Differential activation of hybrid genes containing herpes simplex virus immediate-early or delayed-early promoters after superinfection of stable DNA-transfected cell lines

We compared the levels of gene expression obtained after herpes simplex virus (HSV) superinfection of cell lines containing integrated human beta-interferon (IFN) or chloramphenicol acetyltransferase (CAT) genes under the control of HSV immediate-early (IE) or delayed-early class promoters. DNA-transfected mouse Ltk+ cell lines harboring coselected IE175-IFN or thymidine kinase (TK)-IFN hybrid genes gave only low basal expression of human IFN. However, infection of both cell types with HSV type 1 or HSV type 2 produced abundant synthesis of IFN-specific RNA and biologically active IFN protein product. The IE175-IFN cell lines consistently gave 20- to 150-fold increases in IFN titers, and several TK-IFN cell lines yielded 100- to 500-fold induction. In the IE175-IFN cells, expression of IFN RNA also increased up to 200-fold and was detectable within 30 to 60 min after virus infection. Qualitatively similar results were obtained with hybrid G418-resistant Ltk- or Vero cell lines containing coselected IE175-CAT and TK-CAT constructs, except that there was relatively high basal expression of IE175-CAT. All three sets of IE cell lines (but not the delayed-early cell lines) responded to virus infection both in the presence of cycloheximide and with mutants defective in IE gene expression, demonstrating specific trans-activation by the pre-IE virion factor. In contrast, activation in the TK hybrid cell types required viral gene expression and the presence of a functional IE175 gene product. Up to 30-fold amplification in the copy number of the resident IFN or CAT DNA sequences also occurred within 20 h after HSV infection in IE175 hybrid cells but not in TK hybrid cells. Amplification was abolished either by treatment with phosphonacetate or by superinfection with a ts mutant unable to synthesize viral DNA, demonstrating specific HSV activation of the viral DNA replication origin (oriS) present in the IE hybrid constructs.     

Herpes simplex virus type 1 promoter activity during latency establishment, maintenance, and reactivation in primary dorsal root neurons in vitro

A neonatal rat dorsal root ganglion-derived neuronal culture system has been utilized to study herpes simplex virus (HSV) latency establishment, maintenance, and reactivation. We present our initial characterization of viral gene expression in neurons following infection with replication-defective HSV recombinants carrying beta-galactosidase and/or green fluorescent protein reporter genes under the control of lytic cycle- or latency-associated promoters. In this system lytic virus reporter promoter activity was detected in up to 58% of neurons 24 h after infection. Lytic cycle reporter promoters were shut down over time, and long-term survival of neurons harboring latent virus genomes was demonstrated. Latency-associated promoter-driven reporter gene expression was detected in neurons from early times postinfection and was stably maintained in up to 83% of neurons for at least 3 weeks. In latently infected cultures, silent lytic cycle promoters could be activated in up to 53% of neurons by nerve growth factor withdrawal or through inhibition of histone deacetylases by trichostatin A. We conclude that the use of recombinant viruses containing reporter genes, under the regulation of lytic and latency promoter control in neuronal cultures in which latency can be established and reactivation can be induced, is a potentially powerful system in which to study the molecular events that occur during HSV infection of neurons.     

The checkpoints of viral gene expression in productive and latent infection: the role of the HDAC/CoREST/LSD1/REST repressor complex

At the portal of entry into the body, herpes simplex viruses (HSV) vigorously multiply and spread until curtailed by the adaptive immune response. At the same time, HSV invades nerve ending-abutting infected cells and is transported in a retrograde manner to the neuronal nucleus, where it establishes a latent (silent) infection. At intervals, as a consequence of physical or metabolic stress, the virus is activated and transported in an anterograde manner to the body surface. The progression of infection is regulated at four checkpoints. In cell culture or at the portal of entry into the body, HSV uses components of the HDAC1- or HDAC2/CoREST/LSD1/REST repressor complex to activate α genes (checkpoint 1) and then uses an α protein, ICP0, to suppress the same repressor complex from silencing post-α gene expression (checkpoint 2). In neurons destined to harbor latent virus (checkpoint 3), HSV hijacks the same repressor complex to silence itself as a first step in the establishment of the latent state. Suppression of histone deacetylases (HDACs) plays a key role in the reactivation from latency (checkpoint 4). HSV has evolved a strategy of using the same host repressor complex to meet its diverse lifestyle needs.