Histone deacetylase inhibitors induce reactivation of herpes simplex virus type 1 in a latency-associated transcript-independent manner in neuronal cells

Abstract

Histone acetylation is implicated in the regulation of herpes simplex virus type 1 (HSV-1) latency. However, the role of histone acetylation in HSV-1 reactivation is less clear. In this study, the well-established model system, quiescently infected, neuronally differentiated PC12 (QIF-PC12) cells, was used to address the participation of histone acetylation in HSV-1 reactivation. In this model, sodium butyrate and trichostatin A (TSA), two histone deacetylase inhibitors, stimulated production of infectious HSV-1 progeny from a quiescent state. To identify viral genes responsive to TSA, the authors analyzed representative alpha, beta, and gamma viral genes using quantitative real-time polymerase chain reaction. Only the latency-associated transcript (LAT) accumulated in response to TSA treatment, under culture conditions that restricted virus replication and spread. This led the authors to evaluate the importance of LAT expression on TSA-induced reactivation. In QIF-PC12 cells, the LAT deletion mutant virus dLAT2903 reactivated equivalently with its wild-type parental strain (McKrae) after TSA treatment, as well as forskolin and heat stress treatment. Both viruses also reactivated equivalently from latently infected trigeminal ganglia explants from rabbits. In contrast, there was a marked reduction in the recovery of dLAT2903, as compared to wild-type virus, from the eyes of latently infected rabbits following epinephrine iontophoresis. These combined in vitro, ex vivo, and in vivo data suggest that LAT is not required for reactivation from latently infected neuronal cells per se, but may enhance processes that allow for the arrival of virus at, or close to, the site of original inoculation (i.e., recrudescence).

Fig. 1

Effect of sodium butyrate (NaB) and TSA on HSV-1 strain 17⁺ reactivation from QIF-PC12 cells. Cells, grown in duplicate 12-well plates per treatment group, were neuronally differentiated with nerve growth factor (NGF) then infected with HSV-1 strain 17⁺ at an MOI of 5 in the presence of acycloguanosine (ACV). Seven days after the withdrawal of ACV from the medium (day 17 postinfection), culture medium was changed to medium containing (A) NaB (solid bars), forskolin (striped bar), or no drug (clear bar), or (B) TSA at the indicated concentrations, as well as no treatment, control medium. Reactivation was monitored for 8 days using culture supernatants titered onto Vero cells. Histogram shows results of cumulative wells positive by day 8 post-induction. * P < 0.05 (mock vs. treatment with agent). For all treatment points, the standard deviations were < 10%. Similar results were obtained in duplicate NaB and TSA experiments.

Fig. 2

Location of primers used to assess LAT cDNA levels. (A) Top line represents the region spanning from nucleotide position 117,000 to 124,000 of the prototype viral genome. Locations of the α0 transcripts and 8.3-kb minor LAT and 2.0-kb major LAT are displayed. (B) Nucleotide positions of regions assessed by real-time PCR represent the flanking boundaries of PCR products. The expression of LATs was assessed at three locations. The minor 8.3-kb LAT was assessed at the region 5′ of the major LAT to evaluate changes in expression from the LAP1 promoter (Dobson et al, 1989; Zwaagstra et al, 1990), and also 3′ of the major 2.0-kb LAT to include changes occurring from the LAP2 promoter (Chen et al, 1995; Goins et al, 1994; Nicosia et al, 1993; Wechsler et al, 1988). The third site was within the 2.0-kb major LAT (119,721 to 117,792). Note that primers used to assess levels of α0 cDNA span the first intron of the α0 gene transcript. Transcription from LAP1 initiates at nucleotide 118,801.

Figure 3

HSV-1 strain 17⁺ reactivates after TSA induction, and expression pattern of viral genes following TSA-induced reactivation. (A) QIF-PC12 cells were established and maintained in 12-well plates and assayed for reactivation as described in Figure 1, as a positive control for experiment shown in B. Ten days after ACV was removed, cultures were induced with TSA (400 ng/ml), heat stress (HS, 43°C x 3 h, positive control), or mock medium on day 17 postinfection. (B) QIF-PC12 cells, maintained in medium containing ACV, in parallel 6-well plates, were harvested from duplicate wells and RNA isolated at the times indicated following mock or TSA treatment. RNA was reverse transcribed and the quantity of cDNA synthesized for selected host and viral transcripts was determined by real-time PCR. The average numbers of cDNA copies were determined from triplicate reactions and are presented relative to the levels detected 6 h after mock treatment. The 6 h mock treatment was set at 1 for each transcript. Sufficient amounts of cDNA were used in all PCRs such that all representatives of α, β, and γ class genes examined were readily detected. Signal was rarely detected in controls lacking Rtase (-Rtase). When detected in –Rtase controls, the level was substantially below the levels detected in experimental samples. * P < 0.05 (LAT vs. mock treatment). For all cDNA results, the mean standard deviations were < 17%.

Fig. 4

Reactivation of dLAT2903 and McKrae from QIF-PC12 cells is similar. Cells were grown in duplicate 12-well plates per treatment group, neuronally differentiated with NGF, and infected with the indicated strain at an MOI of 5. On day 17 postinfection, cultures were exposed to maintenance medium (mock), medium containing TSA (400 ng/ml) or forskolin (50 μM). Reactivation was monitored daily for 8 days using culture supernatants titered onto Vero cells. Data represents means from at least two independent experiments. Spontaneous reactivation from mock induced cultures was less than 12%. The mean standard deviations for each time point were less than 15%. Similar results were observed when TSA treatment was performed on day 31 postinfection.