MATERIAS PRIMAS.

Since the ICP27 promoter is unable to drive expression o f ICP4 appropriate for efficient virus growth, further cell lines were generated with the ICP4 coding sequence under the control o f either its own promoter and polyadenylation signal or the mouse mammary tumour virus (MMTV) promoter and SV40 polyadenylation signal.

The MMTV promoter (pMAMneo, Invitrogen) is inducible by IpM

dexamethasone (Lee et a l, 1981). The rationale behind the construction of these cell

lines was that either ICP4 expression would be appropriately regulated by its own promoter or alternatively, expression of ICP4 from the MMTV promoter could be artificially induced by addition o f dexamethasone prior to or at the time o f infection. It is known that EHVl gene 12 can potently transactivate the HSVl ICP4 promoter

(Purewal et a l, 1994), and that the effect is more pronounced on this promoter than on

the ICP27 promoter (Thomas et a l, 1999a). It was therefore a distinct possibility that

the low levels o f EHVl gene 12 constitutively expressed by the 27/12 cell line would continually transactivate the ICP4 promoter, thereby generating unacceptably high background levels of the toxic ICP4 protein. The second cell line, with the inducible MMTV promoter controlling the expression of ICP4 was therefore designed in an attempt to avoid this potential problem.

The plasmids used to make these cell lines were named p4/4/4/zeo and pMAMzeo/4 respectively. Plasmid p4/4/4/zeo was constructed by cloning the zeocin

encoding BamYil fragment from pVgRXR (Invitrogen) into the unique Bglll site in

plasmid p4/4/4 (see section 4.7). The structure o f plasmid p4/4/4zeo is shown in figure 4.8.1 A. The plasmid pMAMzeo/ICP4 was based on the plasmid pMAMneo (Clontech).

The neomycin resistance gene from pMAMneo was removed as a BamHI fragment and

replaced with the zeocin encoding BamHi fragment from pVgRXR (Invitrogen). This

plasmid was named pMAMzeo. The ICP4 coding region (HSVl nt 127167 to 131187,

MseHBstEll) was then excised from p4/4/4 (see section 4.7) and inserted into the unique

Xhol site in pMAMzeo, creating pMAMzeo/ICP4. The structure of pMAMzeo/ICP4 is

and separately transfected into the previously described 27/12 cells (see section 4.2). Zeocin and neomycin resistant colonies were picked and cloned out and cell lines generated as described in section 2.3.7. In the case of the ICP4 promoter driving expression o f ICP4, 138 colonies were picked and screened for their ability to grow viruses deficient in VP 16, ICP27 and ICP4. The best o f these was named 27/12/4:4 and selected for further investigation. In the case o f the MMTV promoter driving expression of ICP4, 88 colonies were picked and screened for their ability to grow viruses deficient in VP 16, ICP27 and ICP4. The best o f these was named 27/12/M:4 and selected for further investigation. When these colonies were undergoing screening, it became apparent that there were marked differences in the proportion of clones which were able to support growth o f a virus deficient in VP16, ICP27 and ICP4. When ICP4 was controlled by its own promoter, the vast majority o f clones were o f only limited permissivity for the disabled virus, with only two facilitating significant productive virus growth (as assayed by the presence o f significant CPE within 48 hours of infection). However, when the MMTV promoter was driving the expression o f ICP4, more than half the colonies picked enabled relatively efficient propagation o f the disabled virus. This observation is summarised in table XI.

Promoter driving ICP4 expression Number of clones picked Number of clones able to complement ICP4 % of clones able to complement ICP4 ICP4 138 2 1.4 MMTV 88 60 68

Table XI: The effect of prom oter choice on the percentage of clones capable of complementing ICP4 deficiencies in a virus lacking functional VP16, ICP27 and ICP4

These results could be related to the integration site o f the plasmid containing ICP4 under the control of its own promoter in the cellular genome. It is possible that in the case o f the 27/12/4:4 cell line, the ICP4 plasmid may have been preferentially inserted

into a site which either does not allow ICP4 to be expressed or in which ICP4 is constitutively expressed. Western blotting for ICP4 levels from this cell line (results presented in figure 4.9.1) suggested that the latter explanation was more likely. Given

the known responsiveness of the ICP4 promoter to EHVl gene 12 (Purewal et a i,

1994), such positional effects are likely to be important in the context of a EHVl gene 12 containing genome. It could be, for example, that in the large percentage o f clones which are unable to support growth o f the disabled virus, integration has occurred at sites which expose the ICP4 promoter to constitutive activation by the low levels Of EHVl gene 12 continually expressed from the cell line. High levels o f toxic ICP4 would occur, selective pressure then causing these clones to lose or prevent expression from the ICP4 containing plasmid. Such positional effects seem not to be important in the case o f the MMTV promoter which appears to provide appropriate regulation of ICP4 regardless o f the insertion site.

Figure 4.8.2 shows growth curves for cell lines 27/12/4:4 and 27/12/M:4. It can be seen that both cell lines support growth o f a virus deficient in VP 16, ICP27 and ICP4 (1764 27- 4-) to nearly equivalent titres as a virus deficient in only VP 16 and ICP27 (1764 27-). Figure 4.8.2 also shows that the effect of 3mM HMBA on viral growth is now negligible, demonstrating the effectiveness o f EHVl gene 12 in complementing the function o f HSVl VP 16 in this system.

A) p4/4/4zeo 55/EII

H SV l nt 131187

Xho\ Hindm PvitW Sphl

ICP4 promoter ICP4 ICP4 pA SV40 prom EM7 zeocin SV40 pA

f^sel EcoKW

H SV l nt 127167

B) pMAMzeoICP4

MMTV LTR SV40 pA SV40 promoter EM-7 zeocin SV40 pA

Barnm Barnm

.MCS.

Nhel Xcyl Sail ~JhcXhol Smal

ICP4 coding region

Msel

(H SV l nt 127167)

AVEll (H SV l nt 131187)

Figure 4.8.1 Maps of plasmids p4/4/4zeo and pMAMzeoICP4

A) Plasmid p4/4/4 was constructed from p4/4/4 (see section 4.7) by inserting a zeocin

resistance cassette into the unique Bglll site derived from the pSP72 polylinker. B)

Plasmid pMAMzeoICP4 was generated from pMAMneo by replacing the neomycin resistance cassette with one encoding zeocin resistance and inserting the ICP4 coding

sequence into the unique Xhol site. Details of the cloning strategies can be found in

section 4.8. Sites indicated in red have been destroyed in the cloning and are indicated for orientation purposes only.

A) 27/12/4:4 (ICP4 under ICP4 promoter) ,1 7 6 4 2 7 - H M B A + 1000000 n ' ..1 7 6 4 2 7 - 4 - H M B A + 100000 ■ 1 7 6 4 2 7 - 4 - H M B A -

I

Time post infection (hours)

B) 27/12/M;4 (ICP4 under MMTV promoter)

1000000 1 1 7 6 4 2 7 - H M B A + ,....1 7 6 4 2 7 - 4 - H M B A + 100000 - 1 7 6 4 2 7 - 4 - H M B A - ^ 10000 ■ Ol, 1000 ■ 100 - 10 - 0 4 8 16 2 4 3 6

Time post infection (hours)

Figure 4.8.2 The expression of ICP4 from either the MMTV promoter or the ICP4 promoter allows effective growth of viruses deficient in VP16, ICP27 and ICP4

Growth curves o f viruses 1764 27- and 1764 27- 4- on cell lines with either A) the ICP4 promoter (cell line 27/12/4:4) or B) the MMTV promoter (cell line 27/12/M:4) are shown. All growth curves were carried out in duplicate at a MOI o f 0.01 in 24-well plates. Yields are given as total values in pfu/well (500pl).

4.9 The MMTV promoter and the ICP4 promoter are induced by viral