Frank J. Jenkins¹, Alyson M. Donoghue² and John R. Martin³

¹ Department of Pathology and Infectious Diseases and Microbiology, University of Pittsburgh, and Division of Behavioral Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213
² Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD. 20814
³ Food and Drug Administration, Division of Antiviral Drug Products, Rockville, MD 20857.

Received 07/30/96; Accepted 08/27/96; On-line 10/04/96

DISCUSSION

In the course of a natural infection, herpes simplex virus encounters a number of different cell types and cellular environments. The virus infects animals at a peripheral site where it replicates locally before traveling through neural tissue to a sensory ganglion. Once at the ganglion the virus establishes a latent state which can, at a later date, become reactivated causing a recrudescent disease. Therefore several areas require investigation in the understanding of viral pathogenicity in the animal, including local replication, neuroinvasiveness (the spread of virus from the local site of replication to sensory ganglion), neurovirulence, and the establishment and maintenance of a latent state.

Studies designed to identify specific viral genes involved in these areas have demonstrated a complex involvement of several different regions of the HSV DNA genome. Centifanto-Fitzgerald et al. (26) reported that the region of the HSV genome defined by map units 0.70-0.83 was responsible for producing a stromal disease in the eyes of rabbits, while Thompson and coworkers have also reported that this region is involved in allowing HSV to replicate in neural tissue in the mouse (27-29). A subset of this region defined by map units 0.761 to 0.796 has recently been reported to be involved in intraperitoneal virulence in mice (17). Day et al. (30) have reported that the 0.40-0.44 region of the viral genome is involved in the spread of virus from corneas to the central nervous system. In addition, Meignier et al. (31) have shown that the deletion of several genes from the unique short region of the HSV genome results in a marked decrease in neurovirulence and latency. Based on these studies, it is clear that HSV neurovirulence, neuroinvasiveness and the ability of HSV to establish and maintain a latent state in animals is not due to a single HSV gene product but can be affected by several viral gene functions.

The results presented in this paper demonstrate that the internal repeat sequences play an essential role in the replication and pathogenesis of HSV in the mouse. The use of three independently isolated recombinant viruses each containing similar but not identical deletions of the internal repeats strengthens our findings. The viral recombinants have a dramatically increased PFU/LD50 ratio by i.c. inoculation and do not establish a reactivable latent infection in the trigeminal ganglion even when inoculated directly into the brain. In addition, the recombinants are not neuroinvasive since they do not spread from the cornea to the trigeminal ganglion, and viral replication in mouse tissues is severely affected. The dramatic decrease in viral titer in both the corneas and brains of infected mice indicates that the recombinants exhibit at best, a limited replication.

The precise function of the internal repeat sequences in HSV pathogenesis is obscure. All of the viral genes and DNA sequences contained within these repeats are also located at the termini of the genome. Therefore deletion of these sequences decreases the copy number of the viral genes and sequences from diploid to haploid without completely removing any known viral gene from the genome. The internal a repeat sequences which have also been deleted in these recombinants are required for the inversion of the viral genome producing the four isomeric forms of viral DNA. However, the construction of the frozen recombinants used in this study clearly demonstrates that inversion per se and the presence of diploid genes is not required for viral replication in cell culture. These results have recently been supported by Harland and Brown (32) who reported the construction of an HSV-2 deletion mutant which is missing the majority of the internal repeat structure and which grows well in cell culture.

The requirement for the internal repeats, therefore, is not obvious from in vitro studies of these recombinant viruses. However, since naturally occurring mutants with large deletions in these sequences or mutants that are frozen and unable to invert have never been described, it would seem likely that the internal repeat sequences are important in the natural environment of the virus.

The recombinant viruses used in this study exhibit high levels of TK activity even though the TK gene is not located in its normal position on the viral genome. Meignier et al. (31) reported that a HSV-1(F) recombinant containing a functional TK gene inserted into the coding region of the immediate early gene ICP47 was capable of establishing latency. Therefore the unusual location of the TK gene in the recombinants used in this study is not likely to be a factor in the decreased pathogenicity.

The DNA sequences deleted in RBMu2, RBMu3 and REMu1 correspond to map units 0.78 - 0.86 on the HSV genome in the prototype arrangement. This region would include some of the sequences identified by Thompson and coworkers (27-29) that are involved in the ability of HSV to replicate in neural tissue. It is possible that the frozen recombinants share defect(s) with the RE6 mutant of Thompson et al. but also must contain additional defects, since the RE6 mutant replicates well at peripheral sites and establishes a latent state in sensory ganglion, while the frozen recombinants used in this study do not replicate well peripherally and do not establish reactivable latent infections.

The work of Leib et al. (25) has indicated roles for ICP0 in viral latency and ICP4 and ICP27 in viral replication and neuroinvasiveness. Since both ICP4 and ICP27 have been shown to be essential for virus replication in cell culture (33-35) it is not surprising that viruses containing mutations in these genes are unable to replicate or exhibit neuroinvasiveness in the mouse. An appealing hypothesis for the results presented in this study is that the reduction of several viral genes, namely ICP0 and ICP4, from diploid to haploid in the viral genome are collectively responsible for the observed effects on pathogenicity. The potential role of inversion of the HSV genome also cannot be ruled out as a factor in the observed decrease in pathogenicity. The important role of the internal repeats in HSV pathogenicity may also explain why naturally occurring mutants containing deletions of the internal repeats have not been described.