e Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/ cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection. Humans are the only natural hosts of hepatitis B virus (HBV), and hepatocytes are the only recognized cells that support productive HBV infection in vivo (1). Viral gene transcription and replication in hepatic cells have been extensively studied (1), and high levels of infectious HBV virions can be produced in cultured cells with relative ease (2). The extremely restricted host and hepatocyte tropism of HBV infection, as well as the possibility of obtaining large amounts of virus in vitro, makes HBV an ideal candidate for the development of hepatocyte-targeting delivery vectors. HBV-based vectors are also invaluable for the study of HBV infection mechanisms.The highly compact HBV genome contains four overlapping open reading frames (ORFs) (preC/C, P, preS1/preS2/S, and X) (Fig. 1A). Multiple essential cis elements overlap these ORFs and function at the DNA or RNA level during different stages of the viral life cycle. Mature virions contain partially double-stranded, relaxed circular DNA (rcDNA) genomes. Upon infection of hepatocytes, rcDNA genomes are converted into covalently closed circular DNA (cccDNA), which serves as a transcription template for viral RNA species. Viral pregenomic RNA (pgRNA), which also functions as mRNA for polymerase, is bound by newly translated polymerase, preferentially in cis. The pgRNA-polymerase complex is then packaged by viral core proteins, also translated using pgRNA as mRNA. Reverse transcription and synthesis of rcDNA take place within the capsids. Mature capsids are subsequently enveloped by membranes containing viral large/middle/small (L/ M/S) surface proteins, encoded by the preS1/preS2/S ORF, to produce progeny virions that bud into the endoplasmic reticulum (ER) lumen to be secreted (1).The compact nature of HBV genome ...

Myeloid differentiation primary response protein 88 (MyD88), which can be induced by alpha interferon (IFN-␣), has an antiviral activity against the hepatitis B virus (HBV). The mechanism of this antiviral activity remains poorly understood. Here, we report that MyD88 inhibited HBV replication in HepG2.2.15 cells and in a mouse model. The knockdown of MyD88 expression weakened the IFN-␣-induced inhibition of HBV replication. Furthermore, MyD88 posttranscriptionally reduced the levels of viral RNA. Remarkably, MyD88 accelerated the decay of viral pregenomic RNA in the cytoplasm. Mapping analysis showed that the RNA sequence located in the 5-proximal region of the pregenomic RNA was critical for the decay. In addition, MyD88 inhibited the nuclear export of pre-S/S RNAs via the posttranscriptional regulatory element (PRE). The retained pre-S/S RNAs were shown to degrade in the nucleus. Finally, we found that MyD88 inhibited the expression of polypyrimidine tract-binding protein (PTB), a key nuclear export factor for PRE-containing RNA. Taken together, our results define a novel antiviral mechanism against HBV mediated by MyD88.Hepatitis B virus (HBV) is a noncytopathic, enveloped virus with a circular, double-stranded DNA genome. It causes both acute and chronic infection of the human liver. Although a highly effective preventive vaccine is now available, HBV infection remains a major health problem worldwide. It is estimated that chronic HBV infection affects 350 to 400 million people globally, about a quarter of whom will eventually develop severe liver diseases, including liver cirrhosis, liver failure, and hepatocellular carcinoma (HCC) (4).Current antiviral therapies involve the use of nucleoside analogs and alpha interferon (IFN-␣) (28). IFN-␣, a type I interferon, engages the IFN-␣ receptor complex to activate the Jak/Stat pathway and trigger the transcription of a diverse set of genes, referred to as IFN-stimulated genes (ISGs) (2, 40). In total, the gene products of ISGs establish an antiviral response in target cells (2, 40). IFN-␣ inhibits HBV replication through a variety of mechanisms. It was reported previously that IFN-␣ can suppress viral gene expression, prevent the formation of viral RNA-containing core particles, and reduce the accumulation of viral replicative intermediates (11,35,37,(46)(47)(48). Importantly, the precise antiviral mechanism of IFN-␣ and the biological functions of many ISGs have not been fully elucidated.Myeloid differentiation primary response protein 88 (MyD88) is a key adaptor in the signaling cascade of the innate immune response (22). We and others have shown that MyD88 expression can be induced by IFN-␣ and that MyD88 has an antiviral activity against HBV in hepatoma cells that is mediated by nuclear factor B (NF-B) activation (12, 25, 51, 52). To counteract its inhibition, the HBV polymerase dampens the activation of the MyD88 promoter by blocking the nuclear translocation of Stat1, thereby reducing IFN-␣-inducible MyD88 expression (50), further suggesting a critical rol...

Signal transducers and activators of transcription 1 (STAT1) is activated by tyrosine phosphorylation upon interferon-gamma (IFN-gamma) stimulation. Phosphorylated STAT1 translocates into nucleus to initiate the transcription of IFN-gamma target genes that are important in mediating antiviral, antiproliferative, and immune response. The inactivation of STAT1 is mainly accomplished via tyrosine dephosphorylation by the nuclear isoform of T cell protein tyrosine phosphatase (TC45) in nucleus. Here we show that beta-arrestin1 directly interacts with STAT1 in nucleus after IFN-gamma treatment and accelerates STAT1 tyrosine dephosphorylation by recruiting TC45. Consequently, beta-arrestin1 negatively regulates STAT1 transcription activity as well as the IFN-gamma-induced gene transcription. Application of beta-arrestin1 siRNA significantly enhances IFN-gamma-induced antiviral response in vesicular stomatitis virus (VSV)-infected cells. Our results reveal that nuclear beta-arrestin1, acting as a scaffold for the dephosphorylation of STAT1, is an essential negative regulator of IFN-gamma signaling and participates in the IFN-gamma-induced cellular antiviral response.

A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W 1 T 2 X 3 W 4 W 5 ) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection.Attachment of virions to human hepatocyte membrane via the interaction of the viral envelope protein with a specific cell surface receptor is considered the initial step of hepatitis B virus (HBV) infection. Besides host-derived phospholipids, the envelope of an HBV virion contains virus-derived small (SHBs), middle (MHBs), and large (LHBs) surface proteins translated from distinct initiation codons while sharing a common carboxyl domain (i.e., SHBs). Consequently, LHBs contains an extra N-terminal PreS domain, which is further divided into the amino-PreS1 and carboxyl-PreS2 domains. MHBs contains the PreS2 domain but lacks the PreS1 domain. LHBs is associated mainly with infectious virions but can also be found in smaller amounts on rod-shaped subviral particles. On the other hand, the spherical subviral particles are composed mainly of SHBs. LHBs plays a pivotal role in the infection and budding stages of the HBV life cycle. LHBs proteins in the virion envelope exhibit mixed topologies, with their PreS domains protruding either inwardly or outwardly (32,35

Prospero-related homeobox protein (Prox1) plays essential roles in the development of many tissues and organs. In the present study, we show that Prox1 is modified by the small ubiquitin-like protein SUMO-1 in cultured cells. Mutation analysis identified at least four potential sumoylation sites within the repression domain of Prox1. Our data indicate that sumoylation of Prox1 reduces its interaction with HDAC3 and as a result downregulates its corepressor activity. These findings suggest that sumoylation may serve as a novel mechanism for the regulation of Prox1Õs corepressor activity. Structured summary:MINT-6787569: PROX1 (uniprotkb:Q92786) physically interacts (MI:0218) with HDAC3 (uniprotkb:O15379) by anti tag coimmunoprecipitation (MI:0007) MINT-6787767: PROX1 (uniprotkb:Q92786) physically interacts (MI:0218) with SUMO-1 (uniprotkb:P63165) by anti tag coimmunoprecipitation (MI:0007)

Hepatitis B virus (HBV) gene transcription is controlled by viral promoters and enhancers, the activities of which are regulated by a number of cellular factors as well as virally encoded proteins. Negative regulation of HBV cis-element activities by cellular factors has been reported less widely than their activation. In this study, we report that nuclear factor Prospero-related homeobox protein (Prox1) represses HBV antigen expression and genome replication in cultured hepatocytes. By using reporter-gene analysis, three of the four HBV promoters, namely the enhancer II/core promoter (ENII/Cp), preS1 promoter (Sp1) and enhancer I/X promoter, were identified as targets for Prox1-mediated repression. Mechanistic analysis then revealed that, for ENII/Cp, Prox1 serves as a corepressor of liver receptor homologue 1 (LRH-1) and downregulates LRH-1-mediated activation of ENII/Cp, whereas for Sp1, Prox1 partially represses Sp1 activity by interacting directly with hepatocyte nuclear factor 1. Identification of Prox1 as an HBV repressor will help in the understanding of detailed interactions between viral cis elements and host cellular factors and may also form the basis for new anti-HBV intervention therapeutics.

No abstract

Co-circulation of subgenotypes IA and IB of hepatitis A virus (HAV) has been reported in South Africa, South America, Europe, and the United States. In this study, phylogenetic and recombination analyses were performed for the first time on 31 complete HAV genomes from infected humans and simians. Three potentially significant intra-genotypic recombination events (I-III) were identified by recombination detection analysis. Recombination events I and II occurred between the lineages represented, respectively, by the Japanese isolate AH2 (AB020565, subgenotype IA) and the North African isolate MBB (M20273, subgenotype IB), giving rise to the recombinant Uruguayan isolate HAV5 (EU131373). Recombination event III occurred between the lineages represented, respectively, by the North African isolate MBB (M20273, subgenotype IB) and the German isolate GBM (X75215, subgenotype IA), resulting in the Italian isolate FG (X83302). The findings demonstrate that humans can be co-infected with different HAV subgenotypes and provide valuable hints for future research on HAV diversity.