Hepatitis B virus nucleocapsid uncoating: biological consequences and regulation by cellular nucleases.

Upon infection of hepatocyte, Hepatitis B virus (HBV) genomic DNA in nucleocapsid is transported into the nucleus and converted into a covalently closed circular (ccc) DNA to serve as the template for transcription of viral RNAs. Viral DNA in the cytoplasmic progeny nucleocapsid is another resource to fuel cccDNA amplification. Apparently, nucleocapsid disassembly, or viral genomic DNA uncoating, is an essential step for cccDNA synthesis from both de novo infection and intracellular amplification pathways, and has a potential to activate DNA sensors and induce an innate immune response in infected hepatocytes. However, where and how the nucleocapsid disassembly occurs is not well understood. The work reported herein showed that the enhanced disassembly of progeny mature nucleocapsids in the cytoplasm supported cccDNA intracellular amplification, but failed to activate the cGAS-STING-mediated innate immune response in hepatocytes. Interestingly, while expression of a cytoplasmic exonuclease TREX1 in human hepatoma cells supporting HBV replication significantly reduced the amounts of cccDNA as well as its precursor, deproteinized relaxed circular (rc) DNA, expression of TREX1 in sodium taurocholate cotransporting polypeptide-expressing human hepatoma cells did not inhibit cccDNA synthesis from de novo HBV infection. The results from this cytoplasmic nuclease protection assay imply that the disassembly of progeny mature nucleocapsids and removal of viral DNA polymerase covalently linked to the 5' end of minus strand of rcDNA take place in the cytoplasm. On the contrary, the disassembly of virion-derived nucleocapsids during de novo infection may occur at a different subcellular compartment and possibly via distinct mechanisms.

Nuclear Sensor Interferon-Inducible Protein 16 Inhibits the Function of Hepatitis B Virus Covalently Closed Circular DNA by Integrating Innate Immune Activation and Epigenetic Suppression.

BACKGROUND AND AIMS: Nuclear-located covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a determining factor for HBV persistence and the key obstacle for a cure of chronic hepatitis B. However, it remains unclear whether and how the host immune system senses HBV cccDNA and its biological consequences. APPROACH AND RESULTS: Here, we demonstrated that interferon-inducible protein 16 (IFI16) could serve as a unique innate sensor to recognize and bind to HBV cccDNA in hepatic nuclei, leading to the inhibition of cccDNA transcription and HBV replication. Mechanistically, our data showed that IFI16 promoted the epigenetic suppression of HBV cccDNA by targeting an interferon-stimulated response element (ISRE) present in cccDNA. It is of interest that this ISRE was also revealed to play an important role in IFI16-activated type I interferon responses. Furthermore, our data revealed that HBV could down-regulate the expression level of IFI16 in hepatocytes, and there was a negative correlation between IFI16 and HBV transcripts in liver biopsies, suggesting the possible role of IFI16 in suppressing cccDNA function under physiological conditions. CONCLUSIONS: The nuclear sensor IFI16 suppresses cccDNA function by integrating innate immune activation and epigenetic regulation by targeting the ISRE of cccDNA, and IFI16 may present as a therapeutic target against HBV infection.

Expression and replication of virus-like circular DNA in human cells.

The consumption of bovine milk and meat is considered a risk factor for colon- and breast cancer formation, and milk consumption has also been implicated in an increased risk for developing Multiple Sclerosis (MS). A number of highly related virus-like DNAs have been recently isolated from bovine milk and sera and from a brain sample of a MS patient. As a genetic activity of these Acinetobacter-related bovine milk and meat factors (BMMFs) is unknown in eukaryotes, we analyzed their expression and replication potential in human HEK293TT cells. While all analyzed BMMFs show transcriptional activity, the MS brain isolate MSBI1.176, sharing homology with a transmissible spongiform encephalopathy-associated DNA molecule, is transcribed at highest levels. We show expression of a replication-associated protein (Rep), which is highly conserved among all BMMFs, and serological tests indicate a human anti-Rep immune response. While the cow milk isolate CMI1.252 is replication-competent in HEK293TT cells, replication of MSBI1.176 is complemented by CMI1.252, pointing at an interplay during the establishment of persistence in human cells. Transcriptome profiling upon BMMF expression identified host cellular gene expression changes related to cell cycle progression and cell viability control, indicating potential pathways for a pathogenic involvement of BMMFs.

HBVcircle: A novel tool to investigate hepatitis B virus covalently closed circular DNA.

BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as a stable episome in infected hepatocytes and serves as a template for the transcription of all viral genes. Due to the narrow host range of HBV, the development of a robust mouse model that supports cccDNA-dependent viral replication is a key hurdle in the development of novel HBV therapeutics. This study aimed to develop a novel tool to investigate HBV cccDNA. METHODS: Through minicircle technology, HBVcircle, a recombinant cccDNA, was easily generated and extracted from a genetically engineered E. coli strain. We characterized the performance of HBVcircle in cell culture by transfection and in immunocompetent mice by hydrodynamic injection (HDI). RESULTS: We demonstrated that HBVcircle formed authentic cccDNA-like molecules in vitro in transiently transfected hepatic cells and in vivo in mouse liver after HDI. HBVcircle supported high levels and persistent HBV replication. In addition, we investigated different factors affecting HBV in vivo replication and persistence, including the host genetic background, vector design and dosage, viral genes and genotypes, and immune activation status. Furthermore, different classes of anti-HBV drugs were also assessed with the HBVcircle system. CONCLUSION: Compared with previous reported HBV mouse models which employ other viral vectors to introduce overlength HBV genomes, viral gene expression and associated phenotypes are entirely driven by cccDNA-like viral genomes in the HBVcircle mouse model. Therefore, the HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery. LAY SUMMARY: To establish a mouse model that supports cccDNA-dependent transcription, a novel tool named HBVcircle, was developed with minicircle technology. HBVcircle formed authentic cccDNA-like molecules in hepatocytes, and supported high levels and persistent HBV replication in vivo. The HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery.

Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus.

Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies.

Associations between activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like cytidine deaminase expression, hepatitis B virus (HBV) replication and HBV-associated liver disease (Review).

The hepatitis B virus (HBV) infection is a major risk factor in the development of chronic hepatitis (CH) and hepatocellular carcinoma (HCC). The activation­induced cytidine deaminase (AID)/apolipoprotein B mRNA editing enzyme, catalytic polypeptide­like (APOBEC) family of cytidine deaminases is significant in innate immunity, as it restricts numerous viruses, including HBV, through hypermutation­dependent and ­independent mechanisms. It is important to induce covalently closed circular (ccc)DNA degradation by interferon­α without causing side effects in the infected host cell. Furthermore, organisms possess multiple mechanisms to regulate the expression of AID/APOBECs, control their enzymatic activity and restrict their access to DNA or RNA substrates. Therefore, the AID/APOBECs present promising targets for preventing and treating viral infections. In addition, gene polymorphisms of the AID/APOBEC family may alter host susceptibility to HBV acquisition and CH disease progression. Through G­to­A hypermutation, AID/APOBECs also edit HBV DNA and facilitate the mutation of HBV DNA, which may assist the virus to evolve and potentially escape from the immune responses. The AID/APOBEC family and their associated editing patterns may also exert oncogenic activity. Understanding the effects of cytidine deaminases in CH virus-induced hepatocarcinogenesis may aid with developing efficient prophylactic and therapeutic strategies against HCC.

A new strategy to deliver synthetic protein drugs: self-reproducible biologics using minicircles.

Biologics are the most successful drugs used in anticytokine therapy. However, they remain partially unsuccessful because of the elevated cost of their synthesis and purification. Development of novel biologics has also been hampered by the high cost. Biologics are made of protein components; thus, theoretically, they can be produced in vivo. Here we tried to invent a novel strategy to allow the production of synthetic drugs in vivo by the host itself. The recombinant minicircles encoding etanercept or tocilizumab, which are synthesized currently by pharmaceutical companies, were injected intravenously into animal models. Self-reproduced etanercept and tocilizumab were detected in the serum of mice. Moreover, arthritis subsided in mice that were injected with minicircle vectors carrying biologics. Self-reproducible biologics need neither factory facilities for drug production nor clinical processes, such as frequent drug injection. Although this novel strategy is in its very early conceptual stage, it seems to represent a potential alternative method for the delivery of biologics.

In vivo electroporation improves therapeutic potency of a DNA vaccine targeting hepadnaviral proteins.

This preclinical study investigated the therapeutic efficacy of electroporation (EP)-based delivery of plasmid DNA (pDNA) encoding viral proteins (envelope, core) and IFN-γ in the duck model of chronic hepatitis B virus (DHBV) infection. Importantly, only DNA EP-therapy resulted in a significant decrease in mean viremia titers and in intrahepatic covalently closed circular DNA (cccDNA) levels in chronic DHBV-carrier animals, compared with standard needle pDNA injection (SI). In addition, DNA EP-therapy stimulated in all virus-carriers a humoral response to DHBV preS protein, recognizing a broader range of major antigenic regions, including neutralizing epitopes, compared with SI. DNA EP-therapy led also to significant higher intrahepatic IFN-γ RNA levels in DHBV-carriers compared to other groups, in the absence of adverse effects. We provide the first evidence on DNA EP-therapy benefit in terms of hepadnaviral infection clearance and break of immune tolerance in virus-carriers, supporting its clinical application for chronic hepatitis B.

Decreased level of recent thymic emigrants in CD4+ and CD8+T cells from CML patients.

BACKGROUND: T-cell immunodeficiency is a common feature in cancer patients, which may relate to initiation and development of tumor. Based on our previous finding, to further characterize the immune status, T cell proliferative history was analyzed in CD4+ and CD8+ T cells from chronic myeloid leukemia (CML) patients. METHODS: Quantitative analysis of deltaRec-psiJalpha signal joint T cell receptor excision circles (sjTRECs) was performed in PBMCs, CD3+, CD4+ and CD8+T cells by real-time PCR, and the analysis of 23 TRBV-D1 sjTRECs was performed by semi-nested PCR. Forty eight CML cases in chronic phase (CML-CP) were selected for this study and 17 healthy individuals served as controls. RESULTS: The levels of deltaRec-psiJalpha sjTRECs in PBMCs, CD3+, CD4+, and CD8+ T cells were significantly decreased in CML patients, compared with control groups. Moreover, the numbers of detectable TRBV subfamily sjTRECs, as well as the frequency of particular TRBV-BD1 sjTRECs in patients with CML were significantly lower than those from healthy individuals. CONCLUSIONS: We observed decreased levels of recent thymic emigrants in CD4+ and CD8+ T cells that may underlay the persistent immunodeficiency in CML patients.

Gut IgA class switch recombination in the absence of CD40 does not occur in the lamina propria and is independent of germinal centers.

Conflicting findings have recently been presented as to the sites and sources of B cells that undergo class switch recombination (CSR) to IgA in the gut. In this study we provide compelling evidence in CD40(-/-) mice demonstrating that IgA CSR can be independent of CD40 signaling and germinal center formation and does not occur in the gut lamina propria (LP) itself. We found that CD40(-/-) mice had near normal levels of gut total IgA despite lacking germinal centers and completely failing to raise specific responses against the T cell-dependent Ags cholera toxin and keyhole limpet hemocyanin. The Peyer's patches in CD40(-/-) mice expressed unexpectedly high levels of activation-induced cytidine deaminase mRNA and germline alpha transcripts, but few postswitch circular DNA transcripts, arguing against significant IgA CSR. Moreover and more surprisingly, wild-type mice exhibited no to low IgA CSR in mesenteric lymph nodes or isolated lymphoid follicles. Importantly, both strains failed to demonstrate any of the molecular markers for IgA CSR in the gut LP itself. Whereas all of the classical sites for IgA CSR in the GALT in CD40(-/-) mice appeared severely compromised for IgA CSR, B cells in the peritoneal cavity demonstrated the expression of activation-induced cytidine deaminase mRNA comparable to that of wild-type mice. However, peritoneal cavity B cells in both strains expressed intermediate levels of the germinal center marker GL7 and exhibited no germline alpha transcripts, and only three of 51 mice analyzed showed the presence of postswitch circular DNA transcripts. Taken together, these findings strongly argue for alternative inductive sites for gut IgA CSR against T cell-independent Ags outside of the GALT and the nonorganized LP.

Screening of an Echinococcus granulosus cDNA library with IgG4 from patients with cystic echinococcosis identifies a new tegumental protein involved in the immune escape.

The worldwide problem of chronic Echinococcus granulosus disease calls for new parasite-derived immunomodulatory molecules. By screening an E. granulosus cDNA library with IgG4 from patients with active cystic echinococcosis, we identified a cDNA that encodes a predicted partial protein that immunofluorescence studies localized in the protoscolex tegument and on the germinal layer of cyst wall. We named this protein EgTeg because the 105 amino acid sequence scored highest against a family of Schistosoma tegumental proteins. Evaluating the role of EgTeg in the human early inflammatory response we found that EgTeg significantly inhibited polymorphonuclear cell (PMN) chemotaxis. Cytometric analysis of intracellular cytokines disclosed a significantly higher percentage of cells producing IL-4 than IFN-gamma (P = 0.001, Student's t-test) in T lymphocytes from patients with cystic echinococcosis stimulated with EgTeg. EgTeg induced weak Th1-dependent proliferation in 42% of patients' peripheral blood mononuclear cells. In immunoblotting (IB) analysis of total IgG and IgG subclass responses to EgTeg in patients with cystic echinococcosis, patients with other parasitoses, patients with cystic lesions and healthy controls, total IgG specific to EgTeg yielded high sensitivity (73%) but low specificity (44%) precluding its use in immunodiagnosis. Conversely, IgG4 specific to EgTeg gave acceptable sensitivity (65%) and high specificity (89%) suggesting its use in immunodiagnosis to confirm ultrasound documented cysts suggestive of E. granulosus. Because the new tegumental antigen EgTeg inhibits chemotaxis, induces IL-4-positive T lymphocytes and noncomplement fixing antibodies (IgG4) it is an immunomodulatory molecule associated with chronic infection.

Restricted cytokine production from mouse peritoneal macrophages in culture in spite of extensive uptake of plasmid DNA.

The production of inflammatory cytokines from macrophages (Mphi), upon stimulation with plasmid DNA (pDNA) containing CpG motifs, is a critical process for DNA-based therapies such as DNA vaccination and gene therapy. We compared Mphi activation, following stimulation with naked pDNA, based on the production of cytokines from cell lines (RAW264.7 and J774A1) and peritoneal Mphis in primary culture. The Mphi cell lines RAW264.7 and J774A1 produced a significant amount of tumour necrosis factor-alpha (TNF-alpha) upon stimulation with naked pDNA and this response required endosomal acidification. On the other hand, peritoneal Mphis (both resident and elicited) in primary culture did not secrete TNF-alpha or interleukin-6, although they contain the mRNA of toll-like receptor-9 (TLR-9) and are able to respond to CpG oligodeoxynucleotides. This unresponsiveness was not a result of impaired cellular uptake of pDNA because the primary cultured Mphis showed a higher uptake of pDNA than the RAW264.7 and J774A1 cell lines. These findings have important implications for Mphi activation by naked pDNA as it has been generally assumed that pDNA that contains CpG motifs is a potent agent for inducing inflammatory cytokines in vivo, based on evidence from in vitro studies using Mphi cell lines.

Assessment of thymic activity in human immunodeficiency virus-negative and -positive adolescents by real-time PCR quantitation of T-cell receptor rearrangement excision circles.

Circular DNA molecules known as T-cell receptor rearrangement excision circles (TREC) arise during T-cell development and are present in cells that have recently emigrated from the thymus. In cross-sectional studies, the number of peripheral blood lymphocytes bearing TREC decreases with age, consistent with an anatomically demonstrated loss of thymic epithelial tissue. TREC numbers increase following hematopoietic stem cell transplantation and during therapy for human immunodeficiency virus (HIV) infection. Quantitation of TREC has therefore been proposed as a parameter of thymic activity. In this study, we used real-time PCR to quantify TREC in peripheral blood samples obtained longitudinally from HIV-seronegative adolescents. TREC values in peripheral blood T cells were very stable throughout adolescence, once thought to be a time of rapid involution of the thymus. In addition, in a cross-sectional analysis, we examined TREC values in a cohort of HIV-positive adolescents and found evidence of ongoing thymopoiesis in perinatally infected individuals, despite lifelong infection. These data demonstrate the utility of TREC assessment in adolescents and that HIV infection does not uniformly result in accelerated thymic involution in childhood.

Quantification of T-cell receptor excision circle DNA using fluorescence resonance energy transfer and the LightCycler system.

T-cell receptor excision circles (TRECs) are circular, stable extrachromosomal DNA fragments and are generated during V(D)J gene recombination, a process responsible for diversity of the T-cell receptor repertoire. Here we describe a sensitive, rapid and easy to perform real-time PCR assay based on the LightCycler technique for the quantification of TRECs among peripheral blood cells and a comparison of this assay with conventional PCR-ELISA. By LightCycler, a sensitivity of 20 copies of plasmid deltaRec-psiJalpha Signal Joint TREC DNA was achieved whereas by PCR-ELISA, a detection limit of 2 copies was demonstrated. In blood samples from healthy individuals (n=52) a median TREC count of 1.6x10(4) copies [range 2x10(1) to 2x10(5)]/2x10(5) peripheral blood mononuclear cells (PBMNCs) and in cord blood, a median TREC count of 1.45x10(5) copies [range 1.2x10(5) to 1.6x10(5)]/2x10(5) PBMNCs) could be detected. No significant difference was found in 15 individuals when unfractionated PBMNCs (median count of 1.1x10(5) copies/2x10(5) PBMNCs) or magnetic associated cell sorter (MACS)-sorted CD45RA+ T-cells (median count of 2.5x10(5) copies/2x10(5) cells) were analyzed for TREC counts. In addition, we examined the number of deltaRec-psiJalpha Signal Joint TREC in paediatric (n=6) and adult patients (n=7) after allogeneic stem cell transplantation. In children, we observed a median TREC count of 5.7x10(4) copies/2x10(5) PBMNCs after 1x10(4) and 5.6x10(4) copies/2x10(5) PBMNCs after 2 years, and in adults, a median count of 3.6x10(4) copies/2x10(5) PBMNCs after 1x10(4) and 1.1x10(4) copies/2x10(5) PBMNCs after 2 years. In conclusion, the LightCycler-based real-time PCR assay described offers a very sensitive and rapid tool for the quantification of TREC DNA.