Cloning, sequencing, and functional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis.

Abstract

The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of oris located within the repeated sequences flanking the short unique arm (Us), and one copy of OriL located within the long unique arm (UL). Precise localization and characterization of oriL have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to oris. In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.The herpes simplex virus type 1 (HSV-1) genome is a 160-kilobase pair (kb) linear duplex DNA molecule consisting of two components, L and S. The L component consists of unique sequences (UL) flanked by the inverted repeated sequences ab and b'a', whereas the S component consists of unique sequences (Us) flanked by inverted repeated sequences ac and c'a' (Fig. 1A) (26,46). The "a" sequence is present as a direct repeat at both molecular termini and in inverted orientation at the L-S junction (9,22,38,56).Attempts to localize the origin(s) of viral DNA synthesis within the structurally complex HSV-1 genome have involved studies of both standard and defective genomes. Electron microscopic studies of replicating standard wildtype viral DNA extracted from infected cells have been interpreted to suggest that the genome contains two origins of viral DNA synthesis, one near the middle of UL and the other near one molecular terminus (20. 27).Indirect evidence supporting the existence of two origins comes from studies of defective molecules of HSV-1 which are generated during serial passage of the virus at high multiplicities of infection. Defective DNA molecules fall into two classes, class I and class II, each consisting of tandem duplications of small subsets of viral DNA sequences. Class I defective genomes contain sequences from the "c" repeats which bracket Us (16-18, 32, 33, 36), whereas class II defective genomes contain sequences from Ul (16,19,32,45) (Fig. IB). Both classes also contain the "a" sequence which specifies a site for the cleavage of viral DNA concatamers during encapsidation (40,55). By analogy with the defective genomes of other DNA viruses, all of which contain origins of DNA synthesis. the existence of two distinct subsets of viral DNA sequences in defective HSV-1 genomes suggests that the genome contains three origins of DNA synthesis: two in diploid "c" sequences (ois), and one in UL (oriL) (Fig. IB).Direct evidence that the repeat units of class I and II * Corresponding author.

Abundant 5 kb RNA of Human Cytomegalovirus without a Major Translational Reading Frame

“…Notable from the sequence composition is the high AT content within the coding region, which is interrupted by short stretches of high GC. A high AT content has been reported for several viral sequences known to be involved in DNA replication, such as simian virus 40 (SV40) (Bergsma et al, 1982), polyoma virus (Soeda et al, 1979), Epstein-Barr virus (Yates et al, 1984) and HSV (Stow & McMonagle, 1983;Weller et al, 1985). These origin (ori) sequences are often located in the vicinity of viral genes that are intensively transcribed into mRNAs before or after the onset of DNA replication.…”

Identification of a Varicella-Zoster Virus Origin of DNA Replication and Its Activation by Herpes Simplex Virus Type 1 Gene Products

“…1 ; for review, see Roizman, 1979). Origins of DNA replication have been located in two distinct regions, namely close to the centre of UL (ORIL; Spaete & Frenkel, 1982;Weller et al, 1985) and within the inverted repeats IRs and TRs (ORIs; Stow, 1982;Mocarski & Roizman, 1982). The cis-acting signals required for ORI s activity reside within a 90 bp segment which contains an almost perfect palindromic sequence of 45 bp (Stow & McMonagle, 1983).…”

Herpes Simplex Virus Causes Amplification of Recombinant Plasmids Containing Simian Virus 40 Sequences

“…This procedure has subsequently been refined by using molecularly cloned restriction fragments (Stow, 1982). Meanwhile, several groups have succeeded in defining very precisely the sequence elements of HSV replication origins Gray & Kaerner, 1984;Weller et al, 1985 ;Lockshon & Galloway, 1986) and cleavage/packaging signals (Spaete & Frenkel, 1982.…”