Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome

Abstract

Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. Recently, murine gammaherpesvirus 68 (MHV-68) infection of mice has been developed as a small animal model of gammaherpesvirus pathogenesis. Efficient generation of mutants of MHV-68 would significantly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model. To this end, we cloned the MHV-68 genome as a bacterial artificial chromosome (BAC) in Escherichia coli. During propagation in E. coli, spontaneous recombination events within the internal and terminal repeats of the cloned MHV-68 genome, affecting the copy number of the repeats, were occasionally observed. The gene for the green fluorescent protein was incorporated into the cloned BAC for identification of infected cells. BAC vector sequences were flanked by loxP sites to allow the excision of these sequences using recombinase Cre and to allow the generation of recombinant viruses with wild-type genome properties. Infectious virus was reconstituted from the BAC-cloned MHV-68. Growth of the BAC-derived virus in cell culture was indistinguishable from that of wild-type MHV-68. To assess the feasibility of mutagenesis of the cloned MHV-68 genome, a mutant virus with a deletion of open reading frame 4 was generated. Genetically modified MHV-68 can now be analyzed in functionally modified mouse strains to assess the role of gammaherpesvirus genes in virus-host interaction and pathogenesis.

FIG. 1

Strategy for cloning and mutagenesis of MHV-68. Viral DNA and the linearized recombination plasmid containing the BAC vector sequences were cotransfected into eukaryotic cells to generate a recombinant virus. Circular DNA of the recombinant virus genome was isolated from cells and electroporated into E. coli. Mutagenesis of the MHV-68 BAC plasmid was performed with E. coli JC8679. The mutated BAC plasmid was retransformed into E. coli DH10B. In E. coli DH10B, the tetracycline resistance gene can be deleted by Flp-mediated recombination. The mutated BAC plasmid was transfected into eukaryotic cells to reconstitute recombinant virus. Propagation of the mutant virus in fibroblasts expressing recombinase Cre results in deletion of the BAC vector sequences. Circled arrows indicate FRT sites. P, loxP site; TR, terminal repeats.

FIG. 2

Construction of the MHV-68 BAC genome and structural analysis of reconstituted virus genomes. (A) The BAC cloned genome was generated in eukaryotic cells by homologous recombination of the MHV-68 DNA with the recombination plasmid pHA2. The recombination plasmid contained 1.5 kbp of flanking homologous sequence (shaded box) as well as the BAC vector, the gpt gene, and the gfp gene, flanked by loxP sites. Electroporation of the circular BAC cloned genome RγHV68A98.01 into E. coli generated the MHV-68 BAC-plasmid pHA3. Integration of the BAC vector into the linear recombinant virus genome resulted in a new EcoRI fragment of 7.4 kbp which is indicated by an arrow. An additional EcoRI fragment of approximately 18 kbp in the BAC plasmid resulted from the fusion of the terminal EcoRI fragments (containing the terminal repeats of the virus genome). P, probe. (B) Structural analysis of BAC plasmids and of reconstituted virus genomes by ethidium bromide-stained agarose gel analysis of EcoRI-digested DNA. The lanes show MHV-68 WT DNA isolated from infected cells (lane 1), MHV-68 BAC plasmid pHA3 DNA isolated from E. coli (lane 2), reconstituted MHV-68 BAC virus RγHV68A98.01 DNA isolated from infected cells (lane 3), and reconstituted MHV-68 BAC virus RγHV68A98.02 DNA (with the BAC vector excised by recombinase Cre) isolated from infected cells (lane 4). The upper arrowhead indicates an additional 18-kbp band present only in lane 2, and the lower arrowhead indicates a 7.4-kbp fragment resulting in a double band in lanes 2 and 3. (C) Southern blot analysis of the gel shown in panel B using a DIG-labeled probe (indicated in panel A). Lanes 3 and 4 were from a longer exposure than lanes 1 and 2. The arrowhead indicates the additional 18-kbp band present only in lane 2. Marker (M) sizes (in kilobase pairs) are indicated on the left.

FIG. 3

Stability of the MHV-68 BAC plasmid pHA3 in E. coli. The BAC plasmid pHA3 was propagated three times in E. coli DH10B. Afterwards, bacteria were plated on agar plates containing chloramphenicol and plasmid DNA isolated from single colonies was analyzed by EcoRI digestion and gel electrophoresis. The analysis of five clones (lanes 1 to 5) is shown on an ethidium bromide-stained agarose gel. The bands representing the EcoRI K fragment which contains the 40-bp internal repeat of MHV-68 are marked by dots. Marker sizes (in kilobase pairs) are indicated on the left.

FIG. 4

Comparison of the in vitro growth properties of several recombinant MHV-68 mutants and WT MHV-68. BHK-21 cells were infected at an MOI of 0.1. Cells and supernatants were harvested at the indicated time points, and viral titers were determined by plaque assay. Titers at 0 h represent input inocula. (A) Growth properties of RγHV68A98.01 compared to WT MHV-68; (B) Expression of gfp in NIH3T3 cells infected with RγHV68A98.01; (C) Growth properties of RγHV68A98.02 compared to RγHV68A98.01; (D) Lack of gfp expression in NIH3T3 cells infected with RγHV68A98.02; (E) Growth properties of RγHV68A98.03 and RγHV68A98.04 compared to RγHV68A98.01; (F) Southern blot analysis of EcoRI-digested DNA isolated from cells infected with WT MHV-68 (lane 1), RγHV68A98.01 (lane 2), RγHV68A98.03 (lane 3), and RγHV68A98.04 (lane 4) with a probe specific for the EcoRI K fragment after digestion of the DNA with EcoRI.

FIG. 5

Construction of the ΔORF4 mutant, structural analysis of the mutated BAC plasmids, and the genomes of reconstituted mutant viruses. (A) Recombination fragment containing the tetracycline resistance gene flanked by FRT sites (circled arrows) and homology regions was generated for mutagenesis in E. coli. Recombination resulted in the deletion of ORF 4 by replacement with the tetracycline resistance gene. Using recombinase Flp, the tetracycline resistance gene was afterwards excised and left one FRT site. (B) Ethidium bromide-stained agarose gel of EcoRI-digested plasmid DNA. MHV-68 BAC plasmid (pHA3) DNA (lane 1), ΔORF4-mutant plasmid (pHA6) DNA containing the tetracycline resistance gene (lane 2), ΔORF4 revertant plasmid (pHA12) DNA (lane 3), ΔORF4 mutant plasmid with the tetracycline resistance gene excised (pHA9) DNA (lane 4). (C) Ethidium bromide-stained agarose gel of EcoRI-digested DNA of reconstituted viruses. BAC MHV-68 (RγHV68A98.01) (lane 5), ORF 4 deletion mutant (RγHV68A98.05) (lane 6), and ORF 4 revertant virus (RγHV68A99.03) (lane 7). (D) Southern blot analysis of the gel shown in panel C with probe P, which is indicated in panel A. As illustrated in panel A, this probe recognizes the 12.7-kbp EcoRI fragment of RγHV68A98.01 and RγHV68A99.03 and the 8.4-kbp EcoRI fragment of RγHV68A98.05. The arrowheads indicate the following bands which are in addition marked by dots: 18-kbp band only present in lane 1; 12.7-kbp band only present in lanes 1 and 3; 8.4-kbp band only present in lanes 2, 4, and 5; 4.8-kbp band present only in lanes 2 and 4; 4.5-kbp band present only in lanes 2, 4 and 5; and 3.3-kbp band present only in lane 5. Marker (M) sizes (in kilobase pairs) are indicated on the left.

FIG. 6

In vitro growth properties of BAC MHV-68 RγHV68A98.01, of ORF 4 deletion mutant RγHV68A98.05, and of revertant virus RγHV68A99.03. NIH3T3 cells were infected at an MOI of 0.1 for 1 h at 4°C to allow adsorption. For penetration, prewarmed medium was added for a 2-h period of incubation at 37°C. Remaining extracellular virus was inactivated by treatment with low-pH citrate buffer for 1 min. Cells and supernatants were harvested at the indicated time points, and viral titers were determined by plaque assay.