ICP0 is not required for efficient stress-induced reactivation of herpes simplex virus type 1 from cultured quiescently infected neuronal cells

Abstract

Viral genes sufficient and required for herpes simplex virus type 1 (HSV-1) reactivation were identified using neuronally differentiated PC12 cells (ND-PC12 cells) in which quiescent infections with wild-type and recombinant strains were established. In this model, the expression of ICP0, VP16, and ICP4 from adenovirus vectors was sufficient to reactivate strains 17+ and KOS. The transactivators induced similar levels of reactivation with KOS; however, 17+ responded more efficiently to ICP0. To identify viral transactivators required for reactivation, we examined quiescently infected PC12 cell cultures (QIF-PC12 cell cultures) established with HSV-1 deletion mutants R7910 (deltaICP0), KD6 (deltaICP4), and in1814, a virus containing an insertion mutation in VP16. Although growth of these mutant viruses was impaired in ND-PC12 cells, R7910 and in1814 reactivated at levels equivalent to or better than their respective parental controls following stress (i.e., heat or forskolin) treatment. After treatment with trichostatin A, in1814 and 17+ reactivated efficiently, whereas the F strain and R7910 reactivated inefficiently. In contrast, KD6 failed to reactivate. In experiments with the recombinant KM100, which contains the in1814 mutation in VP16 and the n212 mutation in ICP0, spontaneous and stress-induced reactivation was observed. However, two strains, V422 and KM110, which lack the acidic activation domain of VP16, did not reactivate above low spontaneous levels after stress. These results demonstrate that in QIF-PC12 cells ICP0 is not required for efficient reactivation of HSV-1, the acidic activation domain of VP16 is essential for stress-induced HSV-1 reactivation, and HSV-1 reactivation is modulated uniquely by different treatment constraints and phenotypes.

FIG. 1.

Outline of QIF-PC12 cell culture model. PC12 cells are neuronally differentiated in medium containing NGF throughout the experiment. ACV is added to the culture medium on day 6, cells are inoculated with HSV-1 the following day, and ACV is maintained in the culture medium through day 16 postplating. Thereafter, HSV-1 remains in a quiescent state in the absence of ACV, until stress treatment on day 17 after infection or spontaneous reactivation occurs.

FIG. 2.

trans expression of ICP0, ICP4, and VP16 from adenovirus vectors reactivates HSV-1 from QIF-PC12 cells. Quiescent infections were established in ND-PC12 cells with strains 17⁺ (A) and KOS (B) at an MOI of 5. On day 17 after infection, cultures free of detectable infectious virus were superinfected with the indicated adenoviruses (MOI of 40) and Ad.C-rtTA (MOI of 10) in the presence of 10 μM DOX. Reactivation was determined from plaque assays of culture supernatants performed on Vero cells. Reactivation frequencies are based on two independent experiments (n = 24 cultures per group per experiment). Standard deviations for all points were less than 15%. The dashed line indicates the level of spontaneous reactivation for 17⁺-established cultures mock treated with maintenance medium. In KOS-established quiescently infected cultures, spontaneous reactivation was not observed.

FIG. 3.

Reactivation kinetics of HSV-1 recombinants lacking ICP0 or ICP4 in QIF-PC12 cells. Quiescent infections were established with indicated strains in 12-well plates. On day 17 after infection, cultures free of detectable infectious virus were subjected to reactivation stimuli. Reactivation was monitored for 8 days by plaque assay of culture supernatants performed on U2OS cells and E5 cells. Results are plotted based on three independent sets of infections. Standard deviations for all points were less than 15%. The levels of spontaneous reactivation for R7910- and F-established cultures mock treated with maintenance medium were equivalent and are indicated by the horizontal dashed lines. Spontaneous reactivation was not detected in KOS- or KD6-established quiescently infected cultures.

FIG. 4.

Reactivation kinetics of in1814 and 17⁺ in QIF-PC12 cells. Quiescent infections were established with indicated strains in 12-well plates. Cultures were induced to reactivate with indicated stressors (n = 24 per treatment group) and monitored on U2OS cells, as described in the legend for Fig. 2. The horizontal dashed lines indicate the levels of spontaneous reactivation for in1814-established cultures mock treated with maintenance medium. The level of spontaneous reactivation was 3.4% for mock-treated 17⁺-established quiescently infected cultures. Standard deviations for all points were less than or equal to 15%. Similar results were seen with duplicate experiments.

FIG. 5.

Reactivation kinetics of KM100 in QIF-PC12 cells. Quiescent infections were established with the indicated viruses in 12-well plates. Cultures were induced to reactivate with indicated stressors (n = 12 per group) and monitored for reactivation on U2OS cells, as described in the legend for Fig. 2. The horizontal dashed lines indicate the levels of spontaneous reactivation for KM100-established cultures mock treated with maintenance medium. The level of spontaneous reactivation was 0% for mock-treated KOS-established cultures and 14.4% for 17⁺-established quiescently infected cultures. Standard deviations for all points were less than or equal to 15%. Similar results were seen with duplicate experiments.

FIG. 6.

Reactivation kinetics of V422 in QIF-PC12 cells. Quiescent infections were established with V422 in 12-well plates. Cultures were induced to reactivate with indicated stressors (n = 24 per group) and monitored for reactivation by plaque assay using culture supernatants on U2OS cells as described in the legend for Fig. 2. Standard deviations for all points were less than or equal to 10%. The parental control (17⁺) reactivated similarly to results shown in Fig. 4. Results obtained from duplicate experiments were similar.

FIG. 7.

Growth of HSV-1 recombinants in ND-PC12 cells. PC12 cultures were neuronally differentiated for 7 days and infected at an MOI of 5. At the indicated times, duplicate cultures were harvested, frozen and thawed three times, and viral yields were measured in duplicate by plaque assay on U2OS and E5 cells. Standard deviations for all points were less than or equal to 15%. Similar findings were observed with a duplicate experiment. hrs, hours.