cis Functions involved in replication and cleavage-encapsidation of pseudorabies virus

Serial passage at high multiplicity of pseudorabies virus generates defective interfering particles (DIPs) whose genomes consist at least in part of reiterations of segments of DNA in which sequences originating from different regions of the genome have become covalently linked (F. J. Rixon and T. Ben-Porat, Virology 97:151-163). To determine whether some cis functions present in these reiterated DNA sequences may be responsible for the amplification of DIP DNA, BamHI restriction fragments of this DNA were cloned. These fragments were analyzed and tested for their ability to promote the amplification of covalently linked pBR325 DNA when cotransfected into cells with helper pseudorabies virus DNA. The cloned DIP BamHI DNA fragments consisted of various combinations of sequences originating from either one or both ends as well as sequences from the middle of the unique long (UL) segment of the genome. Only plasmids with inserts consisting of segments of defective DNA originating from the middle of the UL, as well as from both ends of the genome, were able to replicate and be encapsidated autonomously. This finding indicated that signals present at both ends of the genome may be necessary for efficient cleavage-encapsidation. To confirm this observation, we constructed plasmids in which DNA segments containing an origin of replication and sequences from either one or both ends of the virus genome were linked. These experiments showed that efficient cleavage-encapsidation requires the presence of sequences derived from both ends of the genome. Two origins of replication, one at the end of the UL segment and one in the middle of the UL segment, were also identified.