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.

Protocol of a Seamless Recombination with Specific Selection Cassette in PCR-Based Site-Directed Mutagenesis

“…Since the construction of the first herpesvirus BAC 12 years ago, BACs have been generated for all major human and animal herpesviruses, and this technology has greatly facilitated genetic and functional studies of herpesviruses, because recombinant viruses, especially herpesviruses, were previously difficult to produce due to their large size. Soon, we may have BACs for not only all herpesviruses [27], but all DNA viruses, as well as novel global mutational studies for several virus BACs thanks to accurate and seamless mutagenesis procedures such as the galK recombineering protocol. Global and local studies of virus pathogenesis should help identify new antiviral targets and produce more effective and safe vaccines.…”

“…The BAC clone of a virus can be maintained stably and propagated inside a bacterial cell, which allows for easy manipulation of the viral genome. Any required mutation can be easily and rapidly achieved inside the E. coli cell and this mutation can be verified before making recombinant virus [23][24][25][26][27]. Since the development of this novel method for construction of recombinant viruses, detailed functional study of virus genome has been done using this specific BAC recombinatory mutagenesis approach [23][24][25][26][27].…”

“…The viral BAC and the marker with homologous sequences are then inserted into E. coli via electroporation. Colonies with the BAC recombinant virus can be selected based upon selection markers and verified by PCR [27].…”

Gene Functional Studies Using Bacterial Artificial Chromosome (BACs)

“…Many short reads are assembled to produce the sequence of the BACs, where these large overlapping sequences of the BACs are assembled to produce the who genome sequence (Imelfort, Batley et al 2009). BACs have also been used in mammalian genome mapping (Schalkwyk, Francis et al 1995), genomic imprinting (Tunster, Van De Pette et al 2011), vaccine development and gene therapy (Magin-Lachmann, Kotzamanis et al 2004;Warden, Tang et al 2011). Studies of the evolutionary history and functional dynamics of sex chromosomes have recently been possible using BAC libraries (Janes, Valenzuela et al 2011).…”

“…Therefore the utilization of human herpesvirus BACs to study viral gene function (Warden, Tang et al 2011) has become more and more common. The herpesviruses are a family of DNA viruses which contain large and complex genomes.…”