HSV carrying WT REST establishes latency but reactivates only if the synthesis of REST is suppressed

Abstract

HSVs transit from vigorous replication at the portal of entry into the body to a latent state in sensory neurons in which only noncoding (e.g., latency-associated transcript) and micro-RNAs are expressed. In productive infection, viral genes must be sequentially derepressed at two checkpoints. A leading role in the repression of viral genes is carried out by histone deacetylase (HDAC)/corepressor element-1 silencing transcription factor (CoREST)/lysinespecific demethylase1(LSD1)/RE1-silencing transcription factor (REST) repressor complex (HCLR). Previously, we reported that to define the role of the components of the HCLR complex in the establishment of latency, we constructed recombinant virus (R112) carrying a dominant-negative REST that bound response elements in DNA but could not recruit repressive proteins. This recombinant virus was unable to establish latency. In the current studies, we constructed a virus (R111) carrying WT REST with a WT genome. We report the following findings: (a) R111 readily established latent infection in trigeminal ganglia; however, although the amounts of viral DNAs in latently infected neurons were similar to those of WT virus, the levels of latency-associated transcript and micro-RNAs were 50- to 100-fold lower; (b) R111 did not spontaneously reactivate in ganglionic organ cultures; however, viral genes were expressed if the synthesis of REST was blocked by cycloheximide; and (c) histone deacetylase inhibitors reactivated the WT parent but not the R111 recombinant virus. The results suggest that REST plays a transient role in the establishment of latency but not in reactivation and suggest the existence of at least two phases at both establishment and reactivation.

Fig. 1.

DNA replication and viral gene expression in murine trigeminal ganglia after infection. On the indicated days after inoculation, mouse trigeminal ganglia were removed and extracted. (A) DNA copy numbers normalized with respect to 50 ng of cellular DNA were plotted over days postinoculation. Copies of ICP27 (B), TK (C), VP16 (D), and LAT (E) normalized to 50 ng of cellular RNA and mirH3 (F), mirH5 (G), and mirH6 (H) normalized to 10⁸ copies of cellular miRNA by let-7a were plotted over days postinoculation. The numbers shown hereafter are geometrical means ± SE based on assays of six trigeminal ganglia per group.

Fig. 2.

Viral gene, miRNA, and LAT expression during virus reactivation from latently infected ganglia induced by NGF depletion. On 30 d postinoculation, trigeminal ganglia were excised from WT parent or R111 virus-infected mice, incubated in 199V containing anti-NGF (1 μg/mL) for the indicated time (hours), and then extracted. Normalized copies of ICP27, TK, VP16, and U_L41 mRNAs and of LAT, mirH3, mirH5, and mirH6 were plotted as a function of time after excision.

Fig. 3.

Viral gene expression during virus reactivation from R111 latently infected ganglia incubated in anti-NGF and cycloheximide. On 30 d postinoculation, murine trigeminal ganglia were removed from R111 latently infected mice and incubated in 199V containing anti-NGF with cycloheximide (150 μg/mL) for 24 h. At 0 h (columns 1, 3, 5, and 7) or 24 h (columns 2, 4, 6, and 8), copies of ICP27, TK, VP16, and U_L41 mRNAs were quantified and normalized to 50 ng of cellular RNAs.

Fig. 4.

Expression of human REST in trigeminal ganglia of mice infected with the R111 recombinant virus. (A) Mouse trigeminal ganglia were excised at 1, 3, 7, 11, 14, and 30 d after corneal inoculation and then subjected to analysis of human REST mRNA expression. (B) On day 30, mice ganglia removed were extracted immediately or after 24 h of incubation in medium containing anti-NGF with or without cycloheximide (CHX; 150 μg/mL) and subjected to human REST mRNA expression assay. Copies of human REST detected by primers specific to the human REST gene were normalized to 50 ng of cellular RNA.

Fig. 5.

Viral gene expression in HSV-1(F) latently infected trigeminal ganglia incubated in medium containing dexamethasone (DEX) and cycloheximide (CHX). Trigeminal ganglia excised 30 d after inoculation were processed immediately after excision (blue) or after 24 h of incubation in medium containing NGF plus EGF with dexamethasone (50 μM, purple) or NGF plus EGF with both dexamethasone (50 μM, red) and cycloheximide (100 μg/mL, red). Copies of ICP27, TK, VP16, U_L41, LAT, mirH3, mirH5, and mirH6 were normalized to cellular RNAs.

Fig. 6.

Effect of the HDAC inhibitor TSA on reactivation of HSV-1(F). Trigeminal ganglia excised 30 d after inoculation of HSV-1(F) were processed immediately after excision (columns 1 and 5) or after 24 h of incubation in medium containing anti-NGF antibody (columns 2 and 6), NGF plus EGF (columns 3 and 7), or NGF plus EGF plus TSA (400 nM) (columns 4 and 8). The figure shows the geometrical mean amounts of viral mRNAs or viral miRNAs and LAT normalized with respect to cellular RNAs.

Fig. 7.

Effect of the HDAC inhibitor TSA on the reactivation of R111 recombinant virus. Trigeminal ganglia excised 30 d after inoculation of R111 were processed immediately after excision (blue) or after 24 h of incubation in medium containing anti-NGF antibody (red) or anti-NGF plus TSA (400 nM, green). The figure shows the geometrical mean amounts of viral mRNAs or viral miRNAs and LATs normalized with respect to cellular RNAs.

Fig. 8.

Model of temporal phases in the establishment and reactivation of latent virus. The model postulates phases both at the establishment of latency (Left) and at reactivation (Right), at which time the decision to commit to latency or commit to reactivation take place.