Herpes simplex virus 1 immediate-early and early gene expression during reactivation from latency under conditions that prevent infectious virus production

Abstract

The program of gene expression exhibited by herpes simplex virus during productive infection of cultured cells is well established; however, less is known about the regulatory controls governing reactivation from latency in neurons. One difficulty in examining gene regulation during reactivation lies in distinguishing between events occurring in initial reactivating cells versus events occurring in secondarily infected cells. Thus, two inhibitors were employed to block production of infectious virus: acyclovir, which inhibits viral DNA synthesis, and WAY-150138, which permits viral DNA synthesis but inhibits viral DNA encapsidation. Latently infected murine ganglia were explanted in the presence of either inhibitor, and then amounts of RNA, DNA, or infectious virus were quantified. In ganglia explanted for 48 h, the levels of five immediate-early and early RNAs did not exhibit meaningful differences between acyclovir and WAY-150138 treatments when analyzed by in situ hybridization or quantitative reverse transcription-PCR. However, comparative increases in viral DNA and RNA content in untreated ganglia suggested that virus was produced before 48 h postexplant. This was confirmed by the detection of infectious virus as early as 14 h postexplant. Together, these results suggest that high levels of viral gene expression at 48 h postexplant are due largely to the production of infectious virus and subsequent spread through the tissue. These results lead to a reinterpretation of previous results indicating a role for DNA replication in immediate-early and early viral gene expression; however, it remains possible that viral gene expression is regulated differently in neurons than in cultured cells.

FIG. 1.

Plaque reduction assay with WAY-150138. Patton wild-type (wt); 138^R/5 mutant, which is resistant to WAY-150138; and KOS wt viruses were assayed for plaque formation in the presence of WAY-150138. Average plaque numbers from triplicate wells were calculated as percentages of plaques formed relative to DMSO vehicle treatment.

FIG. 2.

Effects of WAY-150138 and ACV on viral DNA replication. (A) Cells were mock infected (first row) or infected (all other rows) with wild-type virus (left) or 138^R/5 virus (right) in triplicate and incubated for 3 h (second row) or 15 h (all other rows) in media containing DMSO alone, ACV (plus DMSO), or WAY-150138 (plus DMSO), as indicated at the far left. Lysates were applied to a dot blot and hybridized with a probe specific for viral DNA sequences. The mean percentage of hybridization, representing the amount of viral DNA synthesized under each treatment relative to the DMSO-treated control, is labeled to the right of each blot. (B) Amounts of hybridization were quantified using 1:3 serial dilutions of triplicate, untreated, infected cell lysates. For wild-type and 138^R/5 infections, the lowest detectable serial dilutions represented 5% and 4% of the DMSO-treated controls, respectively. h.p.i., hours postinfection.

FIG. 3.

Viral genome copies per TG during explant reactivation. TG from mice infected with 2 × 10⁶ PFU wild-type Patton or 138^R/5 virus were harvested and either frozen directly (latent; 0 h) or incubated for 48 h with 100 μM ACV, 10 μg/ml WAY-150138, or no drug. Viral DNA content was measured by quantitative, real-time PCR and normalized to mouse DNA content. Means ± standard deviations are plotted. For wild type, n = 6 for 0 h, and n = 9 for 48 h; for 138^R/5, n = 3. Mock-infected TG were negative for viral DNA. *, P = 0.0104 by two-sided Student's t test. The nine WAY-150138-treated, wild-type-infected ganglia comprised seven ganglia with viral DNA levels above those present in any of the ACV-treated, wild-type-infected ganglia, and two ganglia whose lower viral DNA contents resulted in relatively large error bars for this group.

FIG. 4.

Quantitative ICP8 RT-PCR. (A) Southern blot of polyacrylamide gel containing ICP8 RT-PCR products. (Left) TG samples (either infected or mock infected, as indicated). (Right) RNA standards representing the indicated number of copies per 90% of a TG. RT indicates whether reverse transcriptase was added (+) to one-half of the cDNA synthesis reaction or omitted (−). The DNA size marker is shown in the center, and the sizes of certain DNA markers are labeled in bp. ntc, no template control for PCR. (B) Portions of dot blots with RT-PCR products from TG samples (either infected or mock infected, as indicated in panel a) and standards. Panels b to e contain replicate sets of RNA standards representing the indicated number of RNA copies per 90% of a TG. RT indicates whether reverse transcriptase was added (+) to one-half of the cDNA synthesis reaction or omitted (−). (C) Standard curve of ICP8 RT-PCR products. Data are derived from panel B in the following manner: filled and open triangles/solid lines represent data from the same cDNA series subjected to separate PCRs and applied to separate blots (panels c and e, respectively); circles/dashed line (panel b) and squares/dotted line (panel d) represent two further, independent cDNA series, each loaded on one of the two blots described above. This standard range encompassed all experimental samples. PIU, phosphorimager units.

FIG. 5.

Viral RNA copies per TG in latent and reactivating TG. TG from mice infected with 2 × 10⁶ PFU wild-type Patton (A) or 138^R/5 (B) were harvested and either frozen directly (latent) or incubated for 48 h with 100 μM ACV, 10 μg/ml WAY-150138, or no drug. Indicated viral RNA species were measured by quantitative RT-PCR. Undetectable samples were calculated as zero. Means ± standard errors of the means are plotted. (A) n = 6 for latent, and n = 11 for others; (B) n = 3. Mock-infected TG were negative for viral RNA. ‡, representation of minimum values, because one or more TG contained RNA levels greater than the linear range of the assay.

FIG. 6.

Infectious virus production during explant reactivation. TG latently infected with wild-type KOS (A) or Patton (B) virus (inoculated with 2 × 10⁶ and 2 × 10⁵ PFU per eye, respectively) were harvested and incubated for the indicated times before being washed, frozen, and processed for infectious virus assay. Circles represent only TG which were positive for infectious virus, but bars represent mean values which include zeros for negative TG. Data are from two experiments for each viral strain.