4.4.1 Visualisation oiiacZ Expression
Figure 4.4.1 illustrates the activity o f P-galactosidase, the protein product o f lacZ m the B 130/2 IE2-expressing cell line infected with the 17+pR16R 70 recombinant HSV- 1 vector. The plaque assay was prepared by titration o f the viruses in serial dilutions of 1:10 on 6 well plates seeded with the B 130/2 cell line as described in Section 2.6.5.
After 48 hours o f incubation, 17+pR16R 70 infected wells were fixed and stained overnight with the chromogenic substrate X-gal to detect p-galactosidase activity as described in Section 2.6.6. P-galactosidase activity was similar in B 130/2 cells infected with the 17+pR16R 90 recombinant HSV-1 vector (data not shown). The recombinant virus was pure (no rephcation was noted on non-complementing cells), yet some viraUy infected cells within the plaque remain unstained. These are most likely cells infected with virus whose LAT P I promoters have undergone homologous recombination, and
‘knocked out’ the lacZ gene.
4.4.2 Characterisation of HSP70 and HSP90 Protein Expression
The 17+pR16 recombinant viruses (17+pR16R 70 and 17+pR16R 90) were each screened for hsp70 and hsp90 protein production. This was achieved by western blot analysis o f extracted protein fi’om infected plates o f B 130/2 cell lines, see Sections 2.5.3-2.5.7. The monoclonal anti-hsp90 antibody, clone AC88 (a kind gift fi*om Dr. David Toft) and the monoclonal anti-hsp70, clone C92F3A-5 (Stressgen) were used for immunodetection o f hsp90 and hsp70 protein, respectively. The X-ray films in Figure 4.4.2 illustrate that 17+pR16R 70 infected B 130/2 ceUs were overexpressing hsp70 against viral and mock infected controls, whereas there was no significant overexpression o f hsp90.
Figure 4.4.1 - Detection of B-Galactosidase Activity in B130/2 Cells Infected with the 17+pR16R 70 Recombinant HSV-1 Vector
Photomicrograph o f B 130/2 cells infected with the 17+pR16R 70 recombinant HSV-1 vector, stained for P-galactosidase activity at lOOx magnification. For details please refer to the text in Section 4.4.1.
Figure 4.4.2 - C haracterisation of H eat Shock Protein Expression in B130/2 Cells Infected w ith the 17+pR16R 70 and 17+pR16R 90 Recom binant HSV-1 Vectors
Western blots o f protein extracted from B 130/2 cells infected with 17+pR16R 70 and 17+pR16R 90 recombinant HSV-1 vectors probed with a) anti-human hsp70 monoclonal (clone C92F3A-5, Stressgen Biotechnologies Ltd, Canada) and b) anti- hsp90 monoclonal (clone AC88, a kind gift from Dr. David Toft). For details, please refer to Section 4.4.2. Numbers on the side indicate molecular weight in kiloDaltons. ECL exposure time was 1 minute for blot a) and 5 minutes for blot b). For both blots: Lane 1 : Rainbow Marker
Lane 2: 17-^R16R 90 infected B 130/2 cells Lane 3: Mock-infected control B 130/2 cells
Lane 4: ICP27-deleted control virus infected B 130/2 cells Lane 5: 17+pR16R 70 infected B 130/2 cells
Figure 4.4.1 * .
A
-220 - 9 7 -66 220- 9 7 - 66- - % Figure 4.4.2 a) 1 2 3 4 5 b) 1 2 3 4 54.4.3 Southern Blot Analysis of 17+pR16R 90 Infected 8130/2 Cells
The western blots demonstrated no signMcant change in hsp90 levels in JE2 coruqplementing B 130/2 cells after infection with 17+pR16R 90 virus. They did not demonstrate whether the hsp90 cDNA had been successfiilly delivered to the cells or whether or not the virus itself contained the hsp90 cDNA. The presence of the hsp90 cDNA in the 17+pR16R 90 virus was confirmed by hybridisation o f a [^^P] radiolabelled hsp90 cDNA random-primed probe to a Southern blot o f DNA extracted fiom 17+pR16R 90 infected B 130/2 cells. For methods, see Section 2.6.13-14. See Figure 4.4.3.
Figure 4.4.3 - Southern Blot of DNA Extracted from B 130/2 Cells Infected with the 17+PR16R 90 and 17+pR16R 70 Recombinant HSV-1 Vectors
Tlie Southern blot was probed with a [^^P] radiolabelled random-piimed cDNA probe generated fiom the full length hsp90 cDNA and exposed for 5 hours and overnight
after washhig. All DNA was digested with EcoKi.
Lane 1; Ikb ladder
Lane 2; 17+ wild-type DNA Lane 3; pR 16R 90 DNA Lane 4: pR16R 70 DNA
Lane 5; 17+pR16R 90 infected B 130/2 cells Lane 6: 17+pR16R 70 infected B 130/2 cells
Lane 7: lCP27-deleted control virus infected B 130/2 cells
Radioactive bands in lanes 3 and 5 correspond with the molecular weight o f the hsp90
EcoKl cDNA fiagments in the pR16R 90 plasmid (1.5kb and L8kb).
5 hours 1 2 3 4 5 6 7 Overnight 1 2 3 4 5 6 7
1 . 8 k b - A I . 8 k b - f
4.5 Discussion
These expression studies show that 17-HpR16R 70 is overexpressing hsp70 protein in B 130/2 cells but 17+pR16R 90 cDNA is not significantly overexpressing hsp90 protein detectable by western blot. The X-ray film exposure time required to detect the overexpression o f hsp 70 was as much as 5 minutes, and therefore the MoMLV-LTR promoter may drive downstream cDNA expression in the context o f virus only weakly. Hsp90 is expressed constitutively at a high level (approximately 1% o f total cell protein), and therefore the overexpression of hsp90 in cells infected with the 17+pR16R 90 virus may not be sufficient in relation to the endogenous gene to be detectable on western blot analysis.
Lokensgard et al., fiised the TATA-less LAT PI promoter with the MoMLV-LTR, and they proposed that elements in the LTR promoter activated latent expression in concert with the LAT P I promoter (Lokensgard et at., 1994). It was hoped in these recombinant vectors that the MoMLV-LTR would be in close enough proximity to the LAT P I promoter (~3.7kb) to confer this effect. This may not have been the case. The authors infected murine dorsal root gangha, whereas in this study BHK-derived cell lines were infected, and therefore expression may be different between neuronal or non-neuronal cell types. Dobson et a l, demonstrated expression of p-galactosidase during latency when the LTR-transgene construct was inserted into the ICP4 region approximately 2kb downstream o f the LAT PI promoter (the majority o f the LAT coding region had been deleted using ^ jffill) (Dobson et a l, 1990). The remaining 5’ fragment o f the LAT P2 promoter in that virus may exert some additional regulatory effect, rather than solely elements located in the LAT PI promoter. Therefore, the proximity o f the LAT P I promoter to the MoMLV-LTR may be crucial to enhance transcription during latency.
The four major problems with the 17+pR16R HSV-1 vectors were considered thus:
1) The lacZ expression under the control o f the LAT PI promoter was weak, being difficult to detect on B 130/2 IE2 com|)lementing cell lines, making purification
difficult. This may render detection of p-galactosidase activity above background in vivo, in non-con^lementing neurons problematic.
2) The recombination frequency between the LAT P I promoter regions in the expression cassette and the RL region of the HSV-1 genome will mean that some non-expressing mutant virus will be present after large scale viral culture, and therefore the resulting preparations will be inq)ure.
3) Purification o f subsequent recombinant viruses containing other transgene cDNAs would be equally as time-consuming as recombination was made into the wild-type HSV-1 genome, which exhibits a much greater advantage in rephcation.
4) The relatively weak overexpression o f the hsp cDNAs as driven by the MoMLV- LTR promoter in the context o f HSV-1 may not be sufficient to produce any physiological and/or protective effect on exposure of neuron-derived cell lines and primary neuronal cultures to various cell stresses.
It was therefore decided to design new recombinant viruses that wiU overexpress the hsp cDNAs at a higher level. The viruses should not undergo homologous recombination that will ‘knock out’ the expression region, and the expression cassette should be recombined into a mutant virus that wiU not exhibit such a growth advantage, making preparation less labour-intensive. These viruses were named the 17+pR19 viruses and their design, construction and characterisation is covered in the following chapter.