Real time PCR for monitoring regulation of host gene expression in herpes simplex virus type 1-infected human diploid cells

Abstract

Herpes simplex virus type 1 (HSV-1) induces prominent shifts in the rates of transcription of host cellular genes of relevance for the outcome of the viral infection. The quantitative analysis of transcription may be obscured by virus-induced alterations in the levels of RNA encoded by cellular housekeeping genes that are used commonly for normalisation of real time reverse transcription PCR (RT-qPCR). In the present study, we analysed β-actin, GAPDH and 18S rRNA for their usefulness in normalisation of RT-qPCR analysis of the transcription of the HSV-1 gamma gB-1 gene and FUT5, a cellular gene induced by viral infection. The transcription of these genes was monitored in a TaqMan-based real time RT-PCR system over a 24 h interval of virus infection of human embryonic lung fibroblasts. The levels of gB-1 and FUT5 RNA were normalised via difference in the threshold cycle (Δ C t) values relative to each and one of the housekeeping genes or calculated in relation to the number of infected cells without any further normalisation. The levels of RNA encoded by β-actin or GAPDH were found to vary by several orders of magnitude during HSV-1 infection, introducing large errors in the estimation of the gB-1 and FUT5 RNA levels. In contrast, the variation of C t values for 18S rRNA was less than one cycle during 24 h period of HSV-1 infection. The FUT5 and gB-1 RNA figures obtained by Δ C t normalisation relative 18S rRNA were identical to those calculated in relation to the number of infected cells. These data recommend 18S rRNA for normalisation in HSV-1-infected human cells but discourage the use of β-actin and GAPDH RNA for this purpose. By applying these procedures, it was shown that the transcription of FUT5 was increased by 50-fold 5–24 h after HSV-1 infection and 200-fold by the inhibition of viral DNA replication in HSV-infected cells.