DNA replication of herpes simplex virus type 1 (HSV-1) is dependent on a virus-encoded sequence-specific origin-binding protein, the product of the UL9 reading frame. We have identified the mutations in the UL9 gene of three temperature-sensitive () mutants of HSV-1 which are responsible for the phenotype (A90T in mutant S and V220M in R and X). The mutations are located in two different conserved helicase sequence motifs of UL9. Two further alterations (I204T and E280D) compared to the published sequence were found in the mutant, revertant and parental wild-type strain 17 sequences and therefore seemed to be irrelevant for the phenotype. The function of the UL9 protein was required at early times during DNA synthesis whereas upward temperature shifts at later times did not considerably inhibit DNA synthesis.


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