Using Vero cells tranformed with the wild-type gene for ICP4 as the permissive host cell, we isolated herpes simplex virus type 1 (HSV-1) mutants containing deletions in both copies of the ICP4 gene. The mutants, d120 and d202, contained deletions of 4.1 and 0.5 kilobases, respectively, in each copy of ICP4. ICP4 mRNA synthesized in d202-infected Vero cells was 0.5 kilobases smaller than that synthesized in cells infected with the wild-type virus. No ICP4 mRNA was detected in d120-infected Vero cells. d120 and d202 specified polypeptides that reacted with ICP4 antiserum and were smaller than the wild-type ICP4 polypeptide by 130 and 30 kilodaltons, respectively. The only other HSV-1 gene products detectable on infection of Vero cells with d120 and d202 were ICP6 (of the early kinetic class of HSV-1 polypeptides), ICPO (immediate early), ICP22 (immediate early), and ICP27 (immediate early). Immediate-early polypeptides ICPO and ICP27 were expressed to a higher level in Vero cells infected with an ICP4 temperature-sensitive (ts) mutant (tsB32) at 39°C, indicating immediate-early stimulatory activity associated with the ts ICP4 polypeptide. In addition, the patterns of complementation of d120, d202, and tsB32 in ICP4-transformed cells also demonstrated inhibitory activity associated with the ts form of the ICP4 polypeptide.

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.

As a first step in identifying the functions and intramolecular functional domains of herpes simplex virus type 1 infected cell protein 0 (ICP0) in productive infection and latency, a series of mutant plasmids specifying varying amounts of the ICP0 primary amino acid sequence were constructed. In transient expression assays with mutant and wild-type plasmids, the N-terminal half of the ICP0 molecule was found to be sufficient to transactivate a variety of viral promoters. Although promoters representing the immediate-early, early, and late kinetic classes were transactivated by wild-type ICP0, individual promoters responded to mutant forms of ICP0 in a manner consistent with the possibility that ICP0 transactivates different promoters by different mechanisms. Unlike infection with virus particles, which contain the 65-kilodalton transcriptional transactiovator, the initiation of viral replication after transfection of cells with purified viral DNA requires de novo protein synthesis. In order to assess the role of ICP0 in the de novo synthesis of infectious virus, Vero cells were transfected with purified DNA of wild-type virus or an ICP0 null mutant and the production of infectious virus was monitored. In cells transfected with mutant DNA, virus production was delayed by 2 days and the level of virus was reduced by several orders of magnitude relative to Vero cells transfected with wild-type viral DNA, suggesting an important role for ICP0 in the de novo synthesis of infectious particles. In cotransfection experiments with infectious DNA of the ICP0 null mutant and a plasmid specifying wild-type ICP0 titers of infectious virus were significantly enhanced relative to transfection with mutant DNA alone, confirming the role of ICP0 in de novo synthesis. These findings are consistent with the proposed role of ICP0 in reactivation of herpes simplex virus from latency (D. A. Leib, D. M. Coen, C. L. Bogard, K. A. Hicks, D. R. Yager, D. M. Knipe, K. L. Tyler, and P. A. Schaffer, J. Virol. 63:759-768, 1989), a process also thought to require de novo protein synthesis. The complementing activities of ICP0 mutant plasmids for ICP0 null mutant DNA in cotransfection assays correlated well with their transactivating activities for viral promoters in transient assays, indicating that the transactivating function of ICP0 is a critical factor in the de novo synthesis of infectious particles.(ABSTRACT TRUNCATED AT 400 WORDS)

SUMMARYA temperature-sensitive mutant of herpes simplex virus type 1, tsQ26, was shown to contain an amino acid substitution in glycoprotein H (gH). The mutant entered cells efficiently at the non-permissive temperature and replicated to give nearly normal yields of intracellular infectivity. The intracellular virions contained, predominantly, an immature form ofgH and no gH was found on the surface of infected cells. Excreted virions were devoid of gH and were not infectious. Virions excreted at the permissive temperature were infectious and contained gH and no loss of gH resulted from incubation of these virions at the non-permissive temperature. The temperaturesensitive phenotype apparently results from the loss of gH from virions during their transport to the cell surface, and since loss of gH is accompanied by loss of infectivity we conclude that gH is an essential component of the infectious virion.

ICPO is a potent activator of herpes simplex virus type 1 gene expression in transient assays and in productive infection. A role for ICPO in reactivation from latency in vivo has also been suggested on the basis of the observation that viruses with mutations in both copies of the diploid gene for ICPO reactivate less efficiently than wild-type virus. Because the ICPO gene is contained entirely within the coding sequences for the latency-associated transcripts (LATs), ICPO mutants also contain mutations in LAT coding sequences. This overlap raises the question of whether mutations in ICPO or the LATs, which have also been implicated in reactivation, are responsible for the reduced reactivation frequencies characteristic of ICPO mutants. Two approaches were taken to examine more definitively the role of ICPO in the establishment and reactivation of latency. First, a series of ICPO nonsense, insertion, and deletion mutant viruses that exhibit graded levels of ICPO-specific transactivating activity were tested for parameters of the establishment and reactivation of latency in a mouse ocular model. Although these mutants are ICPO LAT double mutants, all nonsense mutants induced the synthesis of near-wild-type levels of the 2-kb LAT, demonstrating that the nonsense linker did not disrupt the synthesis of this LAT species. All mutants replicated less efficiently than the wild-type virus in mouse eyes and ganglia during the acute phase of infection. The replication efficiencies of the mutants at these sites corresponded well with the ICPO transactivating activities of individual mutant peptides in transient expression assays. All mutants exhibited reduced reactivation frequencies relative to those of wild-type virus, and reactivation frequencies, like replication efficiencies in eyes and ganglia, correlated well with the level of ICPO transactivating activity exhibited by individual mutant peptides. The amount of DNA of the different mutants varied in latently infected ganglia, as demonstrated by polymerase chain reaction analysis. No correlation was evident between reactivation frequencies and the levels of viral DNA in latently infected ganglia. Thus, replication and reactivation efficiencies of ICPO mutant viruses correlated well with the transactivating efficiency of the corresponding mutant peptides. In a second approach to examining the role of