Herpes simplex virus, predominantly HSV type 2, was the major agent causing benign recurrent lymphocytic meningitis that met our specified diagnostic criteria.

The herpes simplex virus immediate-early gene product ICP4 activates the transcription of viral early and late genes. We characterized the DNA sequence elements of the early glycoprotein D (gD) gene that play a role in the response to ICP4 in vitro. Using gel mobility shift assays and DNase I footprinting, we identified three ICP4-binding sites, two 5' to the mRNA start site and a third within the coding region. Site II, which gave a footprint between nucleotides-75 and-111 relative to the RNA start site, was previously identified by Faber and Wilcox and contained the reported consensus ICP4-binding site. Site III, which was located between nucleotides + 122 and +163, was very similar to the site II sequence, including a core consensus binding sequence, TCGTC. The site I sequence (nucleotides-308 to-282), however, did not share significant homology with either site II or site III. In vitro transcription experiments from mutant constructs of the gD promoter indicated that all three ICP4-binding sites contribute to the stimulation of transcription by ICP4. DNase I footprinting of the gD promoter with uninfected nuclear extracts of HeLa cells showed protection of two very G-rich sequences between nucleotides-33 and-75. We propose that optimal transcription of the gD gene depends on the interaction of ICP4 with multiple binding sites across the gene and cellular factors that recognize specific sequence elements in the promoter.

The herpes simplex virus type 1 regulatory protein ICP4 is a sequence specific DNA binding protein which associates with a number of different sites, some of which include the consensus ATCGTCnnnnYCGRC. In order to investigate the involvement in DNA binding of conserved bases within the consensus, we have synthesised a family of mutant oligonucleotides and tested their ability to form a complex with ICP4. We have also compared the binding specificities of bacterially expressed fragments of ICP4 which include the DNA binding domain. Mutation of most (but not all) bases in the proximal part of the consensus greatly reduced binding by ICP4, as did a mutation affecting the distal part. Most (but not all) G residues identified in methylation interference assays were required for efficient binding. While a bacterially expressed ICP4 peptide encompassing amino acid residues 252-523 bound to DNA with a specificity similar to that of the whole protein, a shorter protein (residues 275-523) had a slightly relaxed DNA binding specificity.

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On the basis of experiments with mutant virus and transfection with isolated genes, the herpes simplex virus immediate-early gene product ICP4 is known to positively regulate the transcription of viral early and late genes and negatively regulate expression from its own promoter. Binding of ICP4 to DNA sequences in several viral genes has been reported, yet the significance of ICP4-DNA interaction in transcriptional activation remains unclear. We have studied this problem by using the early glycoprotein D (gD) gene, which possesses a binding site at approximately-100 relative to the RNA initiation site. We linked this promoter and various mutant constructs to the chloramphenicol acetyltransferase gene in order to measure promoter activity in transient transfections both in the presence and in the absence of an ICP4-encoding plasmid. The natural promoter was activated 3.3-fold, and a deletion construct lacking the binding site was activated minimally (1.7-fold). Constructs containing multiple tandem repeats of the binding site (three or five inserts) demonstrated higher expression in the presence of ICP4 than did the natural promoter while retaining low levels of expression when unstimulated. Gel mobility shift assays and DNase I footprinting analyses indicated that ICP4 associated with multiple binding sites. In vitro transcription from a gD promoter construct containing multiple binding sites showed increased RNA synthesis in the presence of partially purified ICP4. These data provide the first direct evidence that binding of ICP4 to a specific DNA sequence in the gD gene contributes to activation of transcription.

A 47-year-old man had recurrent signs and symptoms of brainstem encephalitis over a 4-year period. Although CSF viral cultures were repeatedly negative, herpes simplex virus type 1 (HSV-1) DNA was detected in CSF by polymerase chain reaction (PCR). HSV-1-specific antibodies were absent at the time of the first positive PCR test, but CSF seroconversion to high HSV-1-specific antibody titer subsequently occurred. CSF antibody to cytomegalovirus (CMV) and varicella-zoster virus (VZV) was not detectable, nor could CMV, VZV, or Epstein-Barr virus nucleic acid be detected by CSF by PCR. This is the first report of the use of CSF PCR for the rapid antemortem diagnosis of herpetic brainstem encephalitis.

In vitro transcription assays were carried out by using as templates DNAs cut from the herpes simplex virus early glycoprotein D gene, the late glycoprotein C gene, the late VP5 gene, and the immediate-early ICP22 gene. Nuclear extracts from suspension cultures of uninfected HeLa cells effectively synthesized RNAs from genes of the immediate-early and delayed-early classes. To a lesser extent, the extracts also used DNAs cut from the late genes as templates. Transcription from the immediate-early gene was inhibited in extracts prepared from infected cells. Analysis of the proteins in infected-cell extracts by gel electrophoresis, transfer to nitrocellulose, and probing with specific antibody demonstrated the presence of the viral regulatory protein ICP4. Chromatographic fractionation of nuclear extract from infected cells yielded a mixture of proteins (fraction VIII) enriched in ICP4 (S.W. Faber and K.W. Wilcox, Nucleic Acids Res., 14:6067-6083, 1986). Addition of fraction VIII to the in vitro assay affected transcription. Depending on the DNA in the assay, an inhibitory or stimulatory effect was observed. Inhibition of RNA synthesis was found when DNA from the immediate-early gene was used as a template, and stimulation was found when DNA from the early or late gene was used.

ABSTRACT. A potential in vitvo model of varicella-zoster virus (VZV) latency was developed. Dissociated human dorsal root ganglion cultures were infected with VZV and maintained for 1 wk in the presence of bromovinyl arabinosy1 uracil, a potent inhibitor of VZV. Seven to 21 d after removing the inhibitor (214 d after infection), the cells were trypsinized, passed to monolayers of human embryonic lung fibroblasts, and observed for VZV reactivation as indicated by typical cytopathic effects and the appearance of VZV antigens. VZV reactivated from 56% of the cultures containing both neurons and satellite cells but not from cultures specifically enriched for either neurons, satellite cells, or ganglion-derived fibroblasts. The failure to isolate VZV from cell suspensions that were sonicated before cocultivation with fibroblasts indicated that infectious VZV was not present before reactivation. Moreover, immunohistochemical and immunoprecipitation studies revealed no VZV-specific antigens in any cultures before the reactivation stimulus. VZV antigens were detected after trypsinization and cocultivation. These findings suggest that cultures containing both neurons and satellite cells provide a model system for VZV persistence that possesses many properties of a latent infection. (Pediatr Res 32: 699-703,1992) Abbreviations VZV, varicella-zoster virus HSV, herpes simplex virus NGF, nerve growth factor HELF, human embryonic lung fibroblast CPE, cytopathic effect DRG, dorsal root ganglion DMEM, Dulbecco's modified essential medium EMEM, Eagle's minimal essential medium FUDR, fluorodeoxyuridine BVaraU, bromovinyl arabinosyl uracil pfu, plaque forming units VZV, which produces latent infection of human DRG during primary infection (varicella), can reactivate many years later to produce herpes zoster. The sites of latency were initially assumed to be the DRG and trigeminal ganglia, on the basis of the characteristic dermatomal distribution of herpes zoster (1-3) and the detection of VZV within the ganglia during the acute phase of herpes zoster (4, 5). However, numerous attempts to recover VZV from DRG and trigeminal ganglia of adult cadavers have all failed, in contrast to the frequent recovery of HSV from trigeminal and sacral ganglia (6, 7).These sites of VZV latency were established, finally, by detecting VZV nucleic acids in human ganglia harvested at autopsy from individuals who died without active infection (8-1 1). However, in situ hybridization studies using VZV-specific probes have yielded conflicting data concerning the cellular location of the latent VZV and the prevalence of infected cells within these ganglia (8,10,12,13).A model of VZV latency was approached in adult rats by S.C. paraspinous injection of virus and its subsequent detection in neurons by in situ hybridization. VZV was also reported to be reactivated from ganglion cells from these rats upon trypsinization and cocultivation with permissive fibroblasts (14). These findings seem surprising because the rat is not readily infected by VZV. Moreover...