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

INTRODUCTION

The herpes simplex virus 1 (HSV-1) DNA genome consists of two covalently linked components designated L and S which are bracketed on each end by inverted repeat sequences. The inverted repeats of the L component have been designated as ab and b'a', while those of the S component have been designated as ac and c'a' (1,2) (Fig 1A). An unusual property of the HSV-1 genome is the ability of the L and S components to invert relative to each other such that viral DNA isolated from either virions or infected cells consists of four equimolar populations each differing in the relative orientation of the L and S components (3).

Figure 1: Sequence arrangement of the wild-type and recombinant HSV-1 virus genomes and of the TK-mM bacteriophage genome. (A) Sequence arrangement of HSV-1(F) DNA. The open boxes represent terminal sequences (ab and ca) of the L and S components, respectively that are inverted internally (bac). (B) BamHI restriction endonuclease map of the termini of HSV-1 305(F), and the recombinant viruses. The location and orientation of the TK-mM insertion in the recombinant viruses is shown within the parentheses. The dashed line represents the junction of the L and S components. The parentheses delineate the location of the deleted sequences in the recombinant viruses. The hatched box represents the TK gene located in the TK-mM insertion. (C) Schematic of the TK-mM bacteriophage genome. c, ner, A and B represent Mu genes; Kan represents the kanamycin resistance marker; TK represents the thymidine kinase gene under the control of the ICP4 promoter.

Earlier studies from our laboratory described the characterization of three independently isolated HSV-1 recombinant viruses frozen in either the P (prototype), IS (inversion of S component), or ILS (inversion of both components) arrangement (4,5). These recombinants were generated by a target-specific mutagenesis procedure utilizing an TK mini-Mu derivative of the transducing bacteriophage Mu (Fig. 1C). Common to all three recombinant viruses is the deletion of approximately 14 kilobases (kb) of viral DNA sequences from the internal repeats and the insertion of a 9.6 kb mini-Mu genome containing a functional thymidine kinase gene under the control of the HSV ICP4 promoter (Fig. 1B). The DNA sequences deleted from each recombinant are not identical, but are very similar, differing by only a few hundred base pairs and represent greater than 95% of the internal repeat DNA sequences. The deletion of these sequences has resulted in the reduction of several HSV genes and DNA sequences from the normal diploid state to a unique haploid state. Thus, these recombinants contain only a single copy of the genes encoding the immediate early peptides ICP0 and ICP4 (6), a single copy of the gene designated ICP34.5 (7), and a single copy of the latency associated transcript (8)(LAT; Fig. 2).

Figure 2: Schematic of the HSV-1 genome showing an enlarged internal repeat region. The arrows indicated the direction of transcription of the indicated genes. The hatched box represents the region of the internal repeats that are deleted in the recombinant viruses.

Our initial studies indicated that these recombinant viruses were capable of independent replication in Vero, rabbit skin, and human 143 TK- cells, producing viral titers comparable to that of the wild-type parent HSV-1(F) (4,5). The cytopathic effect (CPE), plaque size, and morphology of the recombinant viruses were indistinguishable from HSV-1(F). These studies clearly demonstrated that the sequences deleted in these recombinant viruses were not essential for viral replication in cell culture.

Viral gene products or DNA sequences that are not essential for viral replication in cell culture may, nevertheless, still have an important function in either viral replication or pathogenicity in animals. In the animal, HSV infects cells at a peripheral site where it replicates locally, infects local nerve endings and spreads via axons to sensory ganglia establishing a latent state. At later times, the virus can be reactivated whereby it travels back down the axon to produce a recurrent infection at or near the area of the initial primary infection. Several aspects of HSV pathogenesis including neurovirulence, neuroinvasiveness, and the establishment and maintenance of a latent state in neural tissues has been studied for a number of years using a mouse model system.

In this study, we have analyzed the pathogenicity of the recombinant viruses RBMu2, RBMu3 and REMu1 in mice. We show that deletion of the internal repeats from the HSV genome has resulted in a pronounced effect on neurovirulence, neuroinvasiveness and in the ability to establish a reactivable latent infection in mice. Our findings strongly suggest a potential role for the presence of diploid regions and/or inversion of the HSV genome in viral pathogenicity.