Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Oct;73(10):8320-9.
doi: 10.1128/JVI.73.10.8320-8329.1999.

Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants

E M Borst  1 , G Hahn, U H Koszinowski, M Messerle
Affiliations

Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants

E M Borst et al. J Virol. 1999 Oct.

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. Crnkovic, 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.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Strategy for generation of the HCMV BAC plasmid. (A) The structure of the HCMV AD169 genome with the UL and US components is shown at the top. The terminal and internal repeat sequences are indicated as open boxes. (B) The BAC vector and the selection marker gpt were integrated between the US1 and US7 genes in the US region of the viral genome by homologous recombination in fibroblasts with the recombination plasmid pEB1097. (C) Genomic structure of the resulting reconstituted virus RVHB5 and the corresponding BAC plasmid pHB5. The sizes of expected HindIII (H) and EcoRI fragments (E) in the parental virus genome and in the RVHB5 genome and BAC plasmid pHB5 are indicated. b′, a′, and c′ mark components of the internal repeat sequences. The illustration is not drawn to scale.
FIG. 2
FIG. 2
Characterization of HCMV BAC plasmids. (A) BAC plasmids were isolated from six independent E. coli clones, digested with HindIII, and separated by agarose gel electrophoresis. The sizes of some DNA fragments and positions of DNA size markers are indicated. (B) Predicted structures of the two different conformations of the HCMV BAC plasmid. The positions of some HindIII sites (H) and the sizes of the unique HindIII fragments are shown.
FIG. 3
FIG. 3
Structural analysis of the HCMV BAC plasmid pHB5 and of the genome of reconstituted virus RVHB5. (A) DNA of HCMV AD169 (lane 1), BAC plasmid pHB5 (lane 2), and reconstituted virus RVHB5 (lane 3) was digested with EcoRI and separated on a 0.8% agarose gel. Note that the 2.0-kbp fragment resulting from the integration of the BAC vector is present only in the BAC plasmid pHB5 and in the RVHB5 genome (compare to Fig. 1). (B and C) Southern blot analysis was performed with a US1 probe (lanes 4, 5, and 6) and a BAC-specific probe (lanes 7, 8 and 9). The sizes of relevant EcoRI fragments are indicated.
FIG. 4
FIG. 4
Multiple-step growth curve analysis of the reconstituted virus RVHB5 and of the parental HCMV strain AD169. HFF cells seeded in six-well dishes (5 × 105 cells/well) were infected with an MOI of 0.1. At the indicated time points (days postinfection) supernatants from the infected cultures were harvested, and total PFU of infectious virus in the culture supernatants were determined by plaque assay on HFF cells. Each data point represents the average of three independent wells. Day 0 titers represent input inocula.
FIG. 5
FIG. 5
Construction scheme of the gpUL37 mutant (A) and structural analysis of the mutated BAC plasmid and mutant genome (B). (A) The top line depicts the genomic structure of the HCMV BAC plasmid pHB5, with the region encoding the gpUL37 RNA expanded below. Following recombination in E. coli between BAC plasmid pHB5 and recombination plasmid pSH37b, a 382-bp SnaBI fragment in exon 3 of the gpUL37 gene was replaced by a tetracycline resistance marker. The insertion disrupts the gpUL37 ORF after 199 codons and creates a new stop codon after an additional four codons. The sizes of the EcoRI fragments in the parental BAC plasmid pHB5 and mutant BAC plasmid pHBUL37 are indicated. The probe used for Southern blot analysis is depicted as a black bar. (B) DNA of BAC plasmids pHB5 (lane 1) and pHBUL37 (lane 2) and of the reconstituted viruses RVHB5 and RVUL37 (lanes 5 and 6) was digested with EcoRI and separated on a 0.5% agarose gel. Bands were visualized with ethidium bromide, transferred to nylon filters, and hybridized to the radiolabeled probe (lanes 3, 4, 7, and 8). The sizes of the EcoRI Q fragment (6.4 kbp) in the parental genome and of the new EcoRI fragments (3.7 and 4.9 kbp) in the mutant genome are indicated at the left margin.
FIG. 6
FIG. 6
Transcript analysis of the RVUL37 mutant. (A) Genomic structure of the RVHB5 and RVUL37 viruses in the UL37 region. Exons are depicted in gray. In the genome of RVUL37 exon 3 of the UL37 gene is disrupted by the insertion of the tetracycline resistance gene (Tet). The probe used (black bar) is specific for the 1.9-kb UL37 exon 1 transcript and the 3.4-kb gpUL37 transcript (28, 50). (B) Northern blot of whole cell RNA isolated from mock infected HFF cells (lane 1) and from cells 8 h after infection with 3 PFU/cell of the parental virus RVHB5 (lane 2) or the mutant virus RVUL37 (lane 3) in the presence of cycloheximide. Molecular sizes (in kilobases) of the detected transcripts are shown at the right margin. The blot was rehybridized with a probe specific for β-actin as an internal RNA control. The actin band detectable in all lanes is shown on the bottom panel.
FIG. 7
FIG. 7
Multiple-step growth curves of the mutant virus RVUL37 and of the parental virus RVHB5. HFF cells (5 × 105) were infected at an MOI of 0.1. Virus titers in the supernatants of infected cells were determined as described in the legend for Fig. 4.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Al-Barazi H O, Colberg-Poley A M. The human cytomegalovirus UL37 immediate-early regulatory protein is an integral membrane N-glycoprotein which traffics through the endoplasmic reticulum and Golgi apparatus. J Virol. 1996;70:7198–7208. - PMC - PubMed
    1. Angulo A, Messerle M, Koszinowski U H, Ghazal P. Enhancer requirement for murine cytomegalovirus growth and genetic complementation by the human cytomegalovirus enhancer. J Virol. 1998;72:8502–8509. - PMC - PubMed
    1. Baldick C J, Jr, Marchini A, Patterson C E, Shenk T. Human cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle. J Virol. 1997;71:4400–4408. - PMC - PubMed
    1. Borst, E., G. Pósfai, M. Wagner, and M. Messerle. Unpublished data.
    1. Britt W J, Alford C A. Cytomegalovirus. In: Fields B N, Knipe D M, Howley P M, editors. Fields virology. 3rd ed. Philadelphia, Pa: Lippincott-Raven Publishers; 1996. pp. 2493–2523.

Publication types

LinkOut - more resources