Abstract
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-γ 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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Allen RC, Graves G, Budowle B (1989) Polymerase chain reaction amplification products separated on rehydratable polyacrylamide gels and stained with silver. Biotechniques 7:736–744
Andersson G, Svensson AC, Setterblad N, Rask L (1998) Retroelements in the human MHC class II region. Trends Genet 14:109–114
Buzdin AA, Lebedev Iu B, Sverdlov ED (2003) Human genome-specific HERV-K intron LTR genes have a random orientation relative to the direction of transcription, and, possibly, participated in antisense gene expression regulation. Bioorg Khim 29:103–106
Cameron PU, Mallal SA, French MA, Dawkins RL (1990) Major histocompatibility complex genes influence the outcome of HIV infection. Ancestral haplotypes with C4 null alleles explain diverse HLA associations. Hum Immunol 29:282–295
Christy RJ, Huang RC (1988) Functional analysis of the long terminal repeats of intracisternal A-particle genes: sequences within the U3 region determine both the efficiency and direction of promoter activity. Mol Cell Biol 8:1093–1102
Chu X, Rittner C, Schneider PM (1995) Length polymorphism of the human complement component C4 gene is due to an ancient retroviral integration. Exp Clin Immunogenet 12:74–81
Dangel AW, Mendoza AR, Baker BJ, Daniel CM, Carroll MC, Wu LC, Yu CY (1994) The dichotomous size variation of human complement C4 genes is mediated by a novel family of endogenous retroviruses, which also establishes species-specific genomic patterns among old world primates. Immunogenetics 40:425–436
Dangel AW, Baker BJ, Mendoza AR, Yu CY (1995) Complement component C4 gene intron 9 as a phylogenetic marker for primates: long terminal repeats of the endogenous retrovirus ERV-K(C4) are a molecular clock of evolution. Immunogenetics 42:41–52
Domansky AN, Kopantzev EP, Snezhkov EV, Lebedev YB, Leib-Mosch C, Sverdlov ED (2000) Solitary HERV-K LTRs possess bi-directional promoter activity and contain a negative regulatory element in the U5 region. FEBS Lett 472:191–195
Feuchter A, Mager D (1990) Functional heterogeneity of a large family of human LTR-like promoters and enhancers. Nucleic Acids Res 18:1261–1270
Feuchter AE, Freeman JD, Mager DL (1992) Strategy for detecting cellular transcripts promoted by human endogenous long terminal repeats: identification of a novel gene (CDC4L) with homology to yeast CDC4. Genomics 13:1237–1246
Gilman M (1993) In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology, vol 1. Greene and Wiley-Interscience, New York, pp 4.7.1–4.7.6
Kato N, Pfeifer-Ohlsson S, Kato M, Larsson E, Rydnert J, Ohlsson R, Cohen M (1987) Tissue-specific expression of human provirus ERV3 mRNA in human placenta: two of the three ERV3 mRNAs contain human cellular sequences. J Virol 61:2182–2191
Knossl M, Lower R, Lower J (1999) Expression of the human endogenous retrovirus HTDV/HERV-K is enhanced by cellular transcription factor YY1. J Virol 73:1254–1261
Koyanagi Y, O’Brien WA, Zhao JQ, Golde DW, Gasson JC, Chen IS (1988) Cytokines alter production of HIV-1 from primary mononuclear phagocytes. Science 241:1673–1675
Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, et al (2001) Initial sequencing and analysis of the human genome. Nature 409:860–921
Lechler R, Warrens A (2000) HLA in health and disease. Academic, London
Leib-Mosch C, Seifarth W (1995) Evolution and biological significance of human retroelements. Virus Genes 11:133–145
Leib-Mosch C, Haltmeier M, Werner T, Geigl EM, Brack-Werner R, Francke U, Erfle V, Hehlmann R (1993) Genomic distribution and transcription of solitary HERV-K LTRs. Genomics 18:261–269
Lower R, Lower J, Kurth R (1996) The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences. Proc Natl Acad Sci USA 93:5177–5184
McNeil AJ, Yap PL, Gore SM, Brettle RP, McColl M, Wyld R, Davidson S, Weightman R, Richardson AM, Robertson JR (1996) Association of HLA types A1-B8-DR3 and B27 with rapid and slow progression of HIV disease. Q J Med 89:177–185
Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035–7056
Ono M, Yasunaga T, Miyata T, Ushikubo H (1986) Nucleotide sequence of human endogenous retrovirus genome related to the mouse mammary tumor virus genome. J Virol 60:589–598
Prentice HL, Schneider PM, Strominger JL (1986) C4B gene polymorphism detected in a human cosmid clone. Immunogenetics 23:274–276
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor
Samuelson LC, Wiebauer K, Snow CM, Meisler MH (1990) Retroviral and pseudogene insertion sites reveal the lineage of human salivary and pancreatic amylase genes from a single gene during primate evolution. Mol Cell Biol 10:2513–2520
Schneider PM, Carroll MC, Alper CA, Rittner C, Whitehead AS, Yunis EJ, Colten HR (1986) Polymorphism of the human complement C4 and steroid 21-hydroxylase genes. Restriction fragment length polymorphisms revealing structural deletions, homoduplications, and size variants. J Clin Invest 78:650–657
Schneider PM, Witzel-Schlomp K, Rittner C, Zhang L (2001) The endogenous retroviral insertion in the human complement C4 gene modulates the expression of homologous genes by antisense inhibition. Immunogenetics 53:1–9
Stonans I, Stonane E, Russwurm S, Deigner HP, Bohm KJ, Wiederhold M, Jager L, Reinhart K (1999) HepG2 human hepatoma cells express multiple cytokine genes. Cytokine 11:151–156
Sverdlov ED (2000) Retroviruses and primate evolution. Bioessays 22:161–171
Tassabehji M, Strachan T, Anderson M, Campbell RD, Collier S, Lako M (1994) Identification of a novel family of human endogenous retroviruses and characterization of one family member, HERV-K(C4), located in the complement C4 gene cluster. Nucleic Acids Res 22:5211–5217
Urnovitz HB, Murphy WH (1996) Human endogenous retroviruses: nature, occurrence, and clinical implications in human disease. Clin Microbiol Rev 9:72–99
Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, Gocayne JD, Amanatides P, Ballew RM, Huson DH, Wortman JR, Zhang Q, Kodira CD, Zheng XH, Chen L, Skupski M, Subramanian G, Thomas PD, Zhang J, Gabor Miklos GL, Nelson C, Broder S, Clark AG, Nadeau J, McKusick VA, Zinder N, Levine AJ, Roberts RJ, Simon M, Slayman C, Hunkapiller M, Bolanos R, Delcher A, Dew I, Fasulo D, Flanigan M, Florea L, Halpern A, Hannenhalli S, Kravitz S, Levy S, Mobarry C, Reinert K, Remington K, Abu-Threideh J, Beasley E, Biddick K, Bonazzi V, Brandon R, Cargill M, Chandramouliswaran I, Charlab R, Chaturvedi K, Deng Z, Di Francesco V, Dunn P, Eilbeck K, Evangelista C, Gabrielian AE, Gan W, Ge W, Gong F, Gu Z, Guan P, Heiman TJ, Higgins ME, Ji RR, Ke Z, Ketchum KA, Lai Z, Lei Y, Li Z, Li J, Liang Y, Lin X, Lu F, Merkulov GV, Milshina N, Moore HM, Naik AK, Narayan VA, Neelam B, Nusskern D, Rusch DB, Salzberg S, Shao W, Shue B, Sun J, Wang Z, Wang A, Wang X, Wang J, Wei M, Wides R, Xiao C, Yan C, et al (2001) The sequence of the human genome. Science 291:1304–1351
Yu CY (1998) Molecular genetics of the human MHC complement gene cluster. Exp Clin Immunogenet 15:213–230
Yu CY, Chung EK, Yang Y, Blanchong CA, Jacobsen N, Saxena K, Yang Z, Miller W, Varga L, Fust G (2003) Dancing with complement C4 and the RP-C4-CYP21-TNX (RCCX) modules of the major histocompatibility complex. Prog Nucleic Acid Res Mol Biol 75:217–292
Acknowledgements
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Schn 284/6-3). We would like to thank J. Hielscher and T. Beißmann for their technical assistance regarding the real-time PCR experiments.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mack, M., Bender, K. & Schneider, P.M. Detection of retroviral antisense transcripts and promoter activity of the HERV-K(C4) insertion in the MHC class III region. Immunogenetics 56, 321–332 (2004). https://doi.org/10.1007/s00251-004-0705-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00251-004-0705-y