sequence analysis, using the software RECCO, suggested that HERV-KX sequences were produced by recombination, possibly arising <it>ex vivo</it>, between transcripts from different HML-2 proviral loci.</p> <p>Conclusion</p> <p>As RT-PCR probably will be instrumental for isolating an infectious HERV-K(HML-2) variant, generation of "new" HERV-K(HML-2) sequences by <it>ex vivo </it>recombination seems inevitable. Further complicated by an unknown amount of allelic sequence variation in HERV-K(HML-2) proviruses, newly identified HERV-K(HML-2) variants should be interpreted very cautiously.</p

Expression patterns of transcribed human endogenous retrovirus HERV-K(HML-2) loci in human tissues and the need for a HERV Transcriptome Project

Background: A significant proportion of the human genome is comprised of human endogenous retroviruses (HERVs). HERV transcripts are found in every human tissue. Expression of proviruses of the HERV-K(HML-2) family has been associated with development of human tumors, in particular germ cell tumors (GCT). Very little is known about transcriptional activity of individual HML-2 loci in human tissues, though. Results: By employing private nucleotide differences between loci, we assigned ~1500 HML-2 cDNAs to individual HML-2 loci, identifying, in total, 23 transcriptionally active HML-2 proviruses. Several loci are active in various human tissue types. Transcription levels of some HML-2 loci appear higher than those of other loci. Several HML-2 Rec-encoding loci are expressed in GCT and non-GCT tissues. A provirus on chromosome 22q11.21 appears strongly upregulated in pathologic GCT tissues and may explain high HML-2 Gag protein levels in GCTs. Presence of Gag and Env antibodies in GCT patients is not correlated with activation of individual loci. HML-2 proviruses previously reported capable of forming an infectious HML-2 variant are transcriptionally active in germ cell tissue. Our study furthermore shows that Expressed Sequence Tag (EST) data are insufficient to describe transcriptional activity of HML-2 and other HERV loci in tissues of interest. Conclusion: Our, to date, largest-scale study reveals in greater detail expression patterns of individual HML-2 loci in human tissues of clinical interest. Moreover, large-scale, specialized studies are indicated to better comprehend transcriptional activity and regulation of HERVs. We thus emphasize the need for a specialized HERV Transcriptome Project

Development of a LightCycler PCR assay for detection and quantification of Aspergillus fumigatus DNA in clinical samples from neutropenic patients

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 LightCyclerbased 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

Modulation of human endogenous retrovirus (HERV) transcription during persistent and de novo HIV-1 infection

Background: The human genome contains multiple LTR elements including human endogenous retroviruses (HERVs) that together account for approximately 8–9% of the genomic DNA. At least 40 different HERV groups have been assigned to three major HERV classes on the basis of their homologies to exogenous retroviruses. Although most HERVs are silenced by a variety of genetic and epigenetic mechanisms, they may be reactivated by environmental stimuli such as exogenous viruses and thus may contribute to pathogenic conditions. The objective of this study was to perform an in-depth analysis of the influence of HIV-1 infection on HERV activity in different cell types. Results: A retrovirus-specific microarray that covers major HERV groups from all three classes was used to analyze HERV transcription patterns in three persistently HIV-1 infected cell lines of different cellular origins and in their uninfected counterparts. All three persistently infected cell lines showed increased transcription of multiple class I and II HERV groups. Up-regulated transcription of five HERV taxa (HERV-E, HERV-T, HERV-K (HML-10) and two ERV9 subgroups) was confirmed by quantitative reverse transcriptase PCR analysis and could be reversed by knock-down of HIV-1 expression with HIV-1-specific siRNAs. Cells infected de novo by HIV-1 showed stronger transcriptional up-regulation of the HERV-K (HML-2) group than persistently infected cells of the same origin. Analysis of transcripts from individual members of this group revealed up-regulation of predominantly two proviral loci (ERVK-7 and ERVK-15) on chromosomes 1q22 and 7q34 in persistently infected KE37.1 cells, as well as in de novo HIV-1 infected LC5 cells, while only one single HML-2 locus (ERV-K6) on chromosome 7p22.1 was activated in persistently infected LC5 cells. Conclusions: Our results demonstrate that HIV-1 can alter HERV transcription patterns of infected cells and indicate a correlation between activation of HERV elements and the level of HIV-1 production. Moreover, our results suggest that the effects of HIV-1 on HERV activity may be far more extensive and complex than anticipated from initial studies with clinical material

Influence of Antipsychotic Drugs on Human Endogenous Retrovirus (HERV) Transcription in Brain Cells

Human endogenous retroviruses (HERVs) have been associated with various neurological and neuropsychiatric disorders. Transcripts and proteins of at least three HERV groups, HERV-W, ERV9 and HERV-K(HML-2) have been detected repeatedly in brain samples or cerebrospinal fluid of patients with schizophrenia suggesting that alterations in HERV activity may play a role in etiopathogenesis. Current therapies otherwise include neuroleptics and/or antidepressants that may induce epigenetic alterations and thus influence HERV expression. To investigate the effects of these drugs on HERV transcriptional activity, HERV expression profiles of a broad range of human brain cell lines treated with valproic acid (VPA), haloperidol, risperidone, and clozapine were analyzed using a retrovirus-specific microarray and qRT-PCR. Investigation of 52 HERV subgroups revealed upregulation of several class I and class II HERV elements by VPA in a dose-dependent manner. The strongest effect was observed on HERV-W and ERV9 groups in the human glioblastoma cell lines SK-N-SH and SK-N-MC, respectively. The transcript level of HERV-K(HML-2) elements was not influenced. Transcription of HERV-W, ERV9 and HERV-K(HML-2) taxa was further quantified in postmortem brain samples of patients with schizophrenia, bipolar disorders and a healthy control group with regard to their medication. Patients with schizophrenia showed a significantly higher HERV-W transcription associated with VPA treatment. However in case of ERV9, enhanced transcript levels could not be explained solely by VPA treatment, since a slight increase was also found in untreated patients compared to healthy controls. HERV

Identification of S71-Related Human Endogenous Retroviral Sequences with Full-Length pol Genes

AbstractThe human genome contains sequences related to the simian sarcoma-associated virus SSAV. One of these endogenous retrovital elements, S71, is truncated in the pol gene and carries an insertion of a solitary HERV-K LTR. Using a PCR approach we have now identified further S71-related retroviral elements that lack the HERV-K LTR insertion and contain a full-length retroviral reverse transcriptase. Two of these sequences, pCRTK1 and pCRTK6, were cloned and further characterized. Clones pCRTK1 and pCRTK6 showed between 85 and 90% nucleotide hemology to each other and to S71 within the "tether" region of the pol gene, indicating that pCRTK1 and pCRTK6 clearly belong to the S71 subgroup of C-type-related human endogenous retroviral elements. Some point mutations inactivating the reverse transcriptase are located at the same positions in pCRTK1 and pCRTK6. Therefore, we assume that these S71-related elements were dispersed in the human genome by reintegration as defective proviruses, probably using enzymes for retrotransposition provided in trans by other retrotransposons or by cellular genes. Examination of the presence of S71-related elements in apes and Old World monkeys revealed that the deletion of reverse transcriptase sequences in S71 has occurred in the lineage of primates prior to the insertion of the HERV-K LTR