Retroviral persistence in humans
Human endogenous retroviruses
(Prof. Dr. Christine Leib-Mösch)
Development of a retrovirus-specific microarray
To address these questions, we have developed a retrovirus-specific microarray (RetroArray) in collaboration with PD Dr. W. Seifarth, Medical Faculty Mannheim, University of Heidelberg. The DNA chip is based on the reverse transcriptase domain of retroviral pol genes and comprises representatives of all major HERV groups, as well as exogenous human retroviruses and animal retroviruses. The microarray can be used, for example, to determine endogenous and exogenous retroviral activity in human tissues and cells, to assess purity and homogeneity of human and mammalian cell lines, to survey xenotransplants and transplant recipients and to monitor packaging cell lines and vector preparations to exclude cotransfer of recombination competent retroviruses (RCR) and endogenous retroviruses (Seifarth et al., 2003, Zeilfelder et al., 2007, Seifarth et al., 2009).
Evolutionary history of HERVs in primates
HERVs are remnants of ancient germ line infections with exogenous retroviruses that have been genetically fixed and vertically transmitted since millions of years. During the course of evolution they have been amplified and spread throughout the genome by retrotransposition and/or reinfection. We used our retrovirus-specific DNA chip to trace back the most important integration and amplfication events during primate evolution (Greenwood et al. 2005). The results indicate that a major invasion and expansion of pol containing endogenous retroviruses (ERVs) occurred after the platyrrhine (New World Monkeys) lineage separated from the catarrhines (Old World Monkeys and apes) (Fig. 1).
- Fig. 1 Integration and expansion of endogenous retroviruses in Primates
Although the great majority of endogenous retroviruses resides at orthologous integration sites, the pattern of ERV expression is not conserved among Old World monkeys and humans (Stengel et al., 2006). Like many other genes ERVs show a higher transcriptional activity in human brain compared to brains of Old World monkeys.
Transcriptional activity of HERVs in human tissues
Tissue-specific HERV expression profiles could be established for all human tissues investigated so far using the retrovirus-specific microarray (Fig 2). No tissue completely lacking HERV transcription was found, confirming that HERVs are permanent components of the human transcriptome (Seifarth et al., 2005). Most active HERVs were identified in skin, thyroid gland, placenta and in tissues of reproductive organs.
- Fig. 2(a,b) HERV expression profiles in human tissues
A differential HERV transcription pattern was detected in human breast cancer compared to nonmalignant mammary gland tissue. In various subsets of patients increased transcript levels of single HERV groups were observed (Frank et al., 2008).
In brain samples of Patients with Schizophrenia and bipolar disorders a significant prevalence of a subgroup of HERV-K(HML-2) was found (Frank et al., 2005)
Comprehensive microarray-based analyses of further human malignancies, which have been associated with HERV activity previously, are in progress.
Influence of exogenous pathogens on the expression of endogenous retroviruses
Activation of human endogenous retroviral sequences can contribute to diseases by expression of pathogenic viral proteins or dysregulation of cellular genes. We are currently investigating the interaction of various exogenous viruses and other infectious agents with HERVs by transcription profiling and identification of differentially active genomic HERV loci (Fig. 3).
- Fig. 3 Activation of endogenous retroviruses by infectious agents
HERV LTR sequences as cell type-specific promoters in retroviral vectors
Retroviral long terminal repeats (LTRs) contain regulatory elements such as promoter, enhancer and transcription factor binding sites, which are required for gene expression. The human genome harbors more than half a million endogenous retroviral LTR sequences that can be regarded as mobile regulatory modules. A number of HERV-LTRs have been recruited during evolution as transcriptional control elements for cellular gene regulation (For a review see Leib-Mösch et al., 2005). To investigate the possibility of using such elements for the construction of tissue-specific retroviral vectors, we have randomly isolated more than 100 HERV LTR sequences and examined their activity in a transient luciferase assay in various cell lines (Fig. 4).
- Fig. 4 Promoter activity of two HERV LTRs in different cell lines
LTR sequences derived from two HERV groups, HERV-H and HERV-L, differing widely in their range of activity and tissue-specificity were cloned into a murine leukemia virus (MLV)-based promoter conversion vector (Schön et al., 2009). Various human cell lines were infected with the HERV-MLV hybrid vectors and cell type-specific expression of the reporter gene was demonstrated by FACS analysis. Interestingly, the activities of these vectors nearly exactly reflect the cell-type specificity of the corresponding transient transfected LTR containing plasmids and the tissue specificity of endogenous HERV transcription demonstrated by microarray analysis. In summary, HERV LTRs should be a valuable source of new cell type-specific regulatory sequences and represent promising candidates for the construction of retroviral vectors for use in human gene therapy.