Analysis of Herpes Simplex Virus Type 1 DNA Packaging Signal Mutations in the Context of the Viral Genome

The minimal signal required for the cleavage and packaging of replicated concatemeric herpes simplex virus type 1 (HSV-1) DNA corresponds to an approximately 200-bp fragment, Uc-DR1-Ub, spanning the junction of the genomic L and S segments. Uc and Ub occupy positions adjacent to the L and S termini and contain motifs (pac2 and pac1, respectively) that are conserved near the ends of other herpesvirus genomes. We have used homologous Red/ET recombination in Escherichia coli to introduce wild-type and specifically mutated Uc-DR1-Ub fragments into an ectopic site of a cloned HSV-1 genome from which the resident packaging signals had been previously deleted. The resulting constructs were transfected into mammalian cells, and their abilities to replicate and become encapsidated, generate Uc- and Ub-containing terminal fragments, and give rise to progeny virus were assessed. In general, the results obtained agree well with previous observations made using amplicons and confirm roles for the pac2 T element in the initiation of DNA packaging and for the GC-rich motifs flanking the pac1 T element in termination. In contrast to a previous report, the sequence of the DR1 element was also crucial for DNA packaging. Following repair of the resident packaging signals in mammalian cells, recombination occurred at high frequency in progeny virus between the repaired sequences and mutated Uc-DR1-Ub inserts. This restored the ability of mutated Uc-DR1-Ub inserts to generate terminal fragments, although these were frequently larger than expected from simple repair of the original lesion.