HSV-1 Triggers an Antiviral Transcriptional Response during Viral Replication That Is Completely Abrogated in PKR-/- Cells

Abstract

The activation of the innate immune response during HSV-1 infection stimulates several transcription factors, such as NF-κB and IRF3, which are critical regulators of IFN-β expression. The released IFN-β activates the ISGs, which encode antiviral effectors such as the PKR. We found that HSV-1 triggers an antiviral transcriptional response during viral replication by activating TBK1-IRF3-NF-κB network kinetically. In contrast, we reported that infected PKR^-/- cells fail to activate the transcription of TBK1. Downstream, TBK1 was unable to activate the transcription of IRF3 and NF-κB. These data suggested that in PKR^-/- cells, HSV-1 replication counteracts TBK1-IRF3-NF-κB network. In this scenario, a combined approach of gene knockout and gene silencing was used to determine how the lack of PKR facilitates HSV-1 replication. We reported that in HEp-2-infected cells, PKR can influence the TBK1-IRF3-NF-κB network, consequently interfering with viral replication. Otherwise, an abrogated PKR-mediated signaling sustains the HSV-1 replication. Our result allows us to add additional information on the complex HSV-host interaction network by reinforcing the concept of the PKR role in the innate response-related networks during HSV replication in an in vitro model.

Keywords: HSV-1; PKR; innate antiviral response.

Figure 1

Abrogated activation of TBK1, IRF3 and NF-κB during HSV-1 replication in PKR^−/− cells. (a) Immunoblot analyses of lysates of PKR^−/− and wild-type HEp-2. (b–d) Quantitative Real-time PCR was performed to evaluate the transcriptional levels of TBK1 (b), IRF3 (c), and NF-κB (d) in HEp-2 and PKR^−/− cells. The cells were infected at MOI 10 with HSV-1 and harvested at 9 h and 24 h post-infection to total RNA extraction. The transcriptional levels of considered genes were analyzed by calculating the value of 2^−ΔΔCt. The assay was performed as a means of triplicate ± SD and expressed as fold change over the housekeeping genes. **** <0.0001; *** <0.001; * <0.01.

Figure 2

Analysis of viral DNA and representative viral transcripts in PKR^−/− compared to HEp-2 cell lines. PKR^−/− and parental cell lines were infected with HSV-1 at MOI 10 and collected 24 h post-infection. (a) The absolute quantification of Real-time PCR was used to detect the amount of viral DNA in both cell lines by using a specific TaqMan probe. Viral load was derived from the threshold cycle (CT) using the standard curve generated in parallel, and the result is expressed as a concentration in ng of DNA/μL. (b) Expression of viral transcripts cascades in HEp-2 and PKR^−/− cell lines. The mRNA was purified with Trizol according to the manufacturer’s instructions. The measure of changes in the expression level of ICPO, UL42, and US11 genes was analyzed by calculating the value of 2^−ΔΔCt. The assay was performed as a means of triplicate ± SD and expressed as fold change over the housekeeping genes. *** < p < 0.0001 and ** <0.01.

Figure 3

Validation of PKR silencing by western blotting and RT-qPCR. HEp-2 cells were transfected with three different siRNA duplexes at 5 and 10 µM to silencing PKR, and siRNA scrambled was used as negative control (10 µM). Samples were collected at 48 h post-transfection to measure the expression of PKR. (a) Western blot analysis was performed to detect the protein level of PKR following silencing. The GAPDH was used to check the protein loading. Panel (b) shows fold changes in protein quantification. Band density was determined with the Image J program, expressed as fold change over the appropriate housekeeping genes and with respect to siRNA scrambled control. The data were graphically represented by using GraphPad Prism 6 software. (c) Quantitative Real-time PCR was performed to evaluate the transcriptional levels of PKR in HEp-2 transfected with si-PKR#2 (10 µM) and siRNA scrambled. (d) The amount of PKR transcripts obtained following transfection with si-PKR#2 was confirmed by running the amplicons on an agarose gel. Statistical analyses were performed with one-way analysis of variance (ANOVA) in triplicate, and asterisks (** and ***) indicate the significance of p-values less than 0.01 and 0.001, respectively.

Figure 4

Accumulation of HSV-1 DNA and representative viral transcripts following PKR silencing. HEp-2 cells were transfected with si-PKR#2 (10 µM) and infected with HSV-1 (10 MOI). Scrambled siRNA was used as no-target control. The samples were collected 24 h post-infection (a) viral DNA, and (b) viral transcripts were analyzed. The changes levels of ICPO, UL42, and US11 genes were analyzed by calculating the value of 2^−ΔΔCt. The assay was performed as means of triplicate ± SD and expressed as fold change over the housekeeping genes. *** < p <0.0001 and ** <0.01.