DOI: 10.1128/jvi.73.10.8320-8329.1999. Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in <i>Escherichia coli</i> : a New Approach for Construction of HCMV Mutants

Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli : a New Approach for Construction of HCMV Mutants

10.1128/jvi.73.10.8320-8329.1999

Crossref journal-article

American Society for Microbiology

Journal of Virology (235)

Abstract

ABSTRACT We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in Escherichia coli (M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation in E. coli , during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in E. coli . The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.

Bibliography

Borst, E.-M., Hahn, G., Koszinowski, U. H., & Messerle, M. (1999). Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli â¯: a New Approach for Construction of HCMV Mutants. Journal of Virology, 73(10), 8320â8329.

Authors 4

Eva-Maria Borst (first)
Gabriele Hahn (additional)
Ulrich H. Koszinowski (additional)
Martin Messerle (additional)

References 52 Referenced 331

10.1128/jvi.70.10.7198-7208.1996
10.1128/JVI.72.11.8502-8509.1998
10.1128/jvi.71.6.4400-4408.1997
Borst E. G. Pósfai M. Wagner and M. Messerle. Unpublished data.
Britt W. J. Alford C. A. Cytomegalovirus Fields virology 3rd ed. Fields B. N. Knipe D. M. Howley P. M. 1996 2493 2523 Lippincott-Raven Publishers Philadelphia Pa
10.1038/7914
10.1128/jvi.70.1.78-83.1996
Chee M. S. Bankier A. T. Beck S. Bohni R. Brown C. M. Cerny R. Horsnell T. Hutchinson C. A. Kouzarides T. Martignetti J. A. Preddie E. Satchwell S. C. Tomlinson P. Weston K. M. Barrell B. G. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD 169.Curr. Top. Microbiol. Immunol. 154 1990 125 169 / Curr. Top. Microbiol. Immunol. / Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD 169 by Chee M. S. (1990)
10.1016/0378-1119(95)00193-A
10.1159/000150506
10.1006/viro.1998.9212
10.1128/jvi.67.6.3644-3648.1993
10.1128/jvi.71.12.9833-9836.1997
10.1073/pnas.95.14.8245
10.1016/0378-1119(82)90054-3
10.1128/jb.153.3.1424-1431.1983
10.1128/JVI.72.1.366-379.1998
10.1099/0022-1317-76-9-2151
Hahn G. M. A. McVoy and E. S. Mocarski. Unpublished data.
10.1016/0022-2836(67)90307-5
10.1128/jvi.70.1.383-392.1996
10.1128/jvi.59.2.494-499.1986
10.1128/jvi.66.4.2541-2546.1992
10.1128/jvi.65.11.5860-5872.1991
10.1128/jvi.70.3.2044-2048.1996
10.1128/jvi.69.1.231-238.1995
10.1128/jvi.65.10.5184-5189.1991
10.1016/0042-6822(88)90668-X
10.1128/jvi.66.7.4434-4444.1992
Maniatis T. Fritsch E. F. Sambrook J. Molecular cloning: a laboratory manual 2nd ed. 1989 Cold Spring Harbor Laboratory Cold Spring Harbor N.Y
10.1006/viro.1999.9724
10.1073/pnas.94.26.14759
10.1073/pnas.93.21.11321
Mocarski E. S. Cytomegaloviruses and their replication Fields virology 3rd ed. Fields B. N. Knipe D. M. Howley P. M. 1996 2447 2492 Lippincott-Raven Publishers Philadelphia Pa
10.1016/0092-8674(80)90172-5
10.1006/viro.1997.8875
10.1146/annurev.ge.28.120194.000405
10.1126/science.2660262
10.1128/jvi.67.12.6979-6988.1993
10.1128/jvi.67.5.2575-2582.1993
10.1073/pnas.80.9.2690
10.1128/jb.179.13.4426-4428.1997
10.1016/0092-8674(81)90247-6
10.1128/JVI.73.7.5663-5670.1999
Roizman B. Herpesviridae Fields virology 3rd ed. Fields B. N. Knipe D. M. Howley P. M. 1996 2221 2230 Lippincott-Raven Publishers Philadelphia Pa
10.1089/hum.1998.9.18-2787
10.1073/pnas.89.18.8794
10.1073/pnas.84.20.7213
10.1128/JVI.72.9.7137-7143.1998
10.1016/0042-6822(91)90663-V
10.1128/jvi.65.12.6724-6734.1991
10.1128/JVI.73.8.7056-7060.1999

Dates

Type	When
Created	5 years, 8 months ago (Dec. 31, 2019, 1:19 p.m.)
Deposited	3 years, 6 months ago (March 4, 2022, 10:18 p.m.)
Indexed	1 month ago (Aug. 2, 2025, 1:09 a.m.)
Issued	25 years, 11 months ago (Oct. 1, 1999)
Published	25 years, 11 months ago (Oct. 1, 1999)
Published Print	25 years, 11 months ago (Oct. 1, 1999)

Funders 0

None

BibTeX

@article{Borst_1999, title={Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli  : a New Approach for Construction of HCMV Mutants}, volume={73}, ISSN={1098-5514}, url={http://dx.doi.org/10.1128/jvi.73.10.8320-8329.1999}, DOI={10.1128/jvi.73.10.8320-8329.1999}, number={10}, journal={Journal of Virology}, publisher={American Society for Microbiology}, author={Borst, Eva-Maria and Hahn, Gabriele and Koszinowski, Ulrich H. and Messerle, Martin}, year={1999}, month=oct, pages={8320–8329} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        8,
        2
      ]
    ],
    "date-time": "2025-08-02T05:09:36Z",
    "timestamp": 1754111376536
  },
  "reference-count": 52,
  "publisher": "American Society for Microbiology",
  "issue": "10",
  "license": [
    {
      "start": {
        "date-parts": [
          [
            1999,
            10,
            1
          ]
        ],
        "date-time": "1999-10-01T00:00:00Z",
        "timestamp": 938736000000
      },
      "content-version": "tdm",
      "delay-in-days": 0,
      "URL": "https://journals.asm.org/non-commercial-tdm-license"
    }
  ],
  "content-domain": {
    "domain": [
      "journals.asm.org"
    ],
    "crossmark-restriction": true
  },
  "published-print": {
    "date-parts": [
      [
        1999,
        10
      ]
    ]
  },
  "abstract": "<jats:title>ABSTRACT</jats:title>\n          <jats:p>\n            We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in\n            <jats:italic>Escherichia coli</jats:italic>\n            (M. Messerle, I. Crnkovic\u0301, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759\u201314763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation in\n            <jats:italic>E. coli</jats:italic>\n            , during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in\n            <jats:italic>E. coli</jats:italic>\n            . The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.\n          </jats:p>",
  "DOI": "10.1128/jvi.73.10.8320-8329.1999",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2019,
        12,
        31
      ]
    ],
    "date-time": "2019-12-31T18:19:54Z",
    "timestamp": 1577816394000
  },
  "page": "8320-8329",
  "update-policy": "http://dx.doi.org/10.1128/asmj-crossmark-policy-page",
  "source": "Crossref",
  "is-referenced-by-count": 331,
  "title": "Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in\n            <i>Escherichia coli</i>\n            : a New Approach for Construction of HCMV Mutants",
  "prefix": "10.1128",
  "volume": "73",
  "author": [
    {
      "given": "Eva-Maria",
      "family": "Borst",
      "sequence": "first",
      "affiliation": [
        {
          "name": "<!--label omitted: 1-->Max von Pettenkofer-Institut fu\u0308r Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universita\u0308t Mu\u0308nchen, D-81377 Munich, Germany"
        }
      ]
    },
    {
      "given": "Gabriele",
      "family": "Hahn",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "<!--label omitted: 1-->Max von Pettenkofer-Institut fu\u0308r Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universita\u0308t Mu\u0308nchen, D-81377 Munich, Germany"
        }
      ]
    },
    {
      "given": "Ulrich H.",
      "family": "Koszinowski",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "<!--label omitted: 1-->Max von Pettenkofer-Institut fu\u0308r Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universita\u0308t Mu\u0308nchen, D-81377 Munich, Germany"
        }
      ]
    },
    {
      "given": "Martin",
      "family": "Messerle",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "<!--label omitted: 1-->Max von Pettenkofer-Institut fu\u0308r Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universita\u0308t Mu\u0308nchen, D-81377 Munich, Germany"
        }
      ]
    }
  ],
  "member": "235",
  "reference": [
    {
      "key": "e_1_3_2_2_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.70.10.7198-7208.1996"
    },
    {
      "key": "e_1_3_2_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/JVI.72.11.8502-8509.1998"
    },
    {
      "key": "e_1_3_2_4_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.71.6.4400-4408.1997"
    },
    {
      "key": "e_1_3_2_5_2",
      "unstructured": "Borst E. G. P\u00f3sfai M. Wagner and M. Messerle. Unpublished data."
    },
    {
      "key": "e_1_3_2_6_2",
      "unstructured": "Britt\nW. J.\nAlford\nC. A.\nCytomegalovirus\nFields virology\n3rd ed.\nFields\nB. N.\nKnipe\nD. M.\nHowley\nP. M.\n1996\n2493\n2523\nLippincott-Raven Publishers\nPhiladelphia Pa"
    },
    {
      "key": "e_1_3_2_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/7914"
    },
    {
      "key": "e_1_3_2_8_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.70.1.78-83.1996"
    },
    {
      "key": "e_1_3_2_9_2",
      "first-page": "125",
      "article-title": "Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD 169",
      "volume": "154",
      "author": "Chee M. S.",
      "year": "1990",
      "unstructured": "Chee M. S. Bankier A. T. Beck S. Bohni R. Brown C. M. Cerny R. Horsnell T. Hutchinson C. A. Kouzarides T. Martignetti J. A. Preddie E. Satchwell S. C. Tomlinson P. Weston K. M. Barrell B. G. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD 169.Curr. Top. Microbiol. Immunol. 154 1990 125 169",
      "journal-title": "Curr. Top. Microbiol. Immunol."
    },
    {
      "key": "e_1_3_2_10_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0378-1119(95)00193-A"
    },
    {
      "key": "e_1_3_2_11_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1159/000150506"
    },
    {
      "key": "e_1_3_2_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/viro.1998.9212"
    },
    {
      "key": "e_1_3_2_13_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.67.6.3644-3648.1993"
    },
    {
      "key": "e_1_3_2_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.71.12.9833-9836.1997"
    },
    {
      "key": "e_1_3_2_15_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.95.14.8245"
    },
    {
      "key": "e_1_3_2_16_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0378-1119(82)90054-3"
    },
    {
      "key": "e_1_3_2_17_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.153.3.1424-1431.1983"
    },
    {
      "key": "e_1_3_2_18_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/JVI.72.1.366-379.1998"
    },
    {
      "key": "e_1_3_2_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1099/0022-1317-76-9-2151"
    },
    {
      "key": "e_1_3_2_20_2",
      "unstructured": "Hahn G. M. A. McVoy and E. S. Mocarski. Unpublished data."
    },
    {
      "key": "e_1_3_2_21_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0022-2836(67)90307-5"
    },
    {
      "key": "e_1_3_2_22_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.70.1.383-392.1996"
    },
    {
      "key": "e_1_3_2_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.59.2.494-499.1986"
    },
    {
      "key": "e_1_3_2_24_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.66.4.2541-2546.1992"
    },
    {
      "key": "e_1_3_2_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.65.11.5860-5872.1991"
    },
    {
      "key": "e_1_3_2_26_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.70.3.2044-2048.1996"
    },
    {
      "key": "e_1_3_2_27_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.69.1.231-238.1995"
    },
    {
      "key": "e_1_3_2_28_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.65.10.5184-5189.1991"
    },
    {
      "key": "e_1_3_2_29_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0042-6822(88)90668-X"
    },
    {
      "key": "e_1_3_2_30_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.66.7.4434-4444.1992"
    },
    {
      "key": "e_1_3_2_31_2",
      "unstructured": "Maniatis\nT.\nFritsch\nE. F.\nSambrook\nJ.\nMolecular cloning: a laboratory manual\n2nd ed.\n1989\nCold Spring Harbor Laboratory\nCold Spring Harbor N.Y"
    },
    {
      "key": "e_1_3_2_32_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/viro.1999.9724"
    },
    {
      "key": "e_1_3_2_33_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.94.26.14759"
    },
    {
      "key": "e_1_3_2_34_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.93.21.11321"
    },
    {
      "key": "e_1_3_2_35_2",
      "unstructured": "Mocarski\nE. S.\nCytomegaloviruses and their replication\nFields virology\n3rd ed.\nFields\nB. N.\nKnipe\nD. M.\nHowley\nP. M.\n1996\n2447\n2492\nLippincott-Raven Publishers\nPhiladelphia Pa"
    },
    {
      "key": "e_1_3_2_36_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(80)90172-5"
    },
    {
      "key": "e_1_3_2_37_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/viro.1997.8875"
    },
    {
      "key": "e_1_3_2_38_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.ge.28.120194.000405"
    },
    {
      "key": "e_1_3_2_39_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.2660262"
    },
    {
      "key": "e_1_3_2_40_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.67.12.6979-6988.1993"
    },
    {
      "key": "e_1_3_2_41_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.67.5.2575-2582.1993"
    },
    {
      "key": "e_1_3_2_42_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.80.9.2690"
    },
    {
      "key": "e_1_3_2_43_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.179.13.4426-4428.1997"
    },
    {
      "key": "e_1_3_2_44_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(81)90247-6"
    },
    {
      "key": "e_1_3_2_45_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/JVI.73.7.5663-5670.1999"
    },
    {
      "key": "e_1_3_2_46_2",
      "unstructured": "Roizman\nB.\nHerpesviridae\nFields virology\n3rd ed.\nFields\nB. N.\nKnipe\nD. M.\nHowley\nP. M.\n1996\n2221\n2230\nLippincott-Raven Publishers\nPhiladelphia Pa"
    },
    {
      "key": "e_1_3_2_47_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1089/hum.1998.9.18-2787"
    },
    {
      "key": "e_1_3_2_48_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.89.18.8794"
    },
    {
      "key": "e_1_3_2_49_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.84.20.7213"
    },
    {
      "key": "e_1_3_2_50_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/JVI.72.9.7137-7143.1998"
    },
    {
      "key": "e_1_3_2_51_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0042-6822(91)90663-V"
    },
    {
      "key": "e_1_3_2_52_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jvi.65.12.6724-6734.1991"
    },
    {
      "key": "e_1_3_2_53_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/JVI.73.8.7056-7060.1999"
    }
  ],
  "container-title": "Journal of Virology",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://journals.asm.org/doi/pdf/10.1128/JVI.73.10.8320-8329.1999",
      "content-type": "application/pdf",
      "content-version": "vor",
      "intended-application": "text-mining"
    },
    {
      "URL": "https://journals.asm.org/doi/pdf/10.1128/JVI.73.10.8320-8329.1999",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2022,
        3,
        5
      ]
    ],
    "date-time": "2022-03-05T03:18:48Z",
    "timestamp": 1646450328000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://journals.asm.org/doi/10.1128/JVI.73.10.8320-8329.1999"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        1999,
        10
      ]
    ]
  },
  "references-count": 52,
  "journal-issue": {
    "issue": "10",
    "published-print": {
      "date-parts": [
        [
          1999,
          10
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1128/JVI.73.10.8320-8329.1999"
  ],
  "URL": "http://dx.doi.org/10.1128/JVI.73.10.8320-8329.1999",
  "relation": {},
  "ISSN": [
    "0022-538X",
    "1098-5514"
  ],
  "subject": [],
  "container-title-short": "J Virol",
  "published": {
    "date-parts": [
      [
        1999,
        10
      ]
    ]
  },
  "assertion": [
    {
      "value": "1999-04-05",
      "order": 0,
      "name": "received",
      "label": "Received",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    },
    {
      "value": "1999-07-19",
      "order": 1,
      "name": "accepted",
      "label": "Accepted",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    },
    {
      "value": "1999-10-01",
      "order": 2,
      "name": "published",
      "label": "Published",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    }
  ]
}