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The activity of the pseudorabies virus latency-associated transcript promoter is dependent on its genomic location in herpes simplex virus recombinants as well as on the type of cell infected.

ABSTRACT: As do many other alphaherpesviruses, pseudorabies virus (PRV) transcribes a limited portion of its viral genome in latently infected neurons during latency. The sequence of the PRV latency-associated transcript (LAT) is bounded on its 5' end by a putative promoter region which contains sequence elements similar to those characterized for the herpes simplex virus (HSV) LAT promoter. Using the bacterial beta-galactosidase gene as a reporter, we have assayed PRV LAT promoter activity in the genomic environment in recombinant HSVs. The PRV LAT promoter-beta-galactosidase reporter gene was recombined into the terminal and internal long repeat regions (RL regions), replacing the normal HSV LAT promoter, the cap site, and the first 60 bases of the primary transcript. When recombined into the RL region, appreciable reporter gene expression was observed following infection of two cell lines of neuronal origin; little or no activity was seen with these recombinants following infection of rabbit skin or mouse embryo fibroblasts. No significant expression was seen when the promoter was recombined into the gC locus in the long unique region in any of the cell types utilized. Such results suggest that the PRV latency promoter contains neuronal cell-specific elements and that the HSV RL region provides an appropriate genomic environment for the manifestation of that specificity.

SUBMITTER: Huang CJ

PROVIDER: S-EPMC236661 | biostudies-other | 1994 Mar

REPOSITORIES: biostudies-other

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The activity of the pseudorabies virus latency-associated transcript promoter is dependent on its genomic location in herpes simplex virus recombinants as well as on the type of cell infected.

Huang C J CJ Rice M K MK Devi-Rao G B GB Wagner E K EK

Journal of virology 19940301 3

As do many other alphaherpesviruses, pseudorabies virus (PRV) transcribes a limited portion of its viral genome in latently infected neurons during latency. The sequence of the PRV latency-associated transcript (LAT) is bounded on its 5' end by a putative promoter region which contains sequence elements similar to those characterized for the herpes simplex virus (HSV) LAT promoter. Using the bacterial beta-galactosidase gene as a reporter, we have assayed PRV LAT promoter activity in the genomic ...[more]

PMID: 8107257

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Project description:During herpes simplex virus (HSV) latency, most viral genes are silenced, with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long noncoding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilized an adeno-associated virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition, these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness.IMPORTANCE Herpes simplex virus (HSV) establishes a lifelong infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency, with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This work shows that the LAT RNA is important for reactivation and suggests the potential of this treatment as a therapy for treating HSV infections.

Project description:Herpes simplex virus 1 (HSV-1) is a significant pathogen that establishes lifelong latent infections with intermittent episodes of resumed disease. In mouse models of HSV infection, sporadic low-level lytic gene expression has been detected during latency in the absence of reactivation events that lead to production of new viruses. This viral activity during latency has been reported using a sensitive Cre-marking model for several lytic gene promoters placed in one location in the HSV-1 genome. Here, we extend these findings in the same model by examining first, the activity of an ectopic lytic gene promoter in several places in the genome and second, whether any promoters might be active in their natural context. We found that Cre expression was detected during latency from ectopic and native promoters, but only in locations near the ends of the unique long genome segment. This location is significant because it is in close proximity to the region from which latency-associated transcripts (LATs) are derived. These results show that native HSV-1 lytic gene promoters can produce protein products during latency, but that this activity is only detectable when they are located close to the LAT locus.IMPORTANCEHSV is a significant human pathogen and the best studied model of mammalian virus latency. Traditionally, the active (lytic) and inactive (latent) phases of infection were considered to be distinct, but the notion of latency being entirely quiescent is evolving due to the detection of some lytic gene expression during latency. Here, we add to this literature by finding that the activity can be found for native lytic gene promoters as well as for constructs placed ectopically in the HSV genome. However, this activity was only detectable when these promoters were located close by a region known to be transcriptionally active during latency. These data have implications for our understanding of HSV gene regulation during latency and the extent to which transcriptionally active regions are insulated from adjacent parts of the viral genome.

Project description:UnlabelledIn order to understand factors that may influence latency-associated transcription and latency-associated transcript (LAT) phenotypes, we studied the expression of the herpes simplex virus 2 (HSV-2) LAT-associated microRNAs (miRNAs). We mapped the transcription initiation sites of all three primary miRNA transcripts and identified the ICP4-binding sequences at the transcription initiation sites of both HSV-2 LAT (pri-miRNA for miR-I and miR-II, which target ICP34.5, and miR-III, which targets ICP0) and L/ST (a pri-miRNA for miR-I and miR-II) but not at that of the primary miR-H6 (for which the target is unknown). We confirmed activity of the putative HSV-2 L/ST promoter and found that ICP4 trans-activates the L/ST promoter when the ICP4-binding site at its transcription initiation site is mutated, suggesting that ICP4 may play a dual role in regulating transcription of L/ST and, consequently, of miR-I and miR-II. LAT exon 1 (containing LAT enhancer sequences), together with the LAT promoter region, comprises a bidirectional promoter required for the expression of both LAT-encoded miRNAs and miR-H6 in latently infected mouse ganglia. The ability of ICP4 to suppress ICP34.5-targeting miRNAs and to activate lytic viral genes suggests that ICP4 could play a key role in the switch between latency and reactivation.ImportanceThe HSV-2 LAT and viral miRNAs expressed in the LAT region are the most abundant viral transcripts during HSV latency. The balance between the expression of LAT and LAT-associated miRNAs and the expression of lytic viral transcripts from the opposite strand appears to influence whether individual HSV-infected neurons will be latently or productively infected. The outcome of neuronal infection may thus depend on regulation of gene expression of the corresponding primary miRNAs. In the present study, we characterize promoter sequences responsible for miRNA expression, including identification of the primary miRNA 5' ends and evaluation of ICP4 response. These findings provide further insight into the virus' strategy to tightly control expression of lytic cycle genes (especially the neurovirulence factor, ICP34.5) and suggest a mechanism (via ICP4) for the transition from latency to reactivated productive infection.

Dataset Information

The activity of the pseudorabies virus latency-associated transcript promoter is dependent on its genomic location in herpes simplex virus recombinants as well as on the type of cell infected.

Publications

The activity of the pseudorabies virus latency-associated transcript promoter is dependent on its genomic location in herpes simplex virus recombinants as well as on the type of cell infected.

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