Intracellular cre-mediated deletion of the unique packaging signal carriedby a herpes simplex virus type 1 recombinant and its relationship to the cleavage-packaging process

To gain further insight on the function of the herpes simplex virus type 1 (HSV-1) packaging signal (a sequence), eve constructed a recombinant virus containing a unique a sequence, which was flanked by two loxP sites in para llel orientation, The phenotype of this recombinant, named HSV-1 LaL, was s tudied in cell lines which either express or do not express Cre recombinase . Although LaL virus multiplication was only slightly reduced in standard c ell lines, its growth was strongly inhibited in Cre-expressing cells, In th ese cells, a sequences were detected mostly in low-molecular-weight DNA cir cles, indicating that they had been excised from virus DNA by site-specific recombination. Deletion of the a sequences from the viral genome resulted in the accumulation of uncleaved replication intermediates, as observed by pulsed-field gel electrophoresis, B-type capsids also accumulated in these cells, as shown both by electron microscopy and by sucrose gradient sedimen tation. Further examination of the status of a sequences in Cre-expressing cells indicated that high-level amplification of this sequence can occur in the absence of the cleavage-packaging process. Moreover, the amplified a s ignals in small circular DNA molecules remained uncleaved, indicating that these molecules mere not able to efficiently interact with the cleavage-pac kaging machinery, The cleavage-packaging machinery and the structural prote ins required to assemble virions were, however, functional in HSV-1 LaL-inf ected Cre-expressing cells, since this system could be used to package plas mid DNA harboring an origin of virus replication and one normal a signal, T his is the first study in which accumulation both of uncleaved replication intermediates and of B capsids has been obtained in the presence of the ful l set of proteins required to package virus DNA.