Figure 3

Evaluation of reporter gene expression after infection with recombinant vaccinia virus (rVACV). (a) Reverse transcriptase PCR results from mock-infected cells (untreated), GLV-1h68- (68) or GLV-1h43 (43)-infected cells at 6, 12, 24, 48 and 72 hpi using lacZ-, green fluorescent protein (GFP)- and glyceraldehyde 3-phosphate dehydrogenase-specific primers. Infection with GLV-1h68 led to production of Ruc-GFP and lacZ-specific transcripts as early as 6 hpi, whereas in GLV-1h43-infected cells, no lacZ transcript could be detected. Both reporter gene transcripts were not detectable in mock-infected cells. (b) Western blot analysis using β-galactosidase (β-gal) and Ruc-GFP-specific antibodies, showing no expression of β-galactosidase in GLV-1h43-infected cells. In contrast, both proteins could be detected in GLV-h168-infected cells after 12 hpi. β-galactosidase expression increased up to 48 hpi. Equal protein loading was confirmed by detection of β-actin. (c, d) Determination of β-galactosidase activity in cell lysates (c) and supernatants (d) after infection with GLV-1h43, GLV-1h68 and mock infection. Enzyme activity in cell lysates increased up to 48 hpi with GLV-1h68, further supporting the western blot results. In supernatants, no active enzyme could be detected until 12 hpi, but then increased until 72 hpi. As expected, no active enzyme could be detected in mock- or GLV-1h43-infected cells or supernatants at all time periods. PBS, phosphate-buffered saline.