Latency-associated Transcript Promoter Region Research Articles

Characterization of herpes simplex virus 2 primary microRNA Transcript regulation.

In 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. The 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.

Herpes Simplex Virus Latency-Associated Transcript Sequence Downstream of the Promoter Influences Type-Specific Reactivation and Viral Neurotropism

Herpes simplex virus (HSV) establishes latency in sensory nerve ganglia during acute infection and may later periodically reactivate to cause recurrent disease. HSV type 1 (HSV-1) reactivates more efficiently than HSV-2 from trigeminal ganglia while HSV-2 reactivates more efficiently than HSV-1 from lumbosacral dorsal root ganglia (DRG) to cause recurrent orofacial and genital herpes, respectively. In a previous study, a chimeric HSV-2 that expressed the latency-associated transcript (LAT) from HSV-1 reactivated similarly to wild-type HSV-1, suggesting that the LAT influences the type-specific reactivation phenotype of HSV-2. To further define the LAT region essential for type-specific reactivation, we constructed additional chimeric HSV-2 viruses by replacing the HSV-2 LAT promoter (HSV2-LAT-P1) or 2.5 kb of the HSV-2 LAT sequence (HSV2-LAT-S1) with the corresponding regions from HSV-1. HSV2-LAT-S1 was impaired for reactivation in the guinea pig genital model, while its rescuant and HSV2-LAT-P1 reactivated with a wild-type HSV-2 phenotype. Moreover, recurrences of HSV-2-LAT-S1 were frequently fatal, in contrast to the relatively mild recurrences of the other viruses. During recurrences, HSV2-LAT-S1 DNA increased more in the sacral cord compared to its rescuant or HSV-2. Thus, the LAT sequence region, not the LAT promoter region, provides essential elements for type-specific reactivation of HSV-2 and also plays a role in viral neurotropism. HSV-1 DNA, as quantified by real-time PCR, was more abundant in the lumbar spinal cord, while HSV-2 DNA was more abundant in the sacral spinal cord, which may provide insights into the mechanism for type-specific reactivation and different patterns of central nervous system infection of HSV-1 and HSV-2.

CTCF-Dependent Chromatin Boundary Element between the Latency-Associated Transcript and ICP0 Promoters in the Herpes Simplex Virus Type 1 Genome

Cells latently infected with herpes simplex virus (HSV) contain nucleosomal DNA similar to that of host cell chromatin. Recent studies have demonstrated that histones in the latency-associated transcript (LAT) promoter and intron regions contain histone modifications permissive for transcription. However, those histones associated with the lytic-specific ICP0 gene, which lies only 5 kb away, contain modifications typical of silenced chromatin. How this active chromatin is kept separate from the repressed chromatin in the nearby ICP0 region remains crucial to the understanding of the HSV lytic cycle. In this study, we show that the LAT intron region contains an insulator. Specifically, we show that an 800-bp region from the LAT intron can block enhancers in both tissue culture cells and Drosophila melanogaster embryos. Importantly, the 800-bp HSV insulator protects a LAT transgene from positional effects in Drosophila eye tissue. The 800-bp region contains nine copies of 16-bp repeats. In vitro electrophoretic mobility shift assay revealed that CTCF interacts with the CTCCC sequence within the repeats. In vivo chromatin immunoprecipitation assay demonstrated that CTCF interacts with these repeats in latently infected trigeminal ganglion neurons. The deletion of these repeats impaired insulator activity in human K562 cells and Drosophila embryos. Finally, double-spaced RNA knockdown of CTCF disrupts enhancer-blocking activity of the LAT insulator in transfected Drosophila S3 cells. These results strongly support the hypothesis that the 800-bp DNA in the LAT intron region works as a chromatin boundary during latency to separate active chromatin associated with the LAT promoter region from repressed chromatin in the ICP0 gene.

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.

Identification of a Novel 0.7-kb Polyadenylated Transcript in the LAT Promoter Region of HSV-1 That Is Strain Specific and May Contribute to Virulence

Herpes Simplex virus expresses latency-associated transcripts (LATs) the function of which remains obscure despite increasing knowledge of their structure and expression. Upstream of the LAT coding region is a region of the genome that is poorly characterized although it lies in an area that is responsible for modulation of reactivation efficiency in two different animal models. Transcript mapping with strains 17, McKrae, KOS, and F has revealed strain differences in this region of the viral genome. Strain 17 and McKrae expressed a novel polyadenylated 0.7-kb transcript that is absent from KOS and F. This transcript is expressed in the LAT direction and has the kinetics of a true late gene during the lytic cycle of infection. A deletion mutant, 17ΔBsa, which does not express the 0.7-kb RNA, is less virulent than the parental strain 17. A rescuant with F sequence (17ΔBsa/RF) shows virulence similar to F, whereas a rescuant with strain 17 sequence (17ΔBsa/R17) is similar to strain 17. Virulence is altered by deletion or substitution in the region encoding the 0.7-kb transcript (BsaI-BsaI); however, reactivation in the mouse explant cocultivation assay or the adrenergically induced rabbit reactivation model remained unchanged. The importance of this region for virulence is discussed.

The latency-associated promoter of herpes simplex virus type 1 requires a region downstream of the transcription start site for long-term expression during latency.

The latency-associated transcript (LAT) promoter of herpes simplex virus type 1 (HSV-1) is unique among the many promoters on the viral genome in that it remains active during the latent state. We have previously shown that a DNA fragment comprising the LAT promoter element through the cap site, when moved from the LAT locus to the glycoprotein C gene, is capable of only short-term expression. These and other data suggested that an HSV DNA element from the repeat region, not included in the LAT promoter itself, might be needed to preserve long-term expression. Based on a number of recombinant viruses, we narrowed our search for this putative element to a region 3' of the LAT transcription start site. In the present study, we have shown that a 1.1-kb DNA fragment containing the putative long-term expression element (LTE) is able to restore latent-phase gene expression to the LAT promoter. The element appeared to function best when it was placed in its natural location, which is 3' of the LAT promoter; however, partial function was obtained when the LTE was inserted upstream of the LAT promoter in the reverse direction. These data indicate that the LAT promoter region is more complex than originally anticipated and that in addition to requiring both core promoter and neuronal transcription factor binding sites, the promoter requires a specific region of DNA to prevent its shutoff during a latent infection.

Repression of the HSV-1 latency-associated transcript (LAT) promoter by the early growth response (EGR) proteins: involvement of a binding site immediately downstream of the TATA box.

During herpes simplex virus (HSV) latency, in neurons of the nervous system, a single family of viral transcripts (the Latency-Associated Transcripts or LATs) are synthesized. Within the LAT promoter region, we have identified a consensus sequence for the EGR proteins in an unusual position immediately downstream of the TATA box. The early growth response (EGR) proteins are rapidly induced in cells by stimuli which also induce HSV to reactivate from latency. In order to determine if EGR proteins play any role in control of LAT transcription, we have analyzed the interactions between EGR proteins and the LAT promoter. Gel retardation and DNase I protection assays demonstrated that EGR1 zinc finger protein bound specifically to the LAT promoter region EGR consensus sequence. To determine if EGR proteins could modulate transcription through the LAT promoter, cotransfection assays were performed using chloramphenicol acetyltransferase (CAT) reporter constructs driven by either the wild-type LAT promoter or a LAT promoter with a mutated EGR binding site. Contransfection of the wild-type LAT promoter construct with EGR expression plasmids resulted in inhibition of the basal level of CAT activity with EGR-2 but not EGR-1 or 3. However, normal levels of CAT activity were observed in cotransfections using the mutant LAT promoter CAT construct suggesting that repression was mediated by the binding of EGR-2 proteins to the LAT promoter. Furthermore, data from combination binding assays using EGR1 and TATA binding protein (TBP) in vitro support the hypothesis that binding of EGR proteins to the LAT promoter prevents binding of TBP and thus suppresses transcription. These results may provide a link between stress responses in neurons of the CNS which activate the EGR family of proteins and HSV reactivation from latency due to the same stress response.

A structural and functional comparison of the latency-associated transcript promoters of herpes simplex virus type 1 strains KOS and McKrae

The promoters of the latency-associated transcripts (LATs) of herpes simplex virus type 1 (HSV-1) strains KOS and McKrae were compared to examine their influence upon the reactivation phenotypes of these two strains. Unlike strain KOS, McKrae is readily reactivable using in vivo reactivation models. We found greater than 96% sequence conservation between KOS and McKrae in the LATs promoter region, and both promoters showed equivalent basal and inducible activities. An inter-strain recombinant (termed MK13) was constructed in which the LATs promoter of HSV-1 McKrae was recombined into the background of HSV-1 strain KOS. In a murine u.v. light-induced reactivation model, virus shedding was detected by eye swabbing in two of 44 (5%) mice infected with KOS, 20 of 42 (48%) mice infected with McKrae and none of 45 (0%) mice infected with MK13. These data show that the LATs promoters of these viruses are structurally and functionally similar and that transfer of the LATs promoter from McKrae into KOS is insufficient to confer a reactivatable phenotype.

Herpes simplex virus type 1 latency-associated transcript (LAT) promoter deletion mutants can express a 2-kilobase transcript mapping to the LAT region

The results of studies in several laboratories suggest that a TATA box-containing promoter located in the herpes simplex virus type 1 internal long repeat and long terminal repeat elements drives expression of the latency-associated transcripts (LATs). In the present study, we show that expression of a 2-kb LAT-related transcript can occur in the absence of this LAT TATA promoter, indicating the existence of a cryptic promoter. By Northern (RNA) blot analysis, we have examined LAT expression by herpes simplex virus type 1 variant strains KOS/29 and 1704, which contain deletions of the LAT promoter region. Our data indicate that KOS/29, despite lacking the 203-bp fragment which contains the LAT TATA box, can express a 2-kb LAT-related transcript during productive infection in tissue culture and in mouse trigeminal ganglia during acute infection and reactivation. Similarly, strain 1704, which contains a larger deletion in this promoter region, also expresses a 2-kb LAT-related transcript during tissue culture infection and reactivation of latently infected trigeminal ganglia. However, LATs are not expressed with either virus during latency. Northern blot analysis with a single-stranded, oligonucleotide probe demonstrates that the 2-kb LAT and LAT-related transcript are colinear and share a large area of sequence similarity. These findings suggest the existence of a second promoter in the LAT gene which can function during lytic infection and reactivation, at least in the absence of the LAT TATA promoter. We propose that this cryptic promoter is located either in a proximal region approximately 300 bp upstream of the start site of the 2-kb LAT or in a distal region starting over 1,226 bp upstream of this site.

Sequence analysis of the herpes simplex virus type 1 strain 17 variants 1704, 1705 and 1706 with respect to their origin and effect on the latency-associated transcript sequence.

The precise endpoints of the deletions/insertions in three variants (1704, 1705 and 1706) of herpes simplex virus type 1 (HSV-1) strain 17 have been determined by dideoxynucleotide sequence analysis. The analysis was undertaken to discover whether the three variants had arisen from the same initial event and the extent of the deletions with respect to the latency-associated transcripts (LATs) and the proposed LAT promoter region. It is not possible from the deletion boundaries to determine unequivocally whether the three variants had arisen from the same recombination event although 1706 could be descended from 1705 by illegitimate recombination. The results demonstrate that spontaneous deletions can occur at random within RL, the extent of the deletions in UL is constrained by the essential nature of UL genes in vitro but is otherwise arbitrary and deletions in 1704 completely remove both copies of the LAT promoter region and in IRL extend into the 5' end of the LAT sequence.