Comparison of expression strength of GFPSCP driven by the oriLyt system or by the virus

Considering the results that nearly all infected cells express entirely in NIH3T3:gfpscp-ori

rather than the fidelity of activation. As

viral spread completely when encoded as a second gene copy by the SVTgfpscp) [103], the expression strength of both systems w

were infected with wt-MCMV to determine background fluorescence or with the MCMV SVTgfpscp and NIH3T3:gfpscp

Fluorescence pictures were taken

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mCherry, encoded on the virus and GFPSCP, encoded by the host cell should be expressed at approximately the same time. Thus the infection marker mCherry and the

expression can be compared in the infected cells. Fluorescence microscopy revealed an extremely high coincidence of GFPSCP and mCherry fluorescence, whereby GFPSCP is found at different cell compartments depending on the time course of infection and mCherry being localized to the correlation of over 95 % could be detected, which might eventually be fluorescence signal of GFPSCP is weaker than the strong

silenced replicon plasmid is probably reliable in all infected cells residual spread is not due to individual cell fates.

: Fluorescence microscopy of NIH3T3:gfpscp-ori cl. 3 infected with

MCMV-was infected with MCMV-mCherry, expressing the fluorescence marker mCherry under the 0.1. Three days post infection fluorescence microscopy was performed. Infected herry fluorescence. Induction of GFPSCP was produced only in the infected cells, showing the typical ‘speckles’ pattern. A very high (> 95 %) correlation of GFP to mCherry fluorescence could be observed, speaking for the specificity as well as the efficiency of reactivation from the silenced

Comparison of expression strength of GFPSCP driven by the oriLyt system or by

that nearly all infected cells express GFPSCP, the failure

ori cl. 3 might be rather due to the expression strength of the protein rather than the fidelity of activation. As it was found before that the DN GFPSCP

when encoded as a second gene copy by the viral genome (MCMV , the expression strength of both systems was compared. To this end, NIH3T3

MCMV to determine background fluorescence or with the MCMV gfpscp-ori cl. 3 cells were infected with wt-MCMV at

luorescence pictures were taken 16 h p.i. (Figure 32).

should be expressed at Thus the infection marker mCherry and the DN GFPSCP Fluorescence microscopy revealed an extremely herry fluorescence, whereby GFPSCP is found at different cell compartments depending on the time course of infection and mCherry being localized to the could be detected, which might eventually be the strong mCherry signal.

in all infected cells and the

-mCherry

mCherry, expressing the fluorescence marker mCherry under the 0.1. Three days post infection fluorescence microscopy was performed. Infected s produced only in the infected cells, correlation of GFP to mCherry fluorescence could be observed, speaking for the specificity as well as the efficiency of reactivation from the silenced status.

Comparison of expression strength of GFPSCP driven by the oriLyt system or by

failure to block MCMV due to the expression strength of the protein DN GFPSCP could inhibit viral genome (MCMV-as compared. To this end, NIH3T3 MCMV to determine background fluorescence or with the

MCMV-MCMV at an MOI of 1.

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Figure 32: Comparison of GFPSCP expression from the inducible cell line and the inducible virus.

Fluorescence and brightfield microscopy was applied to evaluate the expression strength of the inducible gfpscp-ori cl. 3 cell line in comparison to the infection of NIH3T3 with MCMV-SVTgfpscp, which expresses the inhibitory protein under control of a Tet-ON CMV/SV40enhancer-Promoter. A) As control NIH3T3 were infected with wt-MCMV. B) gfpscp-ori cl.3 does not express GFPSCP in the uninfected state, but GFPSCP is induced upon infection. C) NIH3T3 were infected with an MCMV-SVTgfpscp. Without Doxycyclin (Dox) weak background expression of GFPSCP can be seen, indicating that the system is not absolutely tight. Under induction with Dox, a strong expression of GFPSCP that exceeds the level obtained from the NIH3T3: gfpscp-ori cell line can be seen. All infections were performed with an MOI of 1, fluorescence pictures were taken 16 h p.i..

While a considerable expression of GFPSCP could be already detected from the cells infected with MCMV-SVTgfpscp in absence of doxycyclin needed for gene induction, indicating leaky control of the gene cassette, no expression of GFPSCP was found in uninfected gfpscp-ori cl. 3. However, doxycyclin induction of the viral encoded expression of gfpscp produced a much stronger signal than the viral induced gfpscp-ori cl. 3 cells. Similarly to the data obtained with the luc-ori and MCMV-luc comparison (see Figure 25), the cell line cannot cope with the expression strength of the virus at early time points. The expression strength increases during replication in

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the oriLyt cells, so does expression of the viral-encoded gene. The GFPSCP DN was selected for proof-of-principle of the replicon vector, yet it may not be the optimal DN for intracellular immunization, which is reflected by the fact that the basal leaky expression of GFPSCP in the viral context was not even inhibitory for virus spread [103]. A protein target with a lower constitutive abundance than the wt MCMV SCP protein, close to 900 copy numbers per virion should be a better target for competitive inhibition [16].

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4.3 Trans- complementation of MCMV late viral proteins by the replicon