THE A SEQUENCE IS DISPENSABLE FOR ISOMERIZATION OF THE HERPES-SIMPLEXVIRUS TYPE-1 GENOME

The herpes simplex virus type 1 (HSV-1) genome consists of two compone nts, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of vir al DNA, Recent studies have indicated that this genome isomerization i s the result of DNA replication-mediated homologous recombination betw een the large inverted repeat sequences that exist in the genome, rath er than site-specific recombination through the terminal repeat a sequ ences present at the L-S junctions, However, there has never been an u nequivocal demonstration of the dispensability of the latter element f or this process using a recombinant virus whose genome lacks a sequenc es at its L-S junctions, This is because the genetic manipulations req uired to generate such a viral mutant are not possible using simple ma rker transfer, since the cleavage and encapsidation signals of the a s equence represent essential cia-acting elements which cannot be delete d outright from the viral DNA, To circumvent this problem, a simple tw o-step strategy was devised by which essential cia-acting sites like t he a sequence can be readily deleted from their natural loci in large viral DNA genomes, This method involved initial duplication of the ele ment at a neutral site in the viral DNA and subsequent deletion of the element from its native site, Ey using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication thr ough the insertion of a second copy into the thymidine kinase (TK) gen e of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer, The final recombinant virus, HSV-1::L-S Delta a, was found to be capa ble of undergoing normal levels of genome isomerization on the basis o f the presence of equimolar concentrations of restriction fragments un ique to each of the four isomeric forms of the viral DNA. Interestingl y, only two of these genomic isomers could be packaged into virions. T his restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene i n a packageable orientation, This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1 genome isomerization. Foll owing infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infectio n coincident,vith the onset of DNA replication, indicating that the lo ss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus, These results therefo re validate the homologous recombination model of HSV-1 genome isomeri zation by directly demonstrating that the a sequence at the L-S juncti on is dispensable for this process, The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicab le to studies of essential cia-acting elements in other large viral DN A molecules.