Role of herpes simplex virus ICP0 in the transactivation of genes introduced by infection or transfection: a reappraisal

Abstract

ICP0, a promiscuous transactivator that enhances the expression of genes introduced by infection or transfection, functions in both nucleus and cytoplasm. The nuclear functions include degradation and dispersal of ND10 bodies and suppression of silencing of viral DNA. Subsequently, ICP0 shifts to the cytoplasm. Transfection of DNA prior to infection has no effect on the localization of ICP0 in cells that are efficient expressers of transgenes (e.g., Vero and HEK293) but results in delayed cytoplasmic localization of ICP0 in cells (e.g., HEp-2 and HEL) that are poor transgene expressers. Here, we examined by real-time PCR (qPCR) the accumulation of a transgene and of viral gI mRNAs in Vero or HEp-2 cells that were transfected and then infected with wild-type or DeltaICP0 mutant viruses. The accumulation of transgene mRNA was unaffected by a DeltaICP0 mutant, gradually increased in HEp-2 cells, but increased and then decreased in Vero cells infected with wild-type virus. In both cell lines, accumulation of gI mRNA increased with time and was less affected by the transfected DNA in Vero cells than in HEp-2 cells. The relative kinetics of mRNA accumulation reflected continued synthesis and degradation of the transgene and gI mRNAs. We conclude that the role of ICP0 is to render the DNA templates introduced by transfection or infection accessible by transcriptional factors, that the two cell lines differ with respect to the transcription-ready status of entering foreign DNA in the nucleus, and that ICP0 is not per se the recruiter of transcriptional factors to the accessible DNA templates.

FIG. 1.

Subcellular localization of ICP0 in DNA-transfected cells. Vero (A) or HEp-2 (B) cells were either mock transfected or transfected with an ampicillin-expressing plasmid, as detailed in Materials and Methods. At 18 h after transfection, the cells were exposed to HSV-1(F) at 10 PFU/cell. The cells were fixed at 6 h or 12 h after infection and stained with the rabbit polyclonal antibody to ICP0, as described in Materials and Methods. The images were captured with the same settings of a Zeiss confocal microscope. Approximately 250 cells from sequential nonoverlapping fields were counted to determine the percentage of infected cells with cytoplasmic ICP0. T, transfected; I, infected.

FIG. 2.

Transgene mRNA in transfected and/or infected HEp-2 or Vero cells. (A) Experimental protocol. (B and C) Results of 3 independent experiments with each cell line. The cells were infected 18 h after mock transfection or transfection with an ampicillin-expressing plasmid. The cells were harvested at the indicated times after infection with HSV-1(F) or ΔICP0 (10 PFU/cell). The ampicillin transcript was quantified as detailed in Materials and Methods. All the data were normalized to 18S rRNA. In each plot, the fold change of the respective transcript is expressed relative to the transcript at the earliest time point, either 1.5 h or 3 h after infection, indicated by the green arrows. T, transfected; Exp., experiment.

FIG. 3.

Viral-gene mRNA in transfected or transfected/infected Vero cells. In three independent experiments, Vero cells were either mock infected or infected 18 h after mock transfection or transfection with an ampicillin-expressing plasmid, as detailed in Materials and Methods. The cells were harvested at 1.5 h, 3 h, 6 h, or 12 h after exposure to HSV-1(F) or ΔICP0 (10 PFU/cell). Total RNA was extracted, reverse transcribed, and used as a template for the quantification of the gI transcript accumulating in cells infected with HSV-1(F) (A, C, and E) or the gI transcript accumulating in cells infected with ΔICP0 virus (B and D). All the data were normalized to 18S rRNA. In each plot, the fold change of the respective transcript is expressed relative to the transcript at the earliest time point, indicated by the green arrows. T, transfected.

FIG. 4.

Viral-gene mRNA in transfected or transfected/infected HEp-2 cells. In three independent experiments, HEp-2 cells were either mock infected or infected 18 h after mock transfection or transfection with an ampicillin-expressing plasmid, as detailed in Materials and Methods. The cells were harvested at 1.5 h, 3 h, 6 h, or 12 h after exposure to HSV-1(F) or ΔICP0 (10 PFU/cell). Total RNA was extracted, reverse transcribed, and used as a template for the quantification of the gI transcript accumulating in cells infected with HSV-1(F) (A, C, and E) or the gI transcript accumulating in cells infected with ΔICP0 virus (B and D). All the data were normalized to 18S RNA. In each plot, the fold change of the respective transcript is expressed relative to the transcript at the earliest time point, shown by the arrows. T, transfected.

FIG. 5.

Stabilities of transgene and viral-gene mRNAs in transfected or transfected/infected Vero cells. (A) Vero cells were either mock transfected or transfected with an ampicillin-expressing plasmid 18 h prior to mock infection or infection with HSV-1(F) at 10 PFU/cell. At 2.5 h after infection, the cells were either mock treated or treated with actinomycin D (Act) (10 μg/ml). The cells were harvested 30 min, 1 h, 2.5 h, or 5 h after the addition of actinomycin D. Total RNA was extracted, reverse transcribed, and used for quantification of the transgene mRNA (B) or the viral-gene gI mRNA (C). The data were normalized with respect to 18S rRNA and to the mRNA levels at 0.5 h after the addition of actinomycin D for each group. T, transfected; I, infected.