Figure 2.

Confirmation of recombinant HSV-1 viruses. (A) 2 μg HSV-1 BAC DNA or 1 μg viral genome DNA was digested with HindIII and separated by 0.5% agarose gel. pYEbacPC8 and YE-PC8 showed three unique bands of 6.4 kbp, 2.2 kbp and 2.0 kbp, which resulted from the substitution of ICP6 with the cell cycle regulatory elements. There was also downward shift of the 8.7-kbp band in viral genome DNA compared with BAC DNA, due to the removal of BAC vector. (B) pYEbac102 BAC DNA (lane 1), pYEbacPC8 BAC DNA (lane 2), YE-102 viral genome DNA (lane 3) and YE-PC8 viral genome DNA (lane 4) were digested with HindIII. An ICP6-specific probe hybridised to the 9.4-kbp fragment in pYEbac102 and YE-102, with no hybridisation to pYEbacPC8 or YE-PC8. On the other hand, a luciferase-specific probe recognised the 2.0-kbp fragment in pYEbacPC8 and YE-PC8 but did not hybridise to any fragment in pYEbac102 or YE-102. A BAC vector probe hybridised to the 8.7-kbp fragment in pYEbac102 and pYEbacPC8, with no hybridisation to YE-102 or YE-PC8 viral genome DNA. (C) ΔGli36 cells cultured in standard medium were infected with YE-PC8 at an MOI of 2.0. The luciferase activities of YE-PC8 were measured at different time points after infection. (D) Parental ΔGli36 cells were incubated either in DMEM containing 10% FBS (denoted as control); DMEM containing 10% FBS and 150 μg ml⁻¹ PAA (denoted as PAA) or DMEM containing 0.5% FBS with 50 μM lovastatin to induce G₀/G₁ growth arrest (denoted as Lovastatin). After 40 h of incubation, the respective dishes were infected in triplicate with YE-PC8 for an hour at an MOI of 2.0. The cells were then replaced with specific tissue culture media as previously described, i.e., control, PAA or Lovastatin. At different time points, cells were collected for quantitation of relative copy-number of viral genome by normalising the copy-number of luciferase gene to that of β-actin in each sample or for (E) luciferase reporter activities measurement. (F) In vitro comparison of cell cycle-dependent gene regulation on YE-PC8 with matched control virus (YE-CMV-Luc) under proliferation and serum starvation conditions. ΔGli36 cells were infected with YE-PC8 or YE-CMV-Luc for an hour at MOI of 0.05. The cells were then replaced with either DMEM containing 10% FBS (denoted as control) or DMEM containing 0.1% FBS (denoted as serum starvation). After 48 h post infection, cells were collected and subjected to luciferase assay. All data were presented as mean±s.e.m., n=3.