Cloning human herpes virus 6A genome into bacterial artificial chromosomes and study of DNA replication intermediates

The DR1 and DR6 First Exons of Human Herpesvirus 6A Are Not Required for Virus Replication in Culture and Are Deleted in Virus Stocks That Replicate Well in T-Cell Lines

“…Moreover, the U1102-CB3S 1992 stock, which was passaged recently 8 times in CBMCs followed by 24 passages in SupT1 cells, also showed the ϳ9.5-kb band. In contrast, the digestion of the laboratory strain U1102-N-CB3S with SfiI gave an ϳ4.1-kb fragment, indicating shortening of the DR L by ϳ5.4 kb, as was found in the HHV-6A BAC clones (3).…”

“…We have recently cloned the intact HHV-6A genome into bacterial artificial chromosomes (BACs), by direct cloning of unit-length DNA produced from circular or head-to-tail replication intermediates into modified BAC vectors containing the green fluorescent protein (GFP) marker and ampicillinpuromycin (Amp-Puro) selection cassette (3). Surprisingly, the HHV-6A BAC clones as well as the parental HHV-6A (U1102) propagated in our laboratory in SupT1 cells were found to contain DRs of ϳ2.7 kb instead of the expected ϳ8-to 10-kb DRs, as in the early publications (19,24,27,46) and in the PubMed sequence.…”

A Herpes Simplex Virus 2 Glycoprotein D Mutant Generated by Bacterial Artificial Chromosome Mutagenesis Is Severely Impaired for Infecting Neuronal Cells and Infects Only Vero Cells Expressing Exogenous HVEM

“…Once cloned into a BAC vector, the herpesvirus genome can be mutated in E. coli, and the desired recombinant clone can be selected using prokaryotic genetic screening methods. This approach has been used in several human herpesviruses, including HSV-1 (17,27,48), HSV-2 (34, 41), varicella-zoster virus (VZV) (39,53,63,64), Epstein-Barr virus (EBV) (10,42), cytomegalovirus (CMV) (3,7), human herpesvirus 6 (HHV-6) (2,49), and Kaposi's sarcoma-associated herpesvirus (KSHV) (65), and greatly simplifies herpesvirus mutagenesis. The HSV-2 BAC clones that have been reported are not optimal for pathogenesis studies in animals because the virus backbones in these BAC clones have one or more genes deleted (34,41).…”

Recent advances in cloning herpesviral genomes as infectious bacterial artificial chromosomes

“…This can be achieved through recombination between the viral genome and the BAC vector flanked by viral sequences surrounding the integration site, [26][27][28][29][30][32][33][34][35][36][37][38][39][40][42][43][44][45]47 recombination of overlapping cosmid inserts, 41,56 direct ligation of viral genomic fragments with the BAC vector, 48 and direct in vitro transposition 46,49 ( Fig. 1).…”

“…Human herpesvirus 6A (HHV6A) was cloned as a BAC using this strategy though the infectiousness of the BAC was not confirmed. 48 The reliance of a unique enzyme site in the viral genome determines that this approach requires prior sequence knowledge of the entire viral genome. Because herpesviruses have large complex genomes with rich repeat sequences, this information might not be always available or accurate.…”