Investigation of Endogenous Retrovirus Sequences in the Neighborhood of Genes Up-regulated in a Neuroblastoma Model after Treatment with Hypoxia-Mimetic Cobalt Chloride

Abstract

Human endogenous retroviruses (ERVs) have been found to be associated with different diseases, e.g., multiple sclerosis (MS). Most human ERVs integrated in our genome are not competent to replicate and these sequences are presumably silent. However, transcription of human ERVs can be reactivated, e.g., by hypoxia. Interestingly, MS has been linked to hypoxia since decades. As some patterns of demyelination are similar to white matter ischemia, hypoxic damage is discussed. Therefore, we are interested in the association between hypoxia and ERVs. As a model, we used human SH-SY5Y neuroblastoma cells after treatment with the hypoxia-mimetic cobalt chloride and analyzed differences in the gene expression profiles in comparison to untreated cells. The vicinity of up-regulated genes was scanned for endogenous retrovirus-derived sequences. Five genes were found to be strongly up-regulated in SH-SY5Y cells after treatment with cobalt chloride: clusterin, glutathione peroxidase 3, insulin-like growth factor 2, solute carrier family 7 member 11, and neural precursor cell expressed developmentally down-regulated protein 9. In the vicinity of these genes we identified large (>1,000 bp) open reading frames (ORFs). Most of these ORFs showed only low similarities to proteins from retro-transcribing viruses. However, we found very high similarity between retrovirus envelope sequences and a sequence in the vicinity of neural precursor cell expressed developmentally down-regulated protein 9. This sequence encodes the human endogenous retrovirus group FRD member 1, the encoded protein product is called syncytin 2. Transfection of syncytin 2 into the well-characterized Ewing sarcoma cell line A673 was not able to modulate the low immunostimulatory activity of this cell line. Future research is needed to determine whether the identified genes and the human endogenous retrovirus group FRD member 1 might play a role in the etiology of MS.

Keywords: ERVFRD-1; HERV-FRD; endogenous retroviruses; human endogenous retrovirus group FRD member 1; hypoxia; multiple sclerosis; neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9); open reading frames.

FIGURE 1

A working model for ERV reactivation. ERVs constitute an integral part of our genome. Under normal conditions, expression of ERVs is switched off epigenetically. Triggered by diverse factors like hypoxia, reactivation of ERV expression can be induced. ERV-encoded proteins can act as immunostimulatory superantigens or induce cytopathic effects, e.g., cell fusion.

FIGURE 2

Expression of NEDD9 in SH-SY5Y cells. Expression of NEDD9 was analyzed in SH-SY5Y cells under different culture conditions by qRT-PCR. Cells were cultured in absence of CoCl₂ (water control), with 100 μM of CoCl₂ or with 200 μM of CoCl₂. Presented are means and standard deviations from three independent experiments. For comparative analysis, beta actin was used as housekeeping control and the median expression of all samples was set as one.

FIGURE 3

Organization of the NEDD9/SMIM13/HERV-FRD region in humans and other species. (A) HERV-FRD is located in the intron of SMIM13. SMIM13 and NEDD9 are located tail-to-tail orientated on human chromosome 6. (B) Comparison of the SMIM13/HERV-FRD region in different vertebrate species. Red: exons of SMIM13; green: SMIM13 intron; yellow: HERV-FRD.

FIGURE 4

Absence of immunomodulatory effects of HERV-FRD transfected A673 cells. A673 cells were transfected with HERV-FRD or with empty vector (mock). (A) RNA from transfected cells were analyzed for presence of HERV-FRD and the Ewing sarcoma specific transcripts EWSR1-FLI1 and LIPI. (B) Transfected cells were used as stimulatory cells in mixed lymphocyte/tumor cell cultures. Stimulated cells were analyzed for presence of T cells (CD3), cytotoxic T cells (CD8), and activated lymphocytes (CD25). Presented are means and standard deviations from three independent experiments.