Use of differential display reverse transcription-PCR to reveal cellular changes during stimuli that result in herpes simplex virus type 1 reactivation from latency: upregulation of immediate-early cellular response genes TIS7, interferon, and interferon regulatory factor-1

Abstract

The detailed mechanism which governs the choice between herpes simplex virus (HSV) latency and reactivation remains to be elucidated. It is probable that altered expression of cellular factors in sensory neurons leads to induction of HSV gene expression resulting in reactivation. As an approach to identify novel cellular genes which are activated or repressed by stimuli that reactivate HSV from latency and hence may play a role in viral reactivation, RNA from explanted trigeminal ganglia (TG) was analyzed by differential display reverse transcription-PCR (DDRT-PCR). Nearly 50 cDNAs whose mRNA level was modified by the stress of explantation were isolated and sequenced. We present a listing of a spectrum of altered RNAs, including both known and unknown sequences. Five of those differentially displayed transcripts were identified as interferon-related murine TIS7 mRNA. These results were confirmed in both infected and uninfected ganglia by quantitative RNase protection assay and immunostaining. Alpha and beta interferons and interferon regulatory factor-1 (IRF-1) were also induced by explantation. In addition, we have identified sequences that correspond to IRF-1 consensus binding sites in both HSV type 1 origins of replication. Our findings suggest that physiological pathways that include these cellular factors may be involved in modulating HSV reactivation.

FIG. 1

PCR differential display of cDNA derived from latently infected mouse TG following explantation. In the autoradiograph of radiolabeled DDRT-PCR products, arrows denote PCR products representing band 64 amplified with 3′ primer 2 and 5′ primer 7, band 56 amplified with 3′ primer 2 and 5′ primer 3.

FIG. 2

Sequences of DDRT-PCR products. cDNAs isolated from differential display gels were reamplified by using primers that included the T7 promoter sequence, and PCR products were sequenced. Sequences were analyzed by BLAST searches. (A) BLAST output using band 56 as query sequence (P value = 7.7 × 10⁻⁴⁹) demonstrates sequence identity with murine TIS7. (B) Alignment of each DDRT band with TIS7 mRNA. Four cDNA bands corresponded to mouse IFN-related gene TIS7 mRNA; band 56 (primers 2 and 7), band 64 (primers 2 and 3), band 116 (primers 6 and 2), and band 125 (primers 6 and 7).

FIG. 3

Confirmation of differential display. RNA was prepared from uninfected TG explants at 0, 1, 2, and 4 h p.e. Complementary DNA from differentially displayed band 56 was reamplified by PCR using 3′ primers that included the T7 promoter. PCR products were used as templates to prepare riboprobes labeled with [³²P]UTP and added to each RNA sample. Following hybridization at 37°C and RNase digestion, samples were separated by PAGE. (A) The input probe (P) and protected fragments were visualized using phosphorimager screens. (B) The intensity of each protected fragment was quantitated with ImageQuant software. Fold induction was expressed as the ratio between each band to the zero time point. (C) Complementary DNA from latently infected TG explants at 0 to 24 h p.e. was subjected to PCR using primers specific for TIS7 (TIS7A set). Products were separated on 2.5% agarose gels stained with ethidium bromide and visualized by fluorimager analysis.

FIG. 4

Immunostaining of TIS7 in TG following explantation. Latently infected BALB/c mice were sacrificed, and TG were excised and incubated in culture medium for 0 to 24 h. Paraffin-embedded sections of uninfected TG at 0 (A), 1 (B), 2 (C), and 4 (D) h p.e. were processed as described in Materials and Methods and reacted with rabbit polyclonal antiserum against TIS7. The experiment was repeated twice, and duplicate slides were screened.

FIG. 5

Detection of IFN-β, IRF-1, IFNα/βR, and IRF-2 transcripts in murine TG following explantation. RT-PCR was used to detect IFN-β (A), IRF-1 (B), IFNα/βR (C), and IRF-2 (D), and each was compared to the level of cyclophilin mRNA. Duplicate samples of TG explant RNA from 0, 1, 2, and 4 h p.e. were analyzed. Products were separated by agarose gel electrophoresis, followed by fluorimager scanning and analysis using ImageQuant software. The relative amount of cDNA is expressed in arbitrary units representing the ratio between the intensity of the PCR product band to the intensity of cyclophilin. The ratio at the zero time point is designated 1. L indicates latent.

FIG. 6

Immunostaining of IFN protein in TG following explantation. Latently infected or uninfected BALB/c mice were sacrificed, and TG were excised and incubated in culture medium for 0 to 24 h. Paraffin-embedded sections of latently infected TG at 0 (A), 4 (B), 8 (C), and 24 (D) h p.e. and of uninfected TG 0 (E) and 4 (F) h p.e. were processed as described in Materials and Methods and reacted with rabbit polyclonal antisera against IFN-α and -β. The experiment was repeated twice, and duplicate slides were screened.

FIG. 7

Putative IRF-1 binding sites in the HSV-1 17+ genome, identified by sequence analysis using the Findpatterns function of the Genetics Computer Group (Madison, Wis.) software. The HSV-1 complete genome sequence (GenBank locus HE1CG accession no. X14112) was searched for the consensus IRF-1 binding site in IFN promoters the hexamer AAGTGA (56). Sequence matches are listed by sequence location, and gene name.

FIG. 8

RT-PCR detection of TNF-α, cyclophilin, and β-actin transcripts in murine TG cultured for various times p.e. RNA from latently infected TG explants was prepared and analyzed by RT-PCR for TNF-α (A), and β-actin (B), and cyclophilin as described in Materials and Methods. Products were visualized by ethidium bromide staining as shown in the inserts. The graphs represent the ratio between the PCR product band and the cyclophilin band. The ratio at the time of explant (time zero) was determined as 1. Experiments were done in duplicate in four separate experiments. L indicates latent.