LYMANTRIA-DISPAR NUCLEAR POLYHEDROSIS-VIRUS HOMOLOGOUS REGIONS - CHARACTERIZATION OF THEIR ABILITY TO FUNCTION AS REPLICATION ORIGINS

Homologous regions (his) were identified in the Lymantria dispar nucle ar polyhedrosis virus (LdMNPV) genome. A 1.58-kb region surrounding hr 4 was sequenced and found to have two distinct domains. Domain P (abou t 600 bp) is composed of seven repeats of about 80 bp including a seri es of palindromes containing MluI sites and overlapping XhoI and SacI sites. Domain II (about 700 bp) is composed of eight partially repeate d sequences of 60 to 100 bp containing a 15- to 25-bp sequence that is 80 to 100% A+T in addition to a 6- to 10-bp palindrome containing an NruI site. Hybridization of a domain I sequence to cosmids containing the LdMNPV genome indicated its presence at eight positions (hr1 to -8 ) on the genome. In contrast, hybridization of domain II indicated tha t it was present only at the hr4 locus. A DpnI-based transient-replica tion assay was used to determine if subclones of hr4 transfected into LdMNPV-infected L. dispar cells functioned as replication origins. Sub clones of hr4 containing either domain I or domain II replicated at ve ry low or moderate levels, respectively. However, when domain I and do main II were linked on the same plasmid, high levels of replication we re observed. A 1.4-kb region containing hrl was also sequenced. It lie s immediately upstream of the polyhedrin gene and contains six domain I-type repeats. Four-hundred-base-pair regions of domain I repeats fro m hr1 and hr4 showed 89% sequence identity. Plasmids containing the hr 1 domain I replicated at low levels. However, hybrid plasmids in which the AT-rich hr4 domain II was inserted adjacent to hr1 domain I repli cated to high levels, indicating that the AT-rich domain II greatly en hances replication. The orientation and position of domains I and II r elative to each other did not have major effects on the levels of repl ication.