A herpes simplex virus type 1 ICP22 deletion mutant is altered for virulence and latency in vivo.

The in vivo function of the herpes simplex virus type 1 immediate early gene ICP22 has been investigated in mice and guinea pigs using a deletion mutant (del22Z) of HSV-1(F) that lacks all but 18 nucleotides of the ICP22 coding sequence. This mutant carries the bacterial lacZ gene at the site of the deletion and makes functional beta-galactosidase, but is unable to synthesize any detectable ICP22 messenger RNA or protein in vitro. Del22Z was impaired in its ability to cause death in mice following intracerebral, intraperitoneal, or intravaginal inoculation. The mutant failed to produce lesions or other visible signs of infection after bilateral corneal infection of mice but could be recovered from trigeminal ganglia explanted at day 30 after inoculation. Del22Z replicated poorly after intravaginal inoculation of mice and guinea pigs in comparison to the parental virus, and was not recoverable from the dorsal root ganglia of either species. Nevertheless, del22Z sequences could be detected in the dorsal root ganglia of guinea pigs at day 30 by the polymerase chain reaction. These studies demonstrate that the ICP22 gene product is required for acute infection and virulence in two standard in vivo animal models.

In vitro characterization of a herpes simplex virus type 1 ICP22 deletion mutant.

We report the construction of a deletion mutant (del22Z) that is unable to synthesize any detectable messenger RNA or protein products from the herpes simplex virus type 1 (HSV-1) immediate early ICP22 gene upon infection. The del22Z deletion mutant lacks all but 18 nucleotides of the ICP22 coding sequence and carries the bacterial lacZ gene at the site of the deletion. No other known open reading frames or flanking sequences were disrupted. Del22Z was able to infect Vero cells productively but was severely restricted in human and rodent cells that were permissive for the parental HSV-1(F). The yield of del22Z was not enhanced significantly, either by increasing the multiplicity of infection or by increasing the duration of the infection. There was a prolonged expression of some early gene products and a delayed appearance of some late gene products in both permissive and restrictive cells. This phenotype of cell-line restricted growth and alteration of the normal gene expression cascade maps specifically to the ICP22 coding region.

Deletion of the herpes simplex virus type 1 ribonucleotide reductase gene alters virulence and latency in vivo.

The role of herpes simplex virus type 1 (HSV-1)-encoded ribonucleotide reductase (RR) has been investigated in mice and guinea pigs using a mutant from which 90% of the large subunit of the enzyme was deleted. The RR mutant was extremely impaired in its ability to induce external vaginal lesions or to cause death in mice following intracerebral, intraperitoneal, or intravaginal inoculation, or in guinea pigs following intraperitoneal or intravaginal inoculation. The RR mutant replicated poorly in the vagina of mice and guinea pigs when compared with the parental virus. Neither infectious nor latent virus was recovered from the trigeminal ganglia of mice or from the dorsal root ganglia of mice and guinea pigs after inoculation with the RR mutant. Using the polymerase chain reaction, RR mutant DNA was, nevertheless, detected in the dorsal root ganglia of guinea pigs. These studies suggest that HSV-1 RR is essential for virulence and may also play a role in the recovery of reactivatable latent virus from ganglia in both mice and guinea pigs.

A noninverting genome of a viable herpes simplex virus 1: presence of head-to-tail linkages in packaged genomes and requirements for circularization after infection.

The wild-type herpes simplex virus 1 genome consists of two components, L and S, which invert relative to each other, giving rise to four isomers. Previously we reported the construction of a herpes simplex virus 1 genome, HSV-1(F)I358, from which 15 kilobase pairs of DNA spanning the junction between L and S components were deleted and which no longer inverted (Poffenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2690-2694, 1983). Further studies on the structure of HSV-1(F)I358 revealed the presence of two submolar populations among packaged DNA. The first, comprising no more than 10% of total packaged DNA, consisted of defective genomes with a subunit size of 36 kilobase pairs. The results suggest that this population arose by recombination through a directly repeated sequence inserted in place of the deleted L-S junction. The second minor population consisted of HSV-1(F)I358 DNA linked head-to-tail. Analyses of the structure of HSV-1(F)I358 DNA after infection indicated that the fraction of total DNA linked head-to-tail increased to approximately 40 to 50% within 30 min after exposure of cells to virus. The formation of head-to-tail linkages did not require de novo protein synthesis. Our interpretation of the results is that the termini of full-length DNA molecules are held together during packaging, that a small fraction of the termini is covalently linked during or after packaging, and that the remainder is covalently joined after the release of viral DNA from the infecting virus by either host or viral factors introduced into the cell during infection.

Characterization of a viable, noninverting herpes simplex virus 1 genome derived by insertion and deletion of sequences at the junction of components L and S.

Earlier studies have shown that the DNA of herpes simplex virus 1 consists of two covalently linked components, L and S, each flanked by inverted repeats. The two components can invert, and viral DNA extracted from infected cells or virions consists of equimolar concentrations of four populations differing solely in the orientation of L and S components relative to each other. This paper describes a recombinant virus (1358) generated by an insertion of a chimeric thymidine kinase gene within the reiterated sequences of the S component and deletions that eliminated most of the internal inverted repeats at the junction between the L and S components. A characteristic of 1358 is that the L and S components are frozen in one (prototype) orientation. Inversion of L and S components is therefore not required for the replication of viral DNA.