Isolation of Herpes Simplex Virus Type 1 Variants Devoid of Hind III RE Sites and Their Use in Intrastrain Recombination Studies

The work presented in this thesis describes the construction of Hind III restriction endonuclease (RE) cleavage site-deletion variants of HSV-1 strain 17+ and their use as unselected markers in intrastrain recombination studies. In addition, the isolation of a Bgl II RE site-deletion variant and a HSV-1 genome containing an additional Hind III site along with the use of these extra markers in recombination experiments are also described. Furthermore, several other deletion/insertion variants were isolated and their preliminary characterization was carried out. The HSV-1 strain 17+ Xba I site negative variant 1702 was used as the parental virus to delete the Hind III sites. There are 10 Hind III sites on the wt HSV-1 genome. Site-directed mutagenesis was used to delete the following 7 Hind III sites: the 0.08 m.c. Hind III site, located within the UL5 ORF; the 0.1 m.c. Hind III site, which lies within the UL6 gene; the two Hind III sites at 0.18 m.c., located within the gene UL13; the 0.26 m.c. Hind III site, located within the UL19 ORF; the 0.64 m.c. Hind III site, which lies within the UL46 coding sequences; and, the 0.91 m.c. Hind III site, which lies within the promoter region of gD (US6). The variant (1733) devoid of the 4 Xba I sites plus the 7 Hind III sites showed normal growth properties in vitro. However, like the parental 1702 virus, 1733 is also tk- and produces truncated gC. During the process of Hind III site-deletion variant (the variants 1721 to 1733) isolation, two other variants (1734 and 1739) were spontaneously isolated. The variant 1734 was devoid of the 0.272 m.c. Bgl II site and showed no other alteration in its RE DNA profiles, growth properties or polypeptide profile compared to those of the parental viruses. This variant was recombined with 1733 to generate a variant (1738) lacking 1 Bgl II, 7 Hind III and 4 Xba I sites. The variant 1738 had very similar growth properties and identical polypeptide profile to those of the parental 1702, 1721 and 1733 viruses. An HSV-1 (strain 17+) variant 1708 containing an additional Xba I site at 0.74 m.c. has previously been described. The variant 1739 isolated herein contained an additional Hind III site at 0.374 m.c. However, like the parental 1702 and 1721 viruses, 1739 was devoid of the 4 Xba I sites. The restriction endonuclease analysis revealed no other deletion/insertion within the genome of 1739. In addition, 1739 showed normal growth characteristics in vitro. To obtain extra unselected markers in recombination studies, recombination experiments between the variants 1708 and 1739 were carried out and a variant (1743) containing an additional Xba I site & an additional Hind III site was generated. The variant 1743 also showed normal in vitro growth properties, was tk- and produced truncated gC. Intrastrain recombination experiments used the variants 1738 and 1743 as the parental viruses, differing in 14 unselected markers (1 Bgl II, 8 Hind III and 5 Xba I sites). The 1008 progeny were analysed for the presence or absence of these restriction endonuclease sites. The data obtained from these experiments yielded the following conclusions: (1) HSV is highly recombinogenic; (2) the high recombination frequencies (RFs) obtained herein between tightly linked markers demonstrate the necessity to have more frequent markers within the genomic region(s) of interest to measure correct RF values; (3) possibly all the four genomic isomers (P, lL, ls, ISL) take part in the process of HSV recombination; (4) no 'hot spots' of recombination were discovered. In addition to the isolation of HSV-1 (strain 17+) RE site-deletion/insertion variants, a number of other variants showing DNA deletions or insertions were also isolated. Most of the deletion variants involved the repeat elements of the HSV-1 genome, suggesting that possibly these elements are more prone to deletion among the non-essential viral regions in tissue culture. These variants include : the variant 1714 with a 759 bp deletion from both repeats of the L component (TRL, IRL), thus removing most of the RL1 ORF and making it non-neurovirulent in mice; the variant 1721X193 with a deletion of approximately 9.9 Kbp between 0.74 and 0.83 m.c. involving the UL/IRL sequences; and, the variant 1727X31 with a deletion of approximately 6.6 Kbp between 0.94 and 1.00 m.c., which involved the Us/TRg region. The variants 1721X193 and 1727X31 were not analysed any further because of lack of time. The insertion variants showed a high range of variation among the inserts i.e. from 356 bp to 30 Kbp. The variant 1740/n containing an insert of 356 bp was analysed and sequence of the insert determined. Since purification and characterization of other insertion variants were not carried out, it is not known whether the large inserts are stable or unstable within their genomes. Moreover, the location and/or nature of these inserts was not determined, again due to constraint imposed by time