Detection of retroviral antisense transcripts and promoter activity of the HERV-K(C4) insertion in the MHC class III region

An insertion of 6.4 kb is present in intron 9 of 60% of the human complement C4 genes, as well as in the C4 genes of a number of Old World primates. This insertion has the typical genomic organization of endogenous retroviruses, with the three major genes gag, pol and env flanked by long terminal repeats (LTRs). This human endogenous retrovirus K [HERV-K(C4)] insertion is in reverse orientation to the C4 coding sequence. Using RT-PCR as well as RNase protection assays, retroviral transcripts could be detected in different human cell lines which were only present in the antisense orientation of the retrovirus. Furthermore, C4 expression as well as intermediate transcripts comprising both HERV-K(C4) and C4 coding sequences was observed in these cells. These findings were confirmed using real-time PCR to quantitate the number of specific mRNA transcripts. Using reporter gene assays, it could be demonstrated that only the 3'LTR exhibits promoter activity, but in the sense orientation of the retrovirus. It has been suggested earlier that expression of C4 could lead to the transcription of a retroviral antisense RNA, which might protect against exogenous retroviral infections. In a previous study, it was shown that the expression of retroviral-like constructs was significantly downregulated in mouse cells transfected with human C4 genes, and that this downregulation was further modulated after IFN-gamma stimulation of C4 expression. In a new series of experiments, we have now confirmed these observations, using human hepatoma cells constitutively expressing C4. A dose-dependent downregulation of up to 45% caused by hybridization of retroviral sense and genomic HERV-K(C4) antisense RNA was observed. The functional 3'LTR promoter, the presence of retroviral antisense RNA transcripts and the functional detection of HERV-K(C4)-specific antisense activity provide strong evidence for a major role of the HERV-K(C4) insertion in the control of gene expression, resulting in a selective advantage favouring the presence of this element in human and primate C4 genes.