Latent infections with periodic reactivation are a common outcome after acute infection with many viruses. The latency-associated transcript ( LAT ) gene is required for wild-type reactivation of herpes simplex virus (HSV). However, the underlying mechanisms remain unclear. In rabbit trigeminal ganglia, extensive apoptosis occurred with LAT − virus but not with LAT + viruses. In addition, a plasmid expressing LAT blocked apoptosis in cultured cells. Thus, LAT promotes neuronal survival after HSV-1 infection by reducing apoptosis.
During herpes simplex virus type 1 (HSV-1) neuronal latency, the only viral RNA detected is from the latency-associated transcript (LAT) gene. We have made a LAT deletion mutant of McKrae, an HSV-1 strain with a very high in vivo spontaneous reactivation rate. This mutant (dLAT2903) lacks the LAT promoter and the first 1.6 kb of the 5' end of LAT. dLAT2903 was compared with its parental virus and with a rescued virus containing a restored LAT gene (dLAT2903R). Replication of the LAT mutant in tissue culture, rabbit eyes, and rabbit trigeminal ganglia was similar to that of the rescued and parental viruses. On the basis of semiquantitative PCR analysis of the amount of HSV-1 DNA in trigeminal ganglia, the LAT mutant was unimpaired in its ability to establish latency. In contrast, spontaneous reactivation of dLAT2903 in the rabbit ocular model of HSV-1 latency and reactivation was decreased to approximately 33% of normal. This decrease was highly significant (P < 0.0001) and demonstrates that in an HSV-1 strain with a high spontaneous reactivation rate, deletion of LAT can dramatically decrease in vivo spontaneous reactivation. We also report here that deletion of LAT appeared to eliminate rather than just reduce in vivo induced reactivation.
The latency-related (LR) gene of herpes simplex virus type 1 (HSV-1) is transcriptionally active during HSV-1 latency, producing at least two LR-RNAs. The LR gene partially overlaps the immediate-early gene ICPO and is transcribed in the opposite direction from ICPO, producing LR-RNAs that are complementary (antisense) to ICPO mRNA. The LR gene is thought to be involved in HSV-1 latency. We report here the fine mapping and partial sequence analysis of this HSV-1 LR gene. 32P-labeled genomic DNA restriction fragments and synthetic oligonucleotides were used as probes for in situ hybridizations and Northern (RNA) blot hybridizations of RNA from trigeminal ganglia of rabbits latently infected with HSV-1. The two most abundant LR-RNAs appeared to share their 5' and 3' ends and to be produced by alternative splicing. These LR-RNAs were approximately 2 and 1.3 to 1.5 kilobases in length and were designated LR-RNA 1 and LR-RNA 2, respectively. Their 5' ends started approximately 1,210 nucleotides downstream from the 3' end of the ICPO mRNA. Their 3' ends overlapped ICPO by nearly 1,000 nucleotides. LR-RNA 1 appeared to have at least one intron removed, while LR-RNA 2 appeared to have at least two introns removed. The LR-RNAs contained two potential long open reading frames, suggesting the possibility that one or more of the LR-RNAs may be a functional mRNA.
The latency-associated transcript (LAT) gene the only herpes simplex virus type 1 (HSV-1) gene abundantly transcribed during neuronal latency, is essential for efficient in vivo reactivation. Whether LAT increases reactivation by a direct effect on the reactivation process or whether it does so by increasing the establishment of latency, thereby making more latently infected neurons available for reactivation, is unclear. In mice, LATnegative mutants appear to establish latency in fewer neurons than does wild-type HSV-1. However, this has not been confirmed in the rabbit, and the role of LAT in the establishment of latency remains controversial. To pursue this question, we inserted the gene for the enhanced green fluorescent protein (EGFP) under control of the LAT promoter in a LAT-negative virus (⌬LAT-EGFP) and in a LAT-positive virus (LAT-EGFP). Sixty days after ocular infection, trigeminal ganglia (TG) were removed from the latently infected rabbits, sectioned, and examined by fluorescence microscopy. EGFP was detected in significantly more LAT-EGFP-infected neurons than ⌬LAT-EGFP-infected neurons (4.9% versus 2%, P < 0.0001). The percentages of EGFP-positive neurons per TG ranged from 0 to 4.6 for ⌬LAT-EGFP and from 2.5 to 11.1 for LAT-EGFP (P ؍ 0.003). Thus, LAT appeared to increase neuronal latency in rabbit TG by an average of two-to threefold. These results suggest that LAT enhances the establishment of latency in rabbits and that this may be one of the mechanisms by which LAT enhances spontaneous reactivation. These results do not rule out additional LAT functions that may be involved in maintenance of latency and/or reactivation from latency.Following primary ocular infection, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons of the trigeminal ganglia (TG). At various times throughout the life of the infected individual, the virus can reactivate, return to the eye, be shed in tears, and produce recurrent corneal disease and scarring leading to impaired vision. Recurrent ocular HSV-1 results in over 400,000 doctor visits per year in the United States and is a leading infectious cause of corneal blindness (15).During latency, abundant viral transcription is consistently detected only in the region of the latency-associated transcript (LAT) gene (25,30). LAT is located in the long repeats of the HSV-1 genome and is therefore present in two copies per genome. The primary LAT transcript is 8.3 kb (33, 34). It gives rise to a family of LAT RNAs (LATs) including a very stable 2-kb LAT that appears to be an intron spliced from the primary transcript (4). LAT null mutants (i.e., LAT transcriptionnegative mutants) have been shown to reactivate poorly by explant or induced reactivation in the mouse (9, 10, 28, 29), by induced reactivation in the rabbit (8, 32), and by spontaneous reactivation in the rabbit (16, 19).The molecular mechanisms by which LAT enhances reactivation are not understood. It is also not known (i) whether LAT functions to enhance the establishmen...
Herpes simplex virus type 1 (HSV-1) is a large (152-kb) double-stranded DNA virus with neurotropic properties. HSV-1 establishes lifelong latent infections in host sensory neurons. This virus is wide spread in the general population. When the eye is infected, the virus travels up nerves and establishes latent infection in neurons of the trigeminal ganglia (TG). During neuronal latency, HSV-1 has no apparent impact on the infected individual. The latent virus can reactivate at various times throughout the life of the individual. This occurs through a mechanism(s) that is currently not completely understood. HSV-1 reactivation in the TG results in virus returning to the eye via the same route previously used to get from the eye to the TG. At the eye, reactivated HSV-1 can produce recurrent disease. Recurrent HSV-1 infection in the eye can cause corneal scarring leading to loss of vision. As a result, HSV-1 is one of the most common infectious causes of corneal blindness in the developed world.During neuronal latency, LAT (latency-associated transcript) is the only abundantly transcribed viral gene (30, 37). The primary LAT transcript is ca. 8.3 kb long (7, 45) and overlaps two viral genes, ICP0 and ICP34.5, in an antisense direction (30,37). A very stable intron, the 2-kb LAT is spliced from the primary transcript (9) and is the major LAT RNA detected during latency (7,33,36,(42)(43)(44).LAT enhances the induced and spontaneous reactivation phenotypes in the rabbit ocular model (12, 21) and the induced reactivation phenotype in mice (1a, 6, 19, 26, 31, 35). The reduced reactivation phenotypes of LAT Ϫ mutants does not necessarily imply that LAT is directly involved in the molecular mechanism of HSV-1 reactivation from latency. LAT might enhance reactivation by increasing the initial amount of latency established and/or by maintaining a high level of latently infected neurons. The larger pool of latently infected neurons and/or the larger pool of neurons containing high copy numbers of the HSV-1 genome would be expected to increase reactivation. Several reports have, in fact, shown that in experimentally infected animals more neurons become latently infected with LAT ϩ viruses compared to LAT Ϫ viruses (29,31,40). In addition, we have recently shown that LAT has anti-* Corresponding author. Mailing address:
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