Cloning the complete guinea pig cytomegalovirus genome as an infectious bacterial artificial chromosome with excisable origin of replication

Abstract

Congenital human cytomegalovirus infections are the major infectious cause of birth defects in the United States. How this virus crosses the placenta and causes fetal disease is poorly understood. Guinea pig cytomegalovirus (GPCMV) is a related virus that provides an important model for studying cytomegaloviral congenital transmission and pathogenesis. In order to facilitate genetic analysis of GPCMV, the 232kb GPCMV genome was cloned as an infectious bacterial artificial chromosome (BAC). The BAC vector sequences were flanked by LoxP sites to allow efficient excision using Cre recombinase. All initial clones contained spontaneous deletions of viral sequences and reconstituted mutant viruses with impaired growth kinetics in vitro. The deletions in one BAC were repaired using Escherichia coli genetics. The resulting repaired BAC reconstituted a virus with in vitro replication kinetics identical to the wild type parental virus; moreover, its genome was indistinguishable from that of the wild type parental virus by restriction pattern analysis using multiple restriction enzymes. These results suggest that the repaired BAC is an authentic representation of the complete GPCMV genome. It should provide a valuable tool for evaluating the impact of genetic modifications on the safety and efficacy of live attenuated vaccines and for identifying genes important for congenital transmission and fetal disease.

Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model

“…Cell culture propagation of virus was carried out in guinea pig lung (GPL) fibroblast cells (ATCC CCL158) maintained in F-12 medium supplemented with 10% fetal calf serum (FCS; Fisher Scientific), 10,000 IU/liter penicillin, 10 mg/liter streptomycin (Gibco-BRL), and 0.075% NaHCO 3 (Gibco-BRL) or as described previously (33). Growth curves and viral titers were determined as described previously (39).…”

“…BAC construct N13R10 contains the complete GPCMV strain 22122 genome, while the BAC N2 contains a 17.9-kb deletion (39). BAC N2 with a targeted deletion of gp145 (N2-⌬145) and N13R10 with a targeted deletion of gp145 (N13R10-⌬145 or ⌬145) were constructed using a linear recombination approach to result in the substitution of a kanamycin resistance cassette (Kn) for nucleotides 224206 to 226104 (GenBank accession number KM384022 [25]) comprising the gp145 open reading frame (Fig.…”

“…The BAC clone N13R10 contains the complete GPCMV genome derived from a tissue culture-passaged stock of GPCMV strain 22122 (39). A gp145-null mutant was constructed by insertion of a Kn marker cassette concomitant with deletion of the entire gp145 ORF using bacteriophage lambda Red-mediated recombination.…”

Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection

“…BAC N13R10 contains the complete GPCMV strain 22122 genome (9), with a deletion that disrupts open reading frames (ORFs) GP129 and GP130. BAC N13R10-r129 was constructed using a two-step galactokinase-mediated recombineering approach (19).…”

“…Prior studies reported the successful cloning of the genome of a tissue culture-attenuated variant of GPCMV strain 22122 as a bacterial artificial chromosome (BAC) designated N13R10 (9). Although virus derived from N13R10 grows like wild-type GPCMV in cell culture, it was highly attenuated in vivo compared to pathogenic salivary gland (SG)-adapted virus.…”