Transient Reversal of Episome Silencing Precedes VP16-Dependent Transcription during Reactivation of Latent HSV-1 in Neurons
Figure 5
Elevated Phase II viral transcript levels in neurons expressing human Oct-1.
(A) Schematic of Oct-1 showing the location of the POU DNA-binding domain near the middle of the protein and an alignment of the human, mouse and rat POU homeo (POUH) subdomain sequences. The four variable positions are located in helix-1 and helix-2 and are numbered according to their position within the POUH sequence. A rodent-like Oct-1 derivative (Oct-1E30D/M33L) was constructed by changing glutamic acid-30 and methionine-33 to the aspartic acid and leucine of the mouse/rat sequence. (B) SCG neuron cultures were infected with HSV-1 GFP-Us11, maintained for 5 days in the presence of ACV and then infected with lentiviral vectors encoding GFP or human Oct-1. After a further 5 days, ACV was removed and reactivation induced with 20 µM LY294002. RNA was collected at intervals and analyzed by qRT-PCR using human Oct-1 specific primers. For each time point, values for each transcript were compared to those from the GFP-expressing neurons (set to 1.0). (C) Relative levels of viral transcripts (ICP27, UL5, UL30, VP16 and UL36) after reactivation of HSV-1 GFP-Us11 in neurons expressing GFP, wild type human Oct-1 (WT) and human Oct-1E30D/M33L (MUT). (D) Wild type (WT) and E30D/M33L (MUT) versions of human Oct-1, VP16 (residues 5–412, VP16ΔC) and the β-propeller domain of human HCF-1 (residues 1–380, HCF-1N380) were synthesized by in vitro translation in the presence of 35S-methionine and visualized by 10% SDS-PAGE followed by autoradiography. (E) Assembly of the VP16-induced complex (VIC) by rodent-like (E30D/M33L) Oct-1 is greatly reduced compared to wild type human Oct-1. Recombinant Oct-1, VP16 and HCF-1 proteins were assayed for VIC formation by gel shift assay using a 32P-labeled probe containing an (OCTA+)TAATGARAT element from the HSV-1 ICP0 promoter [23]. The first three lanes are controls showing probe alone (lane 1), un-programmed rabbit reticulocyte lysate (lane 2), and a mix of lysates containing recombinant VP16 and HCF-1 (lane 3). The shift formed by the rabbit Oct-1 present in the lysate is greatly enhanced by the presence of either wild type or mutant human Oct-1 (lanes 4 and 10). A slower migrating complex (VIC) is formed by addition of VP16 and HCF-1 in the presence of wild type Oct-1 (lane 5) and only weakly by the mutant (lane 11). Reducing the amount of wild type Oct-1 by 5, 10, 50 and 100-fold respectively (lanes 6–9) reduces but does not eliminate this complex. No VIC is detected over a similar range of using mutant Oct-1.