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