Functions of the sequences at the ends of the inverted repeats of pseudorabies virus

Two mutants were constructed to explore the functions of the sequence at the end of the S terminus of pseudorabies virus (PrV). In mutant vYa, 17 bp from the internal inverted repeat, as well as adjacent sequences from the L component, were deleted. In mutant v135/9, 143 bp from the internal inverted repeat (including sequences with homology to the pac-1 site of herpes simplex virus), as well as adjacent sequences from the L component, were deleted. Our aim in constructing these mutants was to ascertain whether equalization of the terminal regions of the S component would occur, whether genome termini that lack either the terminal 17 or 143 bp would be generated as a result of equalization of the repeats (thereby identifying the terminal nucleotides that may include cleavage signals), and whether inversion of the S component would occur (thereby ascertaining the importance of the deleted sequences in this process). The results obtained show the following. (i) The removal of the terminal 17 or 143 bp of the internal S component, including the sequences with homology to the pac-1 site, does not affect the inversion of the U(S). (ii) The equalization of both the vYa and the v135/9 inverted repeats occurs at high frequency, the terminal repeats being converted and becoming similar to the mutated internal inverted repeat. (iii) Mutants in which the 17 terminal base pairs (vYa) have been replaced by unrelated sequences are viable. However, the 143 terminal base pairs appear to be essential to virus survival; concatemeric v135/9 DNA with equalized, mutant-type, inverted repeats accumulates, but mature virions with such equalized repeats are not generated at high frequency. Since concatemeric DNA missing the 143 bp at both ends of the S component is not cleaved, the terminal 143 bp that include the sequences with homology to the pac-1 site are necessary for efficient cleavage. (iv) v135/9 intracellular DNA is composed mainly of arrays in which one S component (with two equalized inverted repeats both having the deletion) is bracketed by two L components in opposite orientations and in which two L components are in head-to-head alignment. Because (i) no homology exists in the circularized (or concatemeric) genomes of these mutants between the sequences adjacent to the two ends of the S component which would allow equalization of the repeats by recombination, and (ii) the alternate product of recombination, i.e., DNA with wild-type sequences at the ends of both inverted repeats, does not accumulate, we postulate that equalization of the termini of the repeats occurs by a copying mechanism. Since it is the terminal repeat that is converted consistently and becomes identical to the internal repeat, we propose that the terminal repeat invades the internal repeat, which acts as a template for its conversion. To account for the accumulation in v135/9-infected cells of concatemeric DNA in which the L components are in head-to-head alignment, the model we propose postulates that the sequences with homology to the pac-1 site (present at the ends of the inverted repeats in wild-type but missing from v135/9 intracellular DNA) are somehow responsible for preventing strand elongation beyond the ends of inverted repeats. In the absence of these sequences, strand elongation would continue after the internal end has been copied, and concatemeric molecules in which L components are in head-to-head alignment would by formed by a process akin to that postulated for poxvirus DNA replication.