Replication of herpes simplex virus DNA: localization of replication recognition signals within defective virus genomes.

Abstract

Serially passaged herpes simplex virus type 1 (HSV-1) strain Justin was previously shown to contain defective virus genomes consisting ofhead-to-tail reiterations ofsequences derived from the end ofthe S component ofthe standard virus DNA. Cotransfection of purified monomeric defective genome repeat units with foster helper virus DNAs onto rabbit skin cells resulted in regeneration and replication of concatemeric defective DNA molecules which were successfully encapsidated. Thus, defective HSV-1 (Justin) genomes contain, within their limited DNA sequences, a sufficient set of recognition sites required for HSV DNA replication and packaging. The arrangement of repeat units within the regenerated defective virus genomes was consistent with their replication by a rolling circle mechanism in which a single repeat unit served as the circularized template. This replication occurred most actively late after infection and could be shown to be inhibited by low concentrations of phosphonoacetate known to inhibit the HSV-specified viral DNA polymerase selectively. The resultant concatemers were shown to be cleaved to M 100 X 106 DNA molecules which were terminated at one end with the proper ac end sequence of the parental standard virus DNA.The standard genome of herpes simplex virus type 1 (HSV-1) is a linear, double-stranded DNA molecule with Mr 100 X 106 (1, 2). Topologically, the standard HSV DNA is terminally reiterated and consists oftwo covalently linked components, L and S, each of which contains a stretch of unique viral DNA sequences surrounded by inverted repeats: the unique sequences of L (UL) surrounded by the ab and b'a' inverted repeat sequences, and the unique sequences ofS (Us) bounded by the ac and c'a' inverted repeats (3-6). The L and S components have been shown to invert relative to each other, yielding equimolar amounts of four isomeric structures. (3,(5)(6)(7)(8).Whereas recently plaque-purified virus stocks predominantly contain standard HSV genomes, defective virus populations obtained through serial undiluted propagation of such stocks have been shown to contain variable proportions of variant DNA molecules in which greater than 90% of

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

“…Upon digestion with EcoRI, these molecules yielded fragments which co-migrated with linear pVO2 indicating that they consist of tandem duplications of the complete input plasmid. Replicated pVO2 DNA therefore consists of concatemers similar in structure to the tandemly reiterated defective HSV genomes generated by passage of virus at high multiplicities of infection (for review, see Frenkel et al, 1980), or following transfection with individual fragments or cloned molecules containing functional origins (Vlazny & Frenkel, 1981 ;Stow, 1982). Although other possibilities can not be rigorously excluded, the concatemeric nature of the pVO2 replication products is consistent with replication occurring through a rolling-circle mechanism similar to that previously proposed for defective HSV genomes.…”

Herpes Simplex Virus Non-structural Proteins. III. Function of the Major DNA-binding Protein

“…Since both ICSP 11, 12 and the DNA polymerase have been mapped to the same region of the virus genome the finding that ICSP 11, 12 has an important role in DNA replication suggests that a functional grouping of HSV proteins involved in DNA synthesis may occur around the 0-4 map unit position (Morse et al, 1978;Marsden et al, 1978;Chartrand et al, 1980;Conley et al, 1981). Additional interest in this observation comes from the findings of several groups (Kaerner et al, 1979;Vlazny & Frenkel, 1981) that certain defective strains of HSV appear to include an origin of DNA replication that maps at around this site. In view of the likely importance of the structure of the DNA within this area of the genome it will be of interest to observe the ability of ICSP 11, 12 to bind to specific fragments of DNA derived from it.…”

Sequence of the Putative Origin of Replication in the UL Region of Herpes Simplex Virus Type 1 ANG DNA

“…The deleted sequence thus maps in the same region of the viral standard genome at which various deletions have been mapped by others (Spaete & Frenkel, 1982;Post et al, 1980;Weller et al, 1983). This region was also suspected to contain the UL origin of HSV-1 DNA replication (Vlazny & Frenkel, 1981;Spaete & Frenkel, 1982;Weller et al, 1983).…”

“…One is present in two copies, as it is located in the TRs and in the IRs repeat, whereas the other is located in the UL region between map positions approximately 0.385 and 0.435. It has also been shown that the HSV-1 TRs/IRs origin of replication is contained in the repeat units of repetitive class I defective HSV-1 genomes (Vlazny & Frenkel, 1981). Defective HSV genomes, which accumulate in the virus progeny during serial passages at high multiplicity of infection, have been shown to contain DNA consisting of tandem repetitions of short regions of the parental viral genome (for t Present address: EMBL, Meyerhofstrasse 1, 6900 Heidelberg, F.R.G.…”