During herpes simplex virus type 1 (HSV-1) latency, gene expression is tightly repressed except for the latency-associated transcript (LAT). The mechanistic basis for this repression is unknown, but its global nature suggests regulation by an epigenetic mechanism such as DNA methylation. Previous work demonstrated that latent HSV-1 genomes are not extensively methylated, but these studies lacked the resolution to examine methylation of individual CpGs that could repress transcription from individual promoters during latency. To address this point, we employed established models to predict genomic regions with the highest probability of being methylated and, using bisulfite sequencing, analyzed the methylation profiles of these regions. We found no significant methylation of latent DNA isolated from mouse dorsal root ganglia in any of the regions examined, including the ICP4 and LAT promoters. This analysis indicates that methylation is unlikely to play a major role in regulating HSV-1 latent gene expression. Subsequently we focused on differential histone modification as another epigenetic mechanism that could regulate latent transcription. Chromatin immunoprecipitation analysis of the latent HSV-1 DNA repeat regions demonstrated that a portion of the LAT region is associated with histone H3 acetylated at lysines 9 and 14, consistent with a euchromatic and nonrepressed structure. In contrast, the chromatin associated with the HSV-1 DNA polymerase gene located in the unique long segment was not enriched in H3 acetylated at lysines 9 and 14, suggesting a transcriptionally inactive structure. These data suggest that histone composition may be a major regulatory determinant of HSV latency.

During herpes simplex virus type 1 (HSV-1) latency, only one region of the viral genome is actively transcribed: the region encoding the latency-associated transcript (LAT). A previous study demonstrated that during latency the LAT promoter is hyperacetylated at histone H3 (K9, K14) relative to lytic genes examined. In the present study, we examine the acetylation profile of regions downstream of the LAT promoter during a latent infection of murine dorsal root ganglia. These analyses revealed the following: (i) the region of the genome containing the 5 exon of the LAT primary transcript was at least as enriched in acetylated H3 as the LAT promoter, and (ii) the region of hyperacetylation does not extend to the ICP0 promoter. In order to assess the contribution of LAT transcription to the acetylation of the 5 exon region, the acetylation profile of KOS/29, a recombinant with a deletion of the LAT promoter, was examined. The region containing the 5 exon of KOS/29 was hyperacetylated relative to lytic gene regions in the absence of detectable LAT transcription. These results indicate that the region containing the 5 exon of LAT, known to contain enhancer activities and to be critical for induced reactivation (rcr), exists in a chromatin structure during latency that is distinct from other lytic gene regions. This result suggests a role for the 5 exon LAT enhancer region as a cis-acting regulator of transcription that maintains a transcriptionally permissive chromatin domain in the HSV-1 latent episome.Herpes simplex virus type 1 (HSV-1) establishes latent infections in sensory neurons as a circular episome associated with histones (8,28,37). Active transcription occurs from only one region of this episome: the region encoding the latencyassociated transcript (LAT) (40, 42). The LAT region carries an 8.3-kb polyadenylated RNA that is spliced to yield a 2.0-kb stable intron that accumulates abundantly in a subset of the sensory neurons (10,27,41). This 2.0-kb intron can be alternatively spliced in some neurons to yield a 1.5-kb intron (38,48). While the LAT region has not been shown to encode any proteins, this region has been implicated in a number of pathogenic functions, including neuronal survival and antiapoptosis (32, 45), virulence (34, 45), suppression of latent transcription (5), establishment of latency (35,46), and reactivation from latency (14,22). Whether this region mediates these different functions through one or more distinct genetic elements remains to be determined, but evidence for the existence of multiple promoters attests to the transcriptional complexity of this region (9, 13, 31, 33). A striking feature of HSV-1 latency is the general suppression of lytic transcription, and this suppression seems to correlate with the association of certain histones with specific tail modifications (20).Cellular chromatin is known to be separated into regions that range from permissive to nonpermissive for polymerase II-mediated transcription, with pericentric heterochromatin being the most nonpermissive and ...

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin-proteasome pathway. Here, we identify von HippelLindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel-Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin-VHL-proteasome pathway in the integrationtranscription transition of the viral replication cycle.prefoldin ͉ ubiquitin ͉ VHL ͉ retrovirus ͉ transcription

Only the latency-associated transcript (LAT) of the herpes simplex virus type 1 (HSV-1) genome is transcribed during latency, while the lytic genes are suppressed, possibly by LAT antisense mechanisms and/or chromatin modifications. In the present study, latently infected dorsal root ganglia were explanted to assess both relative levels of LAT and histone H3 (K9, K14) acetylation of the LAT locus and ICP0 promoter at early times postexplant. We observed that a decrease in both LAT enhancer histone H3 (K9, K14) acetylation and LAT RNA abundance occurs prior to an increase in acetylation, or transcriptional permissiveness, at the ICP0 promoter.Herpes simplex virus type 1 (HSV-1) is characterized by its ability to establish latency as an episome in neurons (12). During this time, transcriptional activity is virtually nonexistent, with the exception of the latency-associated transcript (LAT), an 8.3-to 8.5-kb noncoding RNA that can be spliced to yield a 2.0-kb stable intron (5,13,16). One proposed function of the LAT is the suppression of nearby lytic phase transcripts ICP0, ␥34.5, and ICP4 through antisense mechanisms, thereby promoting the establishment and maintenance of latency (3). Studies using LAT promoter and/or 5Ј exon mutants demonstrate impaired establishment of latency and leaky expression of lytic phase transcripts (3). In addition, a recent study has demonstrated that the lytic gene regions of LAT mutants are associated with less of the repressive histone H3 K9 dimethyl, suggesting that the LAT plays a direct role in promoting a transcriptionally nonpermissive environment for lytic genes during latency (18). If the LAT is indeed responsible for suppressing lytic phase transcripts during latency, one might expect reactivation to directly regulate LAT levels.In addition to putative LAT-mediated suppression of lytic transcripts during latency, mounting evidence suggests that latent gene expression is also regulated at the chromatin level. The latent viral genome is known to associate with nucleosomes (4). Investigation of chromatin modification, in particular, the acetylation of histone H3 lysine residues 9 and 14 (K9 and K14, respectively), demonstrates that during latency, the lytic regions of the virus exist in a hypoacetylated, or transcriptionally nonpermissive, state, while the LAT promoter and 5Ј exon/enhancer remain hyperacetylated, or transcriptionally permissive (9) (Fig. 1). However, LAT transcription is not a prerequisite, nor is it necessary to maintain the hyperacetylated, or structurally relaxed, chromatin state (8), suggesting that the enhancer within the LAT region is an important cisacting DNA element.Reactivation of the latent viral genome has been linked to a reactivation critical region (rcr) that encompasses the LAT core promoter through the LAT 5Ј exon/enhancer, since recombinants lacking this region display greatly reduced reactivation phenotypes (2, 6, 7, 10). However, this still does not address whether the regulatory elements in the rcr act at the RNA or DNA/chromatin level. An ...

A number of electromagnetic field-based technologies are available for therapeutic medical applications. These therapies can be broken down into different categories based on technical parameters employed and type of clinical application. Pulsed radio frequency energy (PRFE) therapy is a non invasive, electromagnetic field-based therapeutic that is based on delivery of pulsed, shortwave radio frequency energy in the 13-27.12 MHz carrier frequency range, and designed for local application to a target tissue without the intended generation of deep heat. It has been studied for use in a number of clinical applications, including as a palliative treatment for both postoperative and non postoperative pain and edema, as well as in wound healing applications. This review provides an introduction to the therapy, a summary of clinical efficacy studies using the therapy in specific applications, and an overview of treatment-related safety.

BackgroundPulsed radiofrequency energy (PRFE) fields are being used increasingly for the treatment of pain arising from dermal trauma. However, despite their increased use, little is known about the biological and molecular mechanism(s) responsible for PRFE-mediated analgesia. In general, current therapeutics used for analgesia target either endogenous factors involved in inflammation, or act on endogenous opioid pathways.Methods and ResultsUsing cultured human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK), we investigated the effect of PRFE treatment on factors, which are involved in modulating peripheral analgesia in vivo. We found that PRFE treatment did not inhibit cyclooxygenase enzyme activity, but instead had a positive effect on levels of endogenous opioid precursor mRNA (proenkephalin, pro-opiomelanocortin, prodynorphin) and corresponding opioid peptide. In HEK cells, increases in opioid mRNA were dependent, at least in part, on endothelin-1. In HDF cells, additional pathways also appear to be involved. PRFE treatment was also followed by changes in endogenous expression of several cytokines, including increased levels of interleukin-10 mRNA and decreased levels of interleukin-1β mRNA in both cell types.ConclusionThese findings provide a new insight into the molecular mechanism underlying PRFE-mediated analgesia reported in the clinical setting.

On the basis of statistical evaluation of published clinical efficacy data, there is strong statistical evidence that PRFE therapy is effective in the treatment of postoperative and nonpostoperative pain and edema and in WH applications.

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