Table X shows the viruses for which purification was attempted using the B4/27 cell line. Viruses were plaque purified every two days for approximately 4 months. It can be seen from the table that the majority o f viruses did not reach purity within this time. It was later found that the cell line B4/27 was unable to adequately support the growth of a virus deleted for both ICP27 and ICP4. The result o f this was that the desired recombinant viruses (ICP27 deleted and ICP4 deleted) had a significant growth disadvantage as compared to the backbone viruses (ICP27 deleted only). This meant that the recombinant viruses were only readily propagated in the presence of the backbone virus, the latter providing the ICP4 required for growth. Ultimately, only one ICP27 and ICP4 deleted virus (1764 27- 4- pR20.5) was purified using this cell line.
With hindsight, the attempted generation o f this number of viruses in parallel (both in this section and in section 3.6) should probably not have been carried out until the conditions for viral growth had been optimised.
Virus Cassette O rientation P urity 17+27-4- pR20.5 forward impure 17+27-4- pR20.7 forward impure 1764 27-4- pR20.5 forward pure 1764 27-4- pR20.5 reverse impure 1764 27-4- pR20.6 forward impure 1764 27-4- pR20.6 reverse impure 1764 27-4- pR20.7 forward impure 1764 27-4- pR20.7 reverse impure 1764 27-4- pR20.9 forward impure 1764 27-4- pR20.9 reverse impure
Table X: Attempted purification of viruses with inserts in ICP4
The table shows the list of ICP4 deleted viruses for which purification was attempted using the B4/27 cell line. It can be seen that in the majority of cases the viruses were not successfully purified.
3.10 Viruses with LAT P2 containing cassettes inserted into the ICP4 locus
cannot be stably propagated
Soon after purification of the 1764 27- 4- pR20.5 virus had been achieved, it became apparent that this virus was unstable and undergoing genome rearrangements as had been the problem when the various cassettes were inserted into the ICP27 locus. This was somewhat surprising as the proposed mechanism by which these rearrangements were occurring would in this case have resulted in the deletion of ICPO, which would be expected to significantly impair the growth characteristics of the resulting virus.
Interestingly, the homologous recombination between the inserted and the endogenous LAT P2 regions had again occurred regardless of the orientation of the inserted LAT P2 with respect to the endogenous LAT P2. This was assumed to be either
a result of a three dimensional contortion of the viral genome or recombination in trans between different replicating viral genomes.
The genome instability described here may explain a previous observation by
Lokensgard et al. in 1997. Here the authors attempted to insert a LAT P2-like element
at the 5’ end of LAPl in the gC locus. They report the construction of such a virus when LAPl and LAP2 are running in opposite directions to each other but state that they were unable to obtain an equivalent virus which contained LAP2 in the forward direction. The authors speculated that a third copy o f LAPl and LATP2 in the genome might be in some way undesirable.
Due to these instability problems, the virus 1764 27-4- pR20.5 had to be propagated by continual selection for both marker genes. As soon as this pressure was removed, the unstable viral genomes underwent rearrangements such that the gene for which active selection was not being applied was deleted. The exact recombinational
mechanisms (in cis or in trans) which resulted in such an outcome have not been
elucidated. Viruses resulting from such deletions had a smaller genome, giving them a growth advantage over the desired recombinants. This advantage was then amplified on scale-up such that after three to five serial passages none of the recombinant viruses had
the desired characteristics of expressing both GFP and lacZ and the predominant
population expressed neither. This meant that the 1764 27-4- pR20.5 virus could not be readily grown up in any quantity and stocks had to be prepared by the pooling o f a large number o f individually picked plaques. In order to confirm that the white mutant population was not a result o f the virus regaining the ICP4 gene from the B4/27 cell line, this population was plated on cells expressing ICP27 only. No growth o f the mutant virus was supported by the ICP27-complementing cell line, indicating that the virus had not regained the ICP4 gene.
Although unlikely, it is possible that the problems encountered were not due to homologous recombination between the two LAT P2 elements, but rather a pressure not to have three copies of the LAT P2 region in the viral genome, as was suggested by
Lokensgard et al. in 1997 (Lokensgard et a l, 1997). This pressure could be attributable
to the proposed structure of LAT P2 which was discussed in section 3.1. Multiple copies o f such an unusually structured element might, for example, destabilise the viral
genome. This instability could then lead to the deletion o f either marker gene plus LAT P2 or the entire expression cassette, resulting in populations which were either blue only, green only or white. Consistent with this hypothesis, all these mutant populations were observed to exist. However, many results from ours and other laboratories have indicated that it is possible to generate less disabled viruses with multiple copies of the LAT region and that such viruses can be stably propagated (for example viruses 17+27- pR 20.1 and 20.2 described in section 3.6). Although it is possible that any selection pressure which may be associated with multiple copies o f LAT P2 is slight and therefore only evident in more disabled viruses, the most likely explanation for the observed instability is aberrant homologous recombination between inserted and endogenous LAT P2 regions. It would be surprising if the advantage conferred by having a smaller genome outweighed the growth impairment resulting from the loss of ICPO, but it appears that this may have been the case with the viruses described in this section. However, whether ICPO had in fact been deleted was not examined.
3.11 Attempted purification of a 1764 27- 4- virus without a LAT P2 containing