| BackgroundHuman endogenous retroviruses or HERVs are a family of viruses within our genomewith similarities to present day exogenous retroviruses. HERVs have made a significantcontribution to animal evolution, including that of humans. HERVs representapproximately10%of the human genome. There is growing evidence that HERVs havemade a significant contribution to human evolution, development and physiology, as wellas playing possible roles in human diseases. At the same time there is a dilemma in the factthat viral genes are excluded from standard genetic screening programs in the service ofbiological and medical geneticists. Critical to our growing understanding of the role ofHERVs is the need for a reliable methodology for the expression of virus-derived proteinsin tissues and organs during development, normal physiology and disease. This thesis isfocused on human endogenous retroviruses expression in normal and neoplastic tissueswith special emphasis on HERV-R (ERV3) and HERV-W (Syncytin-1, Syncytin-2).ERV3is an almost complete human endogenous retrovirus. Altogether approximately50copies are present in the human DNA. Previous studies have shown that these HERV-Rsequences are highly expressed on RNA-level in several human tissues especially thoseinvolved in: a. hormonal dependant organ such as: testis, placenta, adrenal glands andcorpus luteum, b. tissues in which cells are undergoing fusion: cyto/syncytiotrophoblasts,macrophages and myoblasts, c. tissues with close contact with external surface e.g.bronchial epithelium, skin (sebaceous glands, salivary and lacrimal gland. Those studieswere based mainly on real time PCR-QPCR, Northern blot and in situ hybridization and insome selected cases also on protein level.The two proteins designated Syncytin-1and Syncytin-2are captured retroviralenvelope proteins from the HERV-W locus. Hard evidences exist that these two proteins (may be in synergy with other active HERVs) are vital in the development of the humanplacenta and they are expressed in the trophoblastic and syncytiotrophoblastic cell layers.Material and MethodsIn this study we have conducted large scale investigation of the endogenous retrovirus,looking for the expression of its Env-gene-coded protein in a wide range of normal anddiseased tissues.For basic protein profiling, formalin-fixed and paraffin-embedded archival specimenswere collected from144individuals to represent histological normal tissues and from216cases of cancer to represent the20most common forms of human cancer.The study was divided into two parts：1. In this study we have performed a large scale investigation of ERV3Env expressionon protein level in normal and diseased tissues. The impetus was initially to confirmprevious ERV-3expression on RNA level and explore if also ERV3encoded proteins areproduced in the corresponding tissues and to find a suitable model tissue in which theretroviral coded protein could be modified for further studies. For these purposes twoaffinity-purified polyclonal antibodies to ERV3env encoded protein were generated f todetect the ERV3envelope (Env) protein expression in specific human cells, tissues andorgans using western blot, immunohistochemistry and immunofluorescence assays.2. We have also developed new monospecific antibodies against Syncytin-1andSyncytin-2proteins. Those reagents were used in an extended investigation of thedistribution of these viral encoded proteins in normal and in a survey of neoplastic tissues.For these purposes specially designed tissue microarrays (TMAs) were used in combinationwith immunohistochemistry.Results1. We concluded that ERV3encoded Env proteins are expressed at significant levels inplacenta, testis, adrenal gland, corpus luteum, Fallopian tubes, sebaceous glands, astrocytes,bronchial epithelium and the ducts of the salivary glands. Moderate to highly significant expression was also seen in a range of other tissues, including a variety of epithelial cells aswell as cells known to undergo fusion in inflammation and in normal physiology, includingfused macrophages, myocardium and striated muscle.2. The expression of these Syncytin-1and Syncytin-2mainly displays the same pattern,with some important differences. Both syncytins are highly expressed in brain andcorresponding malignant glioma, in the placenta the localization is as expected in thecyto/syncytiotrophoblastic layer. Interestingly, a high expression for syncytin-2was seen inLangerhans islets in the pancreas.Conclusion1. These findings confirm that ERV3proteins have made a significant contribution tohuman evolution and in particular to the holobiontic evolution of the human genome.2. The high expression of the two fusogenic proteins in some tissues applying our newantibodies is in high extent in agreement with previous studies but also show majordifferences which could indicate that different HERV-W locus are active were seen.3. We present this as a suitable model for future investigation of the expression ofHERV-derived proteins in normal tissues, which could then be extrapolated to humanevolution, normal development and physiology as well as the potential for extrapolation todisease. |
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