Herpesvirus BACs: past, present, and future

Abstract

The herpesviridae are a large family of DNA viruses with large and complicated genomes. Genetic manipulation and the generation of recombinant viruses have been extremely difficult. However, herpesvirus bacterial artificial chromosomes (BACs) that were developed approximately 10 years ago have become useful and powerful genetic tools for generating recombinant viruses to study the biology and pathogenesis of herpesviruses. For example, BAC-directed deletion mutants are commonly used to determine the function and essentiality of viral genes. In this paper, we discuss the creation of herpesvirus BACs, functional analyses of herpesvirus mutants, and future applications for studies of herpesviruses. We describe commonly used methods to create and mutate herpesvirus BACs (such as site-directed mutagenesis and transposon mutagenesis). We also evaluate the potential future uses of viral BACs, including vaccine development and gene therapy.

Figure 1

Construction of HCMV_BAC. (a) HCMV genome and bacterial plasmid with BAC vector that has been linearized and transfected into human fibroblast cells. (b) BAC vector is inserted into HCMV genome by homologous recombination. The viral genome (now containing the BAC vector) will naturally circularize during replication. (c) HCMV viral BACs are selected based upon expression of a GFP cassette within the BAC vector. (d) Fluorescent plaques are isolated, and viral BAC DNA is extracted. (e) Viral BACs inserted into E. coli cells via electroporation. Successful integration of the BAC vector into the viral genome can be confirmed by (1) selecting colonies with antibiotic resistance resulting in the chloramphenicol-selectable marker in the BAC vector and (2) confirming the BAC genome sequence has not gained any undesired mutations by using restriction digest analysis to compare the BAC DNA to the original viral DNA. (f) If desired, mutagenize the HCMV viral BAC (via site-directed mutagenesis or random transposon mutagenesis). Either way, viral BAC DNA must be isolated via Maxiprep for transfection back into human cells. (g) Transfect BAC DNA into human fibroblast cells in order to produce infectious virus.

Figure 2

Construction of VZV_BAC. (a) Schematic diagram of a VZV pOka genome. The 125-kb genome, VZV, contains a unique long (UL) and a unique short (US) segment. (b) Four cosmids containing overlapping VZV genomic segments are shown. A BAC vector was inserted between ORF60-61 in a VZV cosmid, pvSpe23, by homologous recombination. The BAC vector carries a GFP and a CM^R marker. (c) VZV_BAC construction. (A) the BAC-containing cosmid was cotransfected with the three complementary cosmids into MeWo cells; (B) homologous recombination between these four cosmids forms a circular full-length VZV genome; (C) the recombinant virus replicated and produced a green plaque; (D) the circular DNA was isolated from infected cells and (E) transformed into E. coli and selected for CMR colonies; (F) the VZV_BAC DNA was isolated from E. coli and verified by restriction digestion and partial sequencing; (G) infectivity and integrity of the VZV_BAC were tested by transfecting BAC DNA into MeWo cells and producing VZV virus.

Figure 3

BAC mutagenesis techniques. (a) generation of a deletion mutant via homologous recombination. (1) Amplification of the kan^R expression cassette by PCR using a primer pair adding 40-bp (or longer) homologies flanking ORFX. (2) Viral BAC DNA is introduced into DY380 by electroporation. (3) Homologous recombination between upstream and downstream homologies of Gene X replaces Gene X with a selectable marker (the kan^R cassette), creating the Gene X-deletion viral BAC. (4) The recombinants are selected based upon their ability to grown on LB agar plates containing kanamycin. (5) The viral BAC DNA is isolated, and the deletion of Gene X is confirmed by PCR analysis. The integrity of the viral genome (after homologous recombination) is examined by restriction enzyme digestion. (6) Purified BAC DNA is transfected into a human cell line. (7) Viral proteins are expressed, and a functional virus is created. B. Generation of Rescue Virus. 1. Gene X is amplified by PCR from the wild-type BAC DNA. (2) Gene X is cloned into a bacterial plasmid. (3) Gene X and a selectable maker (zeocin) are amplified via PCR using a primer pair that adds at least 40 bp of nucleotide sequence that is homologous to viral genomic sequence flanking Gene X. (4) The PCR product was transformed via electroporation into DY380, now carrying the Gene X deletion mutant. (5) and (6) Gene X (with a zeocin marker) is inserted back into the BAC by homologous recombination (7) The Zeo^R vector sequence is removed (by cotransfecting a Cre recombinase-expressing plasmid with the prepared viral BAC DNA). Rescue virus DNA is ready to be purified and transfected into human cells. (c) GalK-based Mutagenesis (1). Insert galK sequence flanked by a sequence homologous to the viral BAC sequence flanking Gene X. (2) Gene X is replaced by the galK gene via homologous recombination. (3) Replace the galK gene with PCR product containing desired mutation in Gene X (referred to as Gene Y). (4) Transfect viral BAC into mammalian cells to produce infectious mutant virus. (d) Transposon Mediated Mutagenesis. (1) and (2) A temperature-sensitive plasmid donor containing transposon (Tn) is inserted into E. coli cells already containing viral BAC that was inserted via electroporation). Once the donor plasmid is inside the cell, the transposon will be inserted into the BAC genome. (3) An increase in temperature will remove the donor plasmid. The transposon mutant is now ready to be purified and transfected into human cells. PCR primers pre-engineered into the transposon insertion site can be used to sequence the insertion region of any transposon mutants with interesting phenotypes.