Transcriptional transactivation of HSV-1 IE gene expression by PrV helper virus.

<p>(A) Replication of PrV in TGEs. Groups of three TGEs were either infected in the AC with 5×10⁶ pfu of PrV-KaDgGgfp (grey bars) or in the GC with 10⁴ pfu of PrV-KaDgGgfp (black bars), and harvested at the time points indicated. DNA extracts were pooled, genome levels were determined by qPCR, and the increase in genome levels relative to 1 hpi was calculated. (B) Transcriptional transactivation of HSV-1 by PrV infection of the GC. TGEs were infected in the AC with 5×10⁶ pfu of HSV-1 CMV-IEproEGFP, and infected either simultaneously or at 7 dpi in the GC with 5×10⁶ pfu of PrV-KaDgGgfp. Replication of PrV was suppressed by the addition of 50 µg/ml ACV to the media. Cultures were harvested at 6 hpi after the addition of PrV to the GC, and relative ICP27 transcript levels were determined. Cultures not infected with helper virus served as controls. The statistical significances of differences in the transcript levels are indicated (simultaneous infection, <i>P</i><0.0001; addition of PrV at 7 dpi, <i>P</i><0.0001; unpaired <i>t</i> test with Welch correction). (C) Transcriptional transactivation of HSV-1 by co-infection of the GC with PrV in the presence of CHX. TGEs were infected in the AC with 5×10⁶ pfu of HSV-1 17 CMV-IEproEGFP in the presence of CHX and either co-infected in the GC with 5×10⁶ pfu of PrV-KaΔgGgfp or mock-treated. Cultures were harvested at 6 hpi and ICP27 transcript levels normalized to β-actin transcripts were determined. The statistical significances of differences in the mean relative transcript levels are indicated (<i>P</i> = 0.0186, unpaired <i>t</i> test with Welch correction). (D) Transcriptional transactivation of HSV-1 by co-infection of the AC with PrV. TGEs were infected in the AC with 2.5×10⁶ pfu of HSV-1 17 syn⁺ in the presence or absence of CHX and co-infected in the AC with 2.5×10⁶ pfu of PrV-KaΔgGgfp or mock-treated as indicated. Cultures were harvested at 6 hpi and ICP27 transcript levels normalized to β-actin transcripts were determined. The statistical significances of differences in the mean relative transcript levels are indicated (mock+CHX vs. PrV+CHX, <i>P</i>>0.999; mock+CHX vs. PrV−CHX, <i>P</i><0.0001; PrV+CHX vs. PrV−CHX, <i>P</i><0.0001; Mann-Whitney test), <i>ns</i> (not significant).