VACCINIA VIRUS-DNA REPLICATION - 200 BASE-PAIRS OF TELOMERIC SEQUENCECONFER OPTIMAL REPLICATION EFFICIENCY ON MINICHROMOSOME TEMPLATES

Vaccinia virus is a complex DNA virus that exhibits significant geneti c and physical autonomy from the host cell. Most if not all of the fun ctions involved in replication and transcription of the 192-kb genome are virally encoded. Although significant progress has been made in id entifying trans-acting factors involved in DNA synthesis, the mechanis m of genome replication has remained poorly understood, The genome is a linear duplex with covalently closed hairpin termini, and it has bee n presumed that sequences and/or structures within these termini are i mportant for the initiation of genome replication, In this report we d escribe the construction of minichromosomes containing a central plasm id insert flanked by hairpin termini derived from the viral genome and their use as replication templates. When replication of these minichr omosomes was compared with a control substrate containing synthetic ha irpin termini, specificity for viral telomeres was apparent, Inclusion of greater than or equal to 200 bp from the viral telomere was suffic ient to confer optimal replication efficiency, whereas 65-bp telomeres were not effective, Chimeric 200-bp telomeres containing the 65-bp te rminal element and 135 bp of ectopic sequence also failed to confer ef ficient replication, providing additional evidence that telomere funct ion is sequence-specific. Replication of these exogenous templates was dependent upon the viral replication machinery, was temporally coinci dent with viral replication, and generated covalently closed minichrom osome products, These data provide compelling evidence for specificity in template recognition and utilization in vaccinia virus-infected ce lls.