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Antiviral drug discovery aims to identify novel agents that inhibit viral replication or reduce expression of key pathogenic viral proteins while minimizing adverse effects. Target-based screening has been employed extensively to identify compounds targeting both viral and host proteins. While phenotypic screening has successfully identified antiviral compounds, determining each compound's target is challenging. Approximately 240 million individuals worldwide are chronically infected with hepatitis B virus (HBV), and more than 650 000 people die per year from HBV-associated liver diseases. The hallmarks of chronic HBV infection are high viral load (HBV DNA) and higher levels of non-infectious particles containing the tolerogenic viral S antigen (HBsAg). The current standard of care effectively reduces viremia, but rarely results in a functional cure, defined as sustained HBsAg loss. Recently, a novel, potent, small-molecule inhibitor of HBV gene expression (RG7834) was discovered from a phenotypic screen focused on identifying HBsAg production inhibitors. Target identification efforts led to the discovery of the non-canonical poly(A) RNA polymerases PAPD5 and PAPD7, which are required for HBV RNA stabilization, and are effectively antagonized by RG7834. Thus, we highlight the powerful approach of phenotypic screening for identifying novel antiviral drugs and unraveling key biological mechanisms important for the viral lifecycle.

Hepatitis E virus (HEV) is a 7.2 kb positive-sense, single-stranded RNA virus containing three partially overlapping reading frames, ORF 1-3. All non-structural proteins required for viral replication are encoded by ORF1 and are transcribed as a single transcript. Computational analysis of the complete ORF1 polyprotein identified a previously uncharacterized region of predicted secondary structure bordered by two disordered regions coinciding partially with a region predicted as a putative cysteine protease. Following successful cloning, expression and purification of this region, the crystal structure of the identified protein was determined and identified to have considerable structural homology to a fatty acid binding domain. Further analysis of the structure revealed a metal binding site, shown unambiguously to specifically bind zinc via a non-classical, potentially catalytic zinc-binding motif. We present analysis for the first time of this identified non-structural protein, expanding the knowledge and understanding of the complex mechanisms of HEV biology.ImportanceHepatitis E virus (HEV) is an emerging virus found predominately in developing countries causing an estimated 20 million infections, which result in approximately 57,000 deaths a year. Although it is known that the non-structural proteins of the HEV ORF1 are expressed as a single transcript, there is debate as to whether ORF1 functions as a single polyprotein or if it is processed into separate domains via a viral or endogenous cellular protease. In the following paper, we present the first structural and biophysical characterization of a HEV non-structural protein using a construct that has partially overlapping boundaries with the predicted putative cysteine protease. Based on the structural homology of the HEV protein with known structures, along with the presence of a catalytic zinc-binding motif, it is possible that the identified protein corresponds to the HEV protease, which could require activation or repression through the binding of a fatty acid. This represents a significant step forward in the characterization and the understanding of the molecular mechanisms of the HEV genome.

Latency-associated nuclear antigen (LANA), a multifunctional protein expressed by the Kaposi sarcoma-associated herpesvirus (KSHV) in latently infected cells, is required for stable maintenance of the viral episome. This is mediated by two interactions: LANA binds to specific sequences (LBS1 and LBS2) on viral DNA and also engages host histones, tethering the viral genome to host chromosomes in mitosis. LANA has also been suggested to affect host gene expression, but both the mechanism(s) and role of this dysregulation in KSHV biology remain unclear. Here, we have examined LANA interactions with host chromatin on a genome-wide scale using chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) and show that LANA predominantly targets human genes near their transcriptional start sites (TSSs). These host LANA-binding sites are generally found within transcriptionally active promoters and display striking overrepresentation of a consensus DNA sequence virtually identical to the LANA-binding site 1 (LBS1) motif in KSHV DNA. Comparison of the ChIP-seq profile with whole-transcriptome (high-throughput sequencing of RNA transcripts [RNA-seq]) data reveals that few of the genes that are differentially regulated in latent infection are occupied by LANA at their promoters. This suggests that direct LANA binding to promoters is not the prime determinant of altered host transcription in KSHV-infected cells. Most surprisingly, the association of LANA to both host and viral DNA is strongly disrupted during the lytic cycle of KSHV. This disruption can be prevented by the inhibition of viral DNA synthesis, suggesting the existence of novel and potent regulatory mechanisms linked to either viral DNA replication or late gene expression. IMPORTANCE Here, we employ complementary genome-wide analyses to evaluate the distribution of the highly abundant latency-associated nuclear antigen, LANA, on the host genome and its impact on host gene expression during KSHV latent infection. Combined, ChIP-seq and RNA-seq reveal that LANA accumulates at active gene promoters that harbor specific short DNA sequences that are highly reminiscent of its cognate binding sites in the virus genome. Unexpectedly, we found that such association does not lead to remodeling of global host transcription during latency. We also report for the first time that LANA's ability to bind host and viral chromatin is highly dynamic and is disrupted in cells undergoing an extensive lytic reactivation. This therefore suggests that the association of LANA to chromatin during a productive infection cycle is controlled by a new regulatory mechanism.

Depending on age of acquisition, hepatitis B virus (HBV) can induce a cell-mediated immune response that results in either cure or progressive liver injury. In adult-acquired infection, HBV antigens are usually cleared, whereas in infancy-acquired infection, they persist. Individuals infected during infancy therefore represent the majority of patients chronically infected with HBV (CHB). A therapy that can promote viral antigen clearance in most CHB patients has not been developed and would represent a major health care advance and cost mitigator. Using an age-dependent mouse model of HBV clearance and persistence in conjunction with human blood and liver tissue, we studied mechanisms of viral clearance to identify new therapeutic targets. We demonstrate that age-dependent expression of the costimulatory molecule OX40 ligand (OX40L) by hepatic innate immune cells is pivotal in determining HBV immunity, and that treatment with OX40 agonists leads to improved HBV antigen clearance in young mice, as well as increased strength of T cell responses in young mice and adult mice that were exposed to HBV when they were young and developed a CHB serological profile. Similarly, in humans, we show that hepatic OX40L transcript expression is age-dependent and that increased OX40 expression on peripheral CD4+ T cells in adults is associated with HBV clearance. These findings provide new mechanistic understanding of the immune pathways and cells necessary for HBV immunity and identify potential therapeutic targets for resolving CHB.

Mutations in human (Homo sapiens) ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) result in the complex metabolic disease ethylmalonic encephalopathy, which is characterized in part by brain lesions, lactic acidemia, excretion of ethylmalonic acid, and ultimately death. ETHE1-like genes are found in a wide range of organisms; however, the biochemical and physiological role(s) of ETHE1 have not been examined outside the context of ethylmalonic encephalopathy. In this study we characterized Arabidopsis (Arabidopsis thaliana) ETHE1 and determined the effect of an ETHE1 loss-of-function mutation to investigate the role(s) of ETHE1 in plants. Arabidopsis ETHE1 is localized in the mitochondrion and exhibits sulfur dioxygenase activity. Seeds homozygous for a DNA insertion in ETHE1 exhibit alterations in endosperm development that are accompanied by a delay in embryo development followed by embryo arrest by early heart stage. Strong ETHE1 labeling was observed in the peripheral and chalazal endosperm of wild-type seeds prior to cellularization. Therefore, ETHE1 appears to play an essential role in regulating sulfide levels in seeds.

The protein product of gene At1g53580 from Arabidopsis thaliana possesses 54% sequence identity to a human enzyme that has been implicated in the rare disorder ethylmalonic encephalopathy. The structure of the At1g53580 protein has been solved to a nominal resolution of 1.48 Angstrom. This structure reveals tertiary structure differences between the ETHE1-like enzyme and glyoxalase II enzymes that are likely to account for differences in reaction chemistry and multimeric state between the two types of enzymes. In addition, the Arabidopsis ETHE1 protein is used as a model to explain the significance of several mutations in the human enzyme that have been observed in patients with ethylmalonic encephalopathy.