Induction of apoptosis accelerates reactivation of latent HSV-1 in ganglionic organ cultures and replication in cell cultures

Abstract

Herpes simplex viruses replicate at the portal of entry into the body and are transported retrograde to sensory neurons in which they can establish a silent, latent infection characterized by the expression of a noncoding latency-associated transcript and a set of microRNAs. At the portal of entry into the body and in cell culture a viral protein, VP16, recruits cellular proteins that initiate a sequential derepression of several kinetic classes of viral genes. Earlier studies have shown that upon reactivation of latent virus in ganglionic organ cultures all genes are derepressed at once, thus obviating the need for VP16 to initiate sequential derepression of viral genes. One hypothesis that could explain the data is that the massive reactivation of all classes of viral genes is the consequence of activation of an apoptotic pathway. Here we show that two proapoptotic drugs, dexamethasone and 2[[3-(2,3-dichlorophenoxy)propyl]amino]-ethanol, each accelerates viral gene expression in ganglionic organ cultures. We also show that in cultured cells apoptosis induced by dexamethasone accelerates viral gene expression and accumulation of infectious virus. The results are surprising in light of the relatively large number of viral proteins that independently block apoptosis induced by viral gene products or exogenous agents. The results suggest that the virus may rely on apoptosis to exit from latency but that apoptosis may be detrimental for virus replication or spread at the portal of entry into the body.

Fig. 1.

Incubation of TG in medium containing NGF+EGF delays accumulation of viral mRNAs. TG excised from mice 30 d after corneal inoculation were incubated in medium 199V containing anti-NGF antibody, NGF, or NGF+EGF (300 ng/mL each). The TG were individually processed at times shown and as described in Materials and Methods. The results shown are the geometric mean per groups of six ganglia selected at random.

Fig. 2.

Dexamethasone accelerates the reactivation of latent HSV from TG maintained in medium containing NGF+EGF. TG were excised from mice 30 d after corneal inoculation and incubated in medium 199V containing anti-NGF antibody, NGF+EGF, or NGF+EGF and dexamethasone (50 or 10 μg/mL). At times shown individual ganglia were processed as described in Materials and Methods. The figure shows the geometric mean amounts of viral mRNAs or viral miRNAs and LATs normalized with respect to cellular RNA.

Fig. 3.

The highest concentration of DCPE tested accelerates the reactivation of latent HSV from TG maintained in medium containing NGF+EGF. TG were excised from mice 30 d after corneal inoculation and incubated in medium 199V containing anti-NGF antibody, NGF+EGF, or NGF+EGF and DCPE (50 or 100 nM). At times shown individual ganglia were processed as described in Materials and Methods. The figure shows the geometric mean amounts of viral mRNAs or viral miRNAs and LATs normalized with respect to cellular RNA.

Fig. 4.

Studies on viral DNA synthesis and induction of apoptosis in untreated and dexamethasone treated TG. (A and B) Quantification of viral DNA copies in TG. TG ganglia excised from 30 d after corneal-inoculated mice were incubated in medium 199v containing anti-NGF antibody (A) or medium containing NGF+EGF+dexamethasone (50 μM) (B). The number of viral DNA copies per 50 ng of DNA from TG was determined by qPCR and are shown as geometric mean titers based on assays of individual ganglia. (C and D) Induction of apoptosis in TG following incubation in medium containing dexamethasone. TG were harvested 16 h after excision and processed as described in Materials and Methods. (D) TG were incubated as above but were fixed, sectioned, and reacted with antibody to cleaved caspase 3 as detailed in Materials and Methods.

Fig. 5.

Dexamethasone accelerates the accumulation of viral mRNAs from cultured cells in vitro. (A) Replicate cultures of SK-N-SH and HEp-2 cells were exposed to medium with and without dexamethasone (50 μM). At times shown, they were exposed to 0.5 pfu of HSV-1(F)/cell and then harvested. Total RNA was extracted, reverse transcribed, and used as a template for the quantification of the transcripts accumulating in the cells. The amounts of viral mRNAs were normalized with respect 18S RNA, and the values were obtained at 1 h after infection. (B and C) The results obtained with infected SK-N-SH and HEp-2 cells, respectively.

Fig. 6.

The replication of wild-type HSV-1 in proapoptotic cells. SK-N-SH and HEp-2 cells were exposed to dexamethasone (50 μM) for 4 h before exposure to 0.5 pfu of HSV-1(F) per mL. The inoculum was replaced 1 h after exposure to virus. The cells were harvested 1, 3, 5, 8, and 24 h after infection. Viral progeny were titrated on Vero cells.