Herpes simplex virus type 1 ICP0 localizes in the stromal layer of infected rabbit corneas and resides predominantly in the cytoplasm and/or perinuclear region of rabbit keratocytes.

Herpes stromal keratitis (HSK) results from the reactivation of herpes simplex virus type-1 (HSV-1) in the cornea. The subsequent corneal inflammation and neovascularization may lead to scarring and visual loss. The cellular and molecular mechanisms underlying HSK remain unknown. The presence of stromal HSV-1 viral proteins or antigens in the HSK cornea remains a subject of debate. It was recently reported that HSV-1 ICP0 rapidly diffuses out of infected rabbit corneas. To investigate further the presence of HSV-1 ICP0 in the infected cornea, particularly in the corneal stroma, ex vivo confocal microscopy was used to scan rabbit corneas infected with the virus ICP0-EYFP, an HSV-1 derivative (strain 17+) that expresses ICP0 fused to the enhanced yellow fluorescent protein (EYFP). These results demonstrate that ICP0 is expressed in the corneal epithelium and stromal cells (keratocytes) of infected rabbit corneas throughout acute infection. Furthermore, expression of ICP0-EYFP appears localized to punctate, granular deposits within stromal keratocytes, showing both a cytoplasmic and perinuclear localization. These findings provide new data demonstrating that anterior corneal keratocytes become infected and express ICP0 during acute HSV-1 infection.

A mutant deleted for most of the herpes simplex virus type 1 (HSV-1) UOL gene does not affect the spontaneous reactivation phenotype in rabbits.

The mechanisms involved in the herpes simplex virus type 1 (HSV-1) latency-reactivation cycle are not fully understood. The latency-associated transcript (LAT) is the only HSV-1 RNA abundantly detected during neuronal latency. LAT plays a significant role in latency because LAT(-) mutants have a reduced reactivation phenotype. Several novel viral transcripts have been identified within the LAT locus, including UOL, which is located just upstream of LAT. The authors report here on a mutant, DeltaUOL, which has a 437-nucleotide deletion that deletes most of UOL. DeltaUOL replicated similarly to its wild-type parental McKrae HSV-1 strain in infected cells, the eyes, trigeminal ganglia, and brains of mice and rabbits. It was indistinguishable from wild-type virus as regards explant-induced reactivation in mice, and spontaneous reactivation in rabbits. In contrast, DeltaUOL was significantly less virulent in mice. Thus, UOL appears to be dispensable for the wild-type reactivation phenotype while appearing to play a role in neurovirulence in ocularly infected animals.

Herpes simplex virus type 1 immediate-early protein ICP0 diffuses out of infected rabbit corneas.

Herpes stromal keratitis (HSK) results from infection of herpes simplex virus (HSV) in the cornea. Recurrent HSV infection is a leading cause of corneal scarring and visual loss. Although it is generally thought that HSK is the result of an immune response to one or more viral proteins, no viral proteins have been detected in HSK corneas. Thus, the viral proteins involved in HSK, if any, remain undetermined. In contrast, it is reported here that when HSK corneal buttons from latently infected rabbits were fixed using standard procedures, the important immediate-early HSV-1 protein ICP0 was readily detected in the fixative by Western blotting. Similarly, when HSK corneal buttons were soaked in buffer (rather than fixative), ICP0 was readily detected in the soaking buffer. Other HSV-1 proteins were not detected either in the fixative or in the soaking buffer. It is also reported here that ICP0 was consistently detected in virus-free tears from the eyes of rabbits acutely infected with HSV-1. These results suggest that ICP0 rapidly diffuses out of the cornea and may explain why ICP0 was detected in the fixative of HSK corneas and in the soaking buffer of acutely infected corneas.

Identification of a protein encoded in the herpes simplex virus type 1 latency associated transcript promoter region.

Herpes simplex virus type 1 (HSV-1) establishes a lifelong latency in the neurons of its host. Sporadically, the latent virus reactivates and spreads back to the original site of infection and causes recrudescent diseases. The only gene actively transcribed during neuronal latency is the latency associated transcript (LAT) gene. Several transcripts have been detected in the important LAT promoter region. However, no polypeptides coded by these transcripts are known. In this communication, we reported the cloning, sequencing, and characterization of a transcript immediately upstream of LAT. We designated this gene UOL (Upstream of LAT). The UOL RNA is polyadenylated, expressed as a late gene in infected cells, transcribed in the same direction as LAT, and contains an open reading frame (ORF) capable of encoding a protein of 96 amino acids with a predicted molecular mass of 11 kDa. The UOL transcript contains 466 nucleotides in length. The 5' end of the UOL transcript starts at nucleotide 118,266 and the 3' end of the UOL transcript ends at nucleotide 118,731 based on the published 17syn+ genomic sequence. The UOL protein was detected in infected cell lysates by immunoprecipitation using an antibody raised against UOL ORF synthetic peptide. More importantly, sera from mice infected with wild-type HSV-1 but not sera from mice infected with a mutant with the UOL region deleted recognized the UOL ORF, expressed in Escherichia coli, on Western blots. These results suggest that a UOL protein is in HSV-1 infected tissue culture cells and in mice infected with HSV-1.

The bovine herpesvirus-1 LR ORF2 is critical for this gene's ability to restore the high wild-type reactivation phenotype to a herpes simplex virus-1 LAT null mutant.

During neuronal latency of herpes simplex virus (HSV)-1, the latency-associated transcript (LAT) is the only viral gene readily detectable. LAT is required for the high-level reactivation phenotype in animal models. LAT's anti-apoptotic activity was recently demonstrated by our group and it was proposed that LAT's anti-apoptotic function is involved in enhancing the reactivation phenotype. Recently, using chimeric virus CJLAT, it was shown that the reactivation phenotype of LAT(-) mutant dLAT2903 can be restored to wild-type levels by inserting the bovine herpes virus (BHV)-1 latency-related (LR) gene into the LAT locus of this HSV-1 LAT deletion mutant. Although transcription of the LR gene, like LAT, inhibits apoptosis, LR appears to be multifunctional. To investigate whether the LR gene's anti-apoptotic function was responsible for restoring the high-reactivation phenotype, a mutated BHV-1 LR gene was inserted into the LAT locus of HSV-1 generating the chimeric virus CJLATmut. This mutation consists of three stop codons inserted just after the ATG of the first LR open reading frame (ORF2). In plasmids and in a BHV-1 mutant, this mutation eliminated the LR gene's anti-apoptotic activity, strongly suggesting that ORF2 encodes a protein responsible for LR's anti-apoptotic activity. Reactivation of the CJLATmut virus, in both rabbits and mice, was significantly lower than in wild-type McKrae virus (P=0.0001 and P=0.0003, respectively) and CJLAT virus, containing wild-type LR in place of LAT (P<0.0001) and was similar to LAT(-) dLAT2903 (P=0.8 and P=0.7, respectively). Thus, disruption of BHV-1 LR ORF2 eliminated the high-reactivation phenotype.