Productive herpesvirus infection requires a profound, time-controlled remodeling of the viral transcriptome and proteome. To gain insights into the genomic architecture and gene expression control in Kaposi's sarcoma-associated herpesvirus (KSHV), we performed a systematic genome-wide survey of viral transcriptional and translational activity throughout the lytic cycle. Using mRNA-sequencing and ribosome profiling, we found that transcripts encoding lytic genes are promptly bound by ribosomes upon lytic reactivation, suggesting their regulation is mainly transcriptional. Our approach also uncovered new genomic features such as ribosome occupancy of viral non-coding RNAs, numerous upstream and small open reading frames (ORFs), and unusual strategies to expand the virus coding repertoire that include alternative splicing, dynamic viral mRNA editing, and the use of alternative translation initiation codons. Furthermore, we provide a refined and expanded annotation of transcription start sites, polyadenylation sites, splice junctions, and initiation/termination codons of known and new viral features in the KSHV genomic space which we have termed KSHV 2.0. Our results represent a comprehensive genome-scale image of gene regulation during lytic KSHV infection that substantially expands our understanding of the genomic architecture and coding capacity of the virus.

Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative because it does not directly affect nuclear HBV closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target cccDNA regulation would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report, to our knowledge, the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes, and from HBV-infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome and, surprisingly, very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection.hepatitis B virus | HBV | cccDNA | chromatin | epigenetics H epatitis B virus (HBV) infection is widespread in humans and is a major public health concern. Primary infection outside the newborn period is usually self-limited, but a subset of infected individuals does not eliminate the virus and goes on to a lifelong persistent infection. Worldwide, at least 240 million people are persistently infected, many of whom develop chronic liver injury (chronic hepatitis B or CHB) (1). CHB often progresses to cirrhosis and liver failure, and is also strongly linked to the development of hepatocellular carcinoma (HCC). It is estimated that CHB accounts for more than 80% of HCC cases in areas of high HBV incidence (2).HBV belongs to the family of Hepadnaviridae, a group of small DNA viruses that infect hepatocytes and replicate through the reverse transcription of an RNA intermediate (3). The 3.2-kb HBV genome in viral particles is in a circular, partially doublestranded DNA conformation (relaxed circular DNA or rcDNA), a result of the unusual replication mechanism of HBV. rcDNA is transcriptionally inert and must be converted into covalently closed circular DNA (cccDNA) in the nucleus of infected cells before viral RNAs can be transcribed. cccDNA is the only template for HBV transcription and, because HBV RNA templates genomic reverse transcription, its persistence is required for persistent infection. HBV replication itself is noncytolytic, but it induces an immune response that in the case of CHB leads to persistent liver inflammation. Suppression of HBV re...

The fission yeast Schizosaccharomyces pombe responds to the deprivation of iron by inducing the expression of the php4 ؉ gene, which encodes a negative regulatory subunit of the heteromeric CCAAT-binding factor. Once formed, the Php2/3/4/5 transcription complex is required to inactivate a subset of genes encoding iron-using proteins. Here, we used a pan-S. pombe microarray to study the transcriptional response to iron starvation and identified 86 genes that exhibit php4 ؉ -dependent changes on a genome-wide scale. One of these genes encodes the iron-responsive transcriptional repressor Fep1, whose mRNA levels were decreased after treatment with the permeant iron chelator 2,2-dipyridyl. In addition, several genes encoding the components of iron-dependent biochemical pathways, including the tricarboxylic acid cycle, mitochondrial respiration, amino acid biosynthesis, and oxidative stress defense, were downregulated in response to iron deficiency. Furthermore, Php4 repressed transcription when brought to a promoter using a yeast DNA-binding domain, and iron deprivation was required for this repression. On the other hand, Php4 was constitutively active when glutathione levels were depleted within the cell. Based on these and previous results, we propose that iron-dependent inactivation of Php4 is regulated at two distinct levels: first, at the transcriptional level by the iron-responsive GATA factor Fep1 and second, at the posttranscriptional level by a mechanism yet to be identified, which inhibits Php4-mediated repressive function when iron is abundant.

We have identified genes encoding candidate proteins involved in iron storage (pcl1 ؉ ), the tricarboxylic acid cycle (sdh4 ؉ ), and iron-sulfur cluster assembly (isa1 ؉ ) that are negatively regulated in response to iron deprivation. Promoter deletion and site-directed mutagenesis permitted identification of a new cis-regulatory element in the promoter region of the pcl1 ؉ gene. This cis-acting regulatory sequence containing the pentanucleotide sequence CCAAT is responsible for transcriptional repression of pcl1 ؉ under low iron supply conditions. In Schizosaccharomyces pombe, the CCAAT-binding factor is a heteromeric DNA-binding complex that contains three subunits, designated Php2, Php3, and Php5. Inactivation of the php2 ؉ locus negatively affects the transcriptional competency of pcl1 ؉ . A fourth subunit, designated Php4, is not essential for the transcriptional activation of target genes under basal and iron-replete conditions. We demonstrate that, in response to iron-limiting conditions, Php4 is required for down-regulation of pcl1 ؉ , sdh4 ؉ , and isa1 ؉ mRNA levels. In vivo RNase protection studies reveal that the expression of php4 ؉ is negatively regulated by iron and that this regulated expression requires a functional fep1 ؉ gene. The results of these studies reveal that Fep1 represses php4؉ expression in response to iron. In contrast, when iron is scarce, Fep1 becomes inactive and php4؉ is expressed to act as a regulatory subunit of the CCAAT-binding factor that is required to block pcl1 ؉ , sdh4 ؉ , and isa1 ؉ gene transcription.

In Schizosaccharomyces pombe, the CCAAT-binding factor is a multisubunit complex that contains the proteins Php2, Php3, Php4, and Php5. Under low iron conditions, Php4 acts as a negative regulatory subunit of the CCAAT-binding factor and fosters repression of genes encoding iron-using proteins. Under conditions of iron excess, Php4 expression is turned off by the iron-dependent transcriptional repressor Fep1. In this study, we developed a biological system that allows us to unlink iron-dependent behavior of Php4 protein from its transcriptional regulation by Fep1. Microscopic analyses revealed that a functional GFP-Php4 protein accumulates in the nucleus under conditions of iron starvation. Conversely, in cells undergoing a transition from low to high iron, GFP-Php4 is exported from the nucleus to the cytoplasm. We mapped a leucine-rich nuclear export signal that is necessary for nuclear exclusion of Php4. This latter process was blocked by leptomycin B. By using coimmunoprecipitation analysis, we showed that Php4 and Crm1 physically interact with each other. Although we determined that nuclear retention of Php4 per se is not sufficient to cause a constitutive repression of iron-using genes, we found that deletion of the grx4 ؉ -encoded glutaredoxin-4 renders Php4 constitutively active and invariably localized in the nucleus. Further analysis by bimolecular fluorescence complementation assay and by two-hybrid assays showed that Php4 and Grx4 are physically associated in vivo. Taken together, our findings indicate that Grx4 and Crm1 are novel components involved in the mechanism by which Php4 is inactivated by iron in a Fep1-independent manner.