Summary
Starting from a cosmid library of HCMV Ad169-DNA random fragments of DNA were generated. Fragments about 200 to 600 bp in length were selected and cloned into open reading frame (ORF) expression vectors to create ORF-libraries that represent either the entire viral genome or defined subregions. About 120.000 clones were isolated and screened immunologically for the synthesis of fusion proteins consisting of an antigenic peptide encoded by the CMV sequence coupled to a truncatedE. coli β-galactosidase molecule. Anti-CMV sera raised in animals as well as human hyperimmune globulin were used for colony screening. Distinct sets of antigenic fusion proteins were recognized by different antisera. Ten of the clones giving strong reactions with human immune sera were mapped on the CMV genome and the sequences of the CMV inserts determined. Antibodies against fusion proteins were raised in mice or rabbits to identify the corresponding CMV proteins. Antigenic fusion proteins described here were recognized by most individual human CMV immune sera tested. They allow determination of the humoral immune response to defined determinants and may therefore be particularly useful in diagnosis and vaccine development.
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Alford CA, Britt WJ (1985) Cytomegalovirus. In: Fields BN (ed) Virology. Raven Press, New York, pp 629–657
Baer R, Bankier AT, Biggin MD, Deininger PL, Farrell PJ, Gibson TJ, Hatfull G, Hudson GS, Satchwell SC, Seguin C, Tuffnell PS, Barrell BG (1984) DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 310: 207–211
Bernhard HU, Helsinki DR (1980) Bacterial plasmid cloning vehicles. In: Setlow JK, Holländer A (eds) Genetic engineering: principles and methods. Plenum, New York, pp 133–168
Cranage MP, Kouzarides T, Bankier AT, Satchwell S, Weston K, Tomlinson P, Barrell B, Hart H, Bell SE, Minson AC, Smith GL (1986) Identification of the human cytomegalovirus glycoprotein B gene and induction of neutralizing antibodies via its expression in recombinant vaccinia virus. EMBO J 5: 3057–3063
Cranage MP, Smith GL, Bell SE, Hart H, Brown CA, Bankier T, Tomlinson P, Barrell BG, Minson TC (1988) Identification and expression of a human cytomegalovirus glycoprotein with homology to the Epstein-Barr virus BXLF2 product, varicella-zoster virus gpIII, and herpes simplex virus type 1 glycoprotein H. J Virol 62: 1416–1422
Davison AJ, Scott JE (1986) The complete DNA sequence of varicella-zoster virus. J Gen Virol 67: 1759–1816
Fleckenstein B, Müller I, Collins J (1982) Cloning of the complete human cytomegalovirus genome in cosmids. Gene 18: 39–46
Gibson W (1983) Protein counterparts of human and simian cytomegaloviruses. Virology 128: 391–406
Gottesman ME, Adhya S, Das A (1980) Transcription antitermination by bacteriophage lambda N gene product. J Mol Biol 140: 57–75
Gubler U, Hoffmann BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269
Hattori M, Sakaki Y (1986) Dideoxy sequencing method using denatured templates. Anal Biochem 152: 232–238
Hopp TP, Woods KR (1981) Prediction of antigenic determinants from amino acid sequences. Proc Natl Acad Sci USA 78: 3823–3828
Jahn G, Kouzarides T, Mach M, Scholl BC, Plachter B, Traupe B, Preddie E, Satchwell SC, Fleckenstein B, Barrell BG (1987) Map position and nucleotide sequence of the gene for the large structural phosphoprotein of human cytomegalovirus. J Virol 61: 1358–1367
Kemble GW, McCormick AL, Pereira L, Mocarski ES (1987) A cytomegalovirus protein with properties of herpes simplex virus ICP8: partial purification of the polypeptide and map position of the gene. J Virol 61: 3143–3151
Koenen M, Rüther U, Müller-Hill B (1982) Immunoenzymatic detection of expressed gene fragments cloned in the lac Z gene ofE. coli. EMBO J 4: 509–512
Kouzarides T, Bankier AT, Satchwell SC, Weston K, Tomlinson P, Barrell BG (1987) Large scale rearrangement of homologous regions in the genomes of HCMV and EBV. Virology 157: 397–413
Kouzarides T, Bankier AT, Satchwell SC, Weston K, Tomlinson P, Barrell BG (1987) Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene. J Virol 61: 125–133
Landini MP, Mirolo G, Baldassari B, La Placa M (1985) Human immune response to cytomegaloviral structural polypeptides studied by immuno blotting. J Med Virol 17: 303–311
Lindenmaier W, Dittmar KEJ, Hauser H, Necker A, Sebald W (1985) Isolation of a functional human interleukin 2 gene from a cosmid libary by recombination in vivo. Gene 31: 33–39
Loon AM van, Van der Logt JTM, Van der Vee J (1981) Enzyme linked immunosorbent assay for measurement of antibodies against cytomegalovirus and rubella virus at a single serum dilution. J Clin Pathol 4: 51–60
Mach M, Utz U, Fleckenstein B (1986) Mapping of the major glycoprotein of human cytomegalovirus. J Gen Virol 67: 1461–1467
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratory Cold Spring Harbor, NY
Martinez J, St Jeor SC (1986) Molecular cloning and analysis of three cDNA clones homologous to human cytomegalovirus RNAs present during late infection. J Virol 60: 531–538
Maxam A, Gilbert W (1980) Sequencing end labelled DNA with base specific chemical cleavages. In: Grossman L, Moldave K (eds) Methods of enzymology, vol 65. Academic Press, New York, pp 499–560
McGeoch DJ, Dalrymple MA, Davison AJ, Dolan A, Frame MC, McNab D, Perry LJ, Scott JE, Taylor P (1988) The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J Gen Virol 69: 1531–1574
McGeoch DJ (1989) The genomes of the human herpesviruses: contents, relationships, and evolution. Annu Rev Microbiol 43: 235–265
Meyer H, Bankier AT, Landini MP, Brown CM, Barrell BG, Rüger B, Mach M (1988) Identification and procaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp 28) of human cytomegalovirus. J Virol 62: 2243–2250
Mocarski ES, Pereira L, Michael N (1985) Precise localization of genes on large animal virus genome use of λgt II and monoclonal antibodies to map the gene for a cytomegalovirus protein family. Proc Natl Acad Sci USA 82: 1266–1270
Nagai K, Thøgersen HC (1984) Generation of globin by sequence specific proteolysis of a hybrid protein produced inEscherichia coli. Nature 309: 810–812
Nowak B, Gmeiner A, Sarnow P, Levine AJ, Fleckenstein B (1984) Physical mapping of human cytomegalovirus genes: identification of DNA sequences coding for a virion phosphoprotein of 71-kDa and a viral 65-kDa polypeptide. Virology 134: 91–102
Re MC, Landini MP, Coppolecchia P, Furlini G, La Placa M (1985) A 28.000 molecular weight human cytomegalovirus structural polypeptide studied by means of a specific monoclonal antibody. J Gen Virol 66: 2507–3511
Robson L, Gibson W (1989) Primate cytomegalovirus assembly protein: genome localization and nucleotide sequence. J Virol 63: 669–676
Rüger B, Klages S, Walla B, Albrecht J, Fleckenstein B, Tomlinson P, Barrell B (1987) Primary structure and transcription of the genes coding for the two virion phosphoproteins pp65 and pp71 of human cytomegalovirus. J Virol 61: 446–453
Scholl BC, von Hintzenstern J, Borisch B, Traupe B, Bröker M, Jahn G (1988) Prokaryotic expression of immunogenic polypeptides of the large phosphoprotein (pp150) of human cytomegalovirus. J Gen Virol 69: 1195–1204
Stanley KK, Luzio JP (1984) Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO J 3: 1429–1434
Stinski MF (1977) Synthesis of proteins and glycoproteins in cells infected with human cytomegalovirus. J Virol 23: 751–767
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354
Verweij CL, de Vries CJM, Distel B, van Zonneveld AJ, van Kessel AG, van Mourik JA, Pannekoek H (1985) Construction of a cDNA coding for human von Willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene. Nucleic Acids Res 13: 4699–4717
Weston K, Barrell BG (1986) Sequence of the short unique region, short repeats, and part of the long repeats of human cytomegalovirus. J Mol Biol 192: 177–208
Wingender E, Bercz G, Blöcker H, Frank R, Mayer H (1989) Expression of human parathyroid hormone in Escherichia coli. J Biol Chem 264: 4367–4373
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Lindenmaier, W., Necker, A., Krause, S. et al. Cloning and characterization of major antigenic determinants of human cytomegalovirus Ad169 seen by the human immune system. Archives of Virology 113, 1–16 (1990). https://doi.org/10.1007/BF01318349
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DOI: https://doi.org/10.1007/BF01318349