HSV-1-inducible proteins bind to NF-kappa B-like sites in the HSV-1 genome.

Several putative NF-kappa B-binding sites in the ICP0 and Vmw65 herpes simplex virus type-1 (HSV-1) genes have been identified. Oligonucleotides encoding some of these sites bind specifically to purified NF-kappa B protein and an NF-kappa B-like protein in nuclear extracts of phorbol ester- or cycloheximide-induced human embryonic lung (HEL) cells. HSV-1 infection of HEL cells induced a nuclear factor that binds specifically to kappa B sites in the ICP0 and Vmw65 gene regions and comigrates with complexes formed by purified NF-kappa B. The HSV-1-inducible nuclear factor bound to the authentic immunoglobulin (Ig) kappa B site. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing two copies of the Ig kappa B site upstream of the c-fos promoter (kappa B2-CAT) showed activity in HEL cells. HSV-1 infection of kappa B2-CAT-transfected HEL cells, however, induced a dramatic increase in CAT activity; mutation in the NF-kappa B-binding site of kappa B2-CAT abolished the inducibility of CAT gene expression. Our results demonstrate that the HSV-1 ICP0 and Vmw65 gene regions contain binding sites for NF-kappa B, and that HSV-1-inducible proteins bind to NF-kappa B-like sites in the HSV-1 genome.

Detection of herpes simplex virus thymidine kinase and latency-associated transcript gene sequences in human herpetic corneas by polymerase chain reaction amplification.

Herpes simplex virus (HSV) latency in sensory ganglion neurons is well documented, but the existence of extraneuronal corneal latency is less well defined. To investigate the possibility of extraneuronal latency during ocular HSV infection, corneal specimens from 18 patients with quiescent herpes simplex keratitis (HSK) were obtained at the time of keratoplasty. Polymerase chain reaction (PCR) amplification followed by southern blot hybridization with a radiolabeled oligonucleotide probe was done to detect the presence of HSV-1 genome in these human corneal samples. Two pairs of oligonucleotides from the region of the HSV thymidine kinase (TK) gene and the latency-associated transcript (LAT) gene were used as primers in the PCR amplification. The DNA sequences from either the TK or the LAT gene were identified in 15 of 18 HSK corneas (83%). These results demonstrate that the HSV genome was retained, at least in part, in human corneas during quiescent HSV infection, giving further support to the concept of corneal extraneuronal latency.

Extraneuronal herpetic latency: animal and human corneal studies.

HSV DNA has been previously detected by both in situ and dot blot hybridization in neuronal tissues latently infected with herpes simplex virus (HSV), but not in extraneuronal tissues. The present study, using dot blot hybridization with a cloned full-length HSV DNA probe and subtractive hybridization assays for detecting HSV RNA, reveals both the presence and activity of the HSV genome in 100% of HSV latently infected rabbit corneas tested. Studies on human herpetic corneas taken at keratoplasty using slot blot hybridization with a cloned full-length HSV DNA probe demonstrated positive binding (hybridization) to the probe in 50% of samples tested but no binding to normal human control DNA. These studies confirm earlier, less sensitive virus recovery assays and implicate the cornea as an extraneuronal site of HSV latency and reactivation.

Molecular analysis of acyclovir-induced suppression of HSV-1 replication in rabbit corneal cells.

An in vitro HSV drug-suppression model has been established in rabbit corneal cell (SIRC) monolayers. Confluent SIRC monolayers were inoculated with McKrae strain HSV-1 (0.014 PFU/cell) and subsequently suppressed with acyclovir (ACV) (40 micrograms/ml) after adsorption for 1 hr at 37 degrees C. On days 0-5 postinoculation (PI), infectious HSV-1 was detected in approximately 50% of the SIRC cell cultures by standard supernatant and cell-free homogenate co-culture assays. On days 6-10 postsuppression, infectious HSV was detected in only 7-19% of the cultures. After removal of ACV suppression in the cultures (day 11 PI), HSV cytopathology developed to a 3-4+ level within 2-5 days. Dot blot hybridization of DNA extracted from the cultures indicated that the HSV genome was retained consistently in SIRC cells at a level of 0.0015-0.015 copies per cell during active ACV-suppression. During ACV-suppressed infection, approximately 0.9% of the SIRC cells contained the HSV genome as demonstrated by blot hybridization. After removal of ACV, a 1000-fold increase in HSV DNA concentration in SIRC cell cultures was detected.

Varicella-zoster virus infection of human sensory neurons.

Primary varicella-zoster virus (VZV) infection of humans may result in latent infection of sensory neurons in the peripheral nervous system. To examine the interaction of VZV with the sensory neuron we infected immunochemically defined human neurons with cell-associated VZV. Utilizing double-label immunofluorescence technology, a VZV-specific glycoprotein and a nonglycosylated phosphoprotein were detected in human fetus dorsal root ganglion (DRG) neurons, as defined by the presence of the neuron-specific enolase isoenzyme and the A2B5 ganglioside antigen, respectively. In addition to VZV antigen expression, progressive virus-induced cytopathic damage (neuronal enlargement and nuclear granulation of a fraction of the neuron population) was evident. As determined by transmission electron microscopy, VZV-infected human fetus DRG neurons contained empty and complete nucleocapsids with numerous pleomorphic virus particles in the cytoplasm, often in association with vacuoles. Although virus-specific antigen expression, particle synthesis, and cytopathic effects were observed in the human neuron population, neurons were less susceptible to VZV-induced cytopathic damage than supporting nonneuronal cells, suggesting neuronal modulation of VZV infection in vitro. This system provides the first model to examine the neuron- and virus-specific gene(s) and gene product(s) pertinent to the interaction of VZV with the human neuron.