Serum hepatitis B virus RNA is encapsidated pregenome RNA that may be associated with persistence of viral infection and rebound.

BACKGROUND & AIMS: Hepatitis B virus (HBV) RNA in serum has recently been linked to efficacy and prognosis of chronic hepatitis B (CHB) treatment. This study explored the nature, origin, underlying mechanisms, and potential clinical significance of serum HBV RNA. METHODS: The levels of HBV DNA and RNA were determined in the supernatant of induced HepAD38, HBV-expressing HepG2.2.15 cells and primary human hepatocytes (PHH), and in the serum of transgenic mice and CHB patients. NP-40 and proteinase K treatment, sucrose density gradient centrifugation, electron microscopy, northern blot, multiple identification PCRs and 5' rapid-amplification of cDNA ends were performed to identify the nature of serum HBV RNA. RESULTS: Although significantly lower than HBV DNA levels, abundant HBV RNA was present in the serum of CHB patients. A series of experiments demonstrated that serum HBV RNA was pregenome RNA (pgRNA) and present in virus-like particles. HBV pgRNA virion levels increased after blocking the reverse transcription activity of HBV DNA polymerase, and decreased after blocking the encapsidation of pgRNA. Furthermore, the presence of HBV pgRNA virion was associated with risk of viral rebound after discontinuation of nucleot(s)ide analogues (NAs) therapy in CHB patients. CONCLUSIONS: Serum HBV RNA was confirmed to be pgRNA present in virus-like particles. HBV pgRNA virions were produced from encapsidated particles in which the pgRNA was non- or partially reverse transcribed. Clinically, HBV pgRNA virion might be a potential biomarker for monitoring safe discontinuation of NA-therapy. LAY SUMMARY: HBV may have another virion form in which the nucleic acid is composed of RNA, not DNA. The level of HBV RNA virion in serum may be associated with risk of HBV viral rebound after withdrawal of treatment, and therefore, a potential predictive biomarker to monitor the safe discontinuation of nucleot(s)ide analogues-therapy.

Hepatitis B Virus X Protein Stabilizes Cyclin D1 and Increases Cyclin D1 Nuclear Accumulation through ERK-Mediated Inactivation of GSK-3ß.

The Hepatitis B virus X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). It has been suggested that the transcriptional activation of cyclin D1 by HBx is implicated in the development of HCC. However, numerous studies have shown that overexpression of cyclin D1 alone is not sufficient to drive oncogenic transformation. Herein, we investigated whether HBx can stabilize cyclin D1 and induce cyclin D1 protein nuclear accumulation, and thereby accelerate hepatocarcinogenesis. The effects of HBx on cyclin D1 stabilization were assessed in cell-based transfection, Western blot, immunoprecipitation, immunocytofluorescence staining, and flow-cytometric assays. The results demonstrated that ectopic expression of HBx in HCC cells could extend the half-life of cyclin D1 protein from 40-60 minutes to 80-110 minutes. HBx stabilized cyclin D1 primarily in the S phase of the cell cycle, in a manner dependent on the inactivation of GSK-3ß, which was mediated by ERK activation. HBx also prompted the nuclear accumulation of cyclin D1, and cotransfection of the constitutively active mutant of GSK-3ß along with HBx could reverse the nuclear accumulation and subsequent cell proliferation induced by HBx. Further, a positive correlation between HBx and nuclear cyclin D1 level was established in HCC specimens detected by an immunohistochemical assay. Taken together, our results indicated that HBx could stabilize and increase cyclin D1 nuclear accumulation through ERK-mediated inactivation of GSK-3ß. This HBx-induced cyclin D1 upregulation might play an important role in HCC development and progression.

Genetic polymorphisms of CXCR5 and CXCL13 are associated with non-responsiveness to the hepatitis B vaccine.

A cohort based study has been undertaken to investigate the possible association of genetic polymorphisms in genes functionally related to follicular T helper (TfH) cells with non-responsiveness to hepatitis B virus (HBV) vaccination. A total of 24 single nucleotide polymorphisms (SNPs) in 6 TfH related genes (CXCR5, ICOS, CXCL13, IL-21, BCL6 and CD40L) were investigated in 20 non-responders and 45 responders to HBV vaccination. Genetic association analysis revealed that three SNPs (rs497916, rs3922, rs676925) in CXCR5 and one SNP (rs355687) in CXCL13 were associated with hepatitis B vaccine efficacy. In addition, significantly unbalanced distributions of two haplotypes, defined by three SNPs (rs497916, rs3922, rs676925) within CXCR5, were also seen between non-responders and responders. Furthermore, we demonstrated that the rs3922 "GG" genotype was associated with higher levels of CXCR5 than the "AG" and "AA" genotype in a group of healthy volunteers. A dual luciferase report assay was used to confirm that the "G" allele in rs3922 may lead to higher gene expression than the "A" allele, implicating that rs3922 might be a functional SNP affecting CXCR5 expression. These results indicated that polymorphism associated changes in CXCR5 expression in TfH cells may be associated with non-responsiveness to hepatitis B vaccination.

Prognostic value of M30/M65 for outcome of hepatitis B virus-related acute-on-chronic liver failure.

AIM: To determine the prognostic value of circulating indicators of cell death in acute-on-chronic liver failure (ACLF) patients with chronic hepatitis B virus (HBV) infection as the single etiology. METHODS: Full length and caspase cleaved cytokeratin 18 (detected as M65 and M30 antigens) represent circulating indicators of necrosis and apoptosis. M65 and M30 were identified by enzyme-linked immunosorbent assay in 169 subjects including healthy controls (n = 33), patients with chronic hepatitis B (CHB, n = 55) and patients with ACLF (n = 81). According to the 3-mo survival period, ACLF patients were defined as having spontaneous recovery (n = 33) and non-spontaneous recovery which included deceased patients and those who required liver transplantation (n = 48). RESULTS: Both biomarker levels significantly increased gradually as liver disease progressed (for M65: P < 0.001 for all; for M30: control vs CHB, P = 0.072; others: P < 0.001 for all). In contrast, the M30/M65 ratio was significantly higher in controls compared with CHB patients (P = 0.010) or ACLF patients (P < 0.001). In addition, the area under receiver operating characteristic curve (AUC) analysis demonstrated that both biomarkers had diagnostic value (AUC ≥ 0.80) in identifying ACLF from CHB patients. Interestingly, it is worth noting that the M30/M65 ratio was significantly different between spontaneous and non-spontaneous recovery in ACLF patients (P = 0.032). The prognostic value of the M30/M65 ratio was compared with the Model for End-Stage Liver Disease (MELD) and Child-Pugh scores at the 3-mo survival period, the AUC of the M30/M65 ratio was 0.66 with a sensitivity of 52.9% and the highest specificity of 92.6% (MELD:AUC = 0.71; sensitivity, 79.4%; specificity, 63.0%; Child-Pugh: AUC = 0.77; sensitivity, 61.8%; specificity, 88.9%). CONCLUSION: M65 and M30 are strongly associated with liver disease severity. The M30/M65 ratio may be a potential prognostic marker for spontaneous recovery in patients with HBV-related ACLF.

The function of targeted host genes determines the oncogenicity of HBV integration in hepatocellular carcinoma.

BACKGROUND & AIMS: Although hepatitis B virus (HBV) integration into the human genome has been considered as one of the major causative factors to hepatocarcinogenesis, the underlying mechanism(s) was still elusive. Here we investigate the essential difference(s) of HBV integration between HCC tumor and adjacent non-tumor tissues and explore the factor(s) that determine the oncogenicity of HBV integration. METHODS: 1115 HBV integration sites were collected from four recent studies. Functional annotation analysis of integration targeted host genes (ITGs) was performed using DAVID based on Gene Ontology and KEGG pathway databases. Array-based expression profiles, real-time qPCR and western blot were used to detect the expression of recurrent integration targeted genes (RTGs). The biological consequences of the overexpression of UBXN8 in 8 HCC cell lines were studied in vitro. RESULTS: HBV is prone to integrate in genic regions (exons, introns, and promoters) and gene-dense regions. Functional annotation analysis reveals that, compared to those in adjacent non-tumor tissues, ITGs in HCC tumor tissues were significantly enriched in functional terms related to negative regulation of cell death, transcription regulation, development and differentiation, and cancer related pathways. 32% of the 75 RTGs identified in this analysis expressed abnormally in HCC tissues. UBXN8, one of the RTGs, was identified as a new tumor suppressor candidate which functions in a TP53 dependent manner. CONCLUSIONS: The oncogenicity of HBV integration was determined, to some extend by the function of HBV integration targeted host genes in HCC.

Re-evaluation of the carcinogenic significance of hepatitis B virus integration in hepatocarcinogenesis.

To examine the role of hepatitis B virus (HBV) integration in hepatocarcinogenesis, a systematic comparative study of both tumor and their corresponding non-tumor derived tissue has been conducted in a cohort of 60 HBV associated hepatocellular carcinoma (HCC) patients. By using Alu-polymerase chain reaction (PCR) and ligation-mediated PCR, 233 viral-host junctions mapped across all human chromosomes at random, no difference between tumor and non-tumor tissue was observed, with the exception of fragile sites (P = 0.0070). HBV insertions in close proximity to cancer related genes such as hTERT were found in this study, however overall they were rare events. No direct correlation between chromosome aberrations and the number of HBV integration events was found using a sensitive array-based comparative genomic hybridization (aCGH) assay. However, a positive correlation was observed between the status of several tumor suppressor genes (TP53, RB1, CDNK2A and TP73) and the number of chromosome aberrations (r = 0.6625, P = 0.0003). Examination of the viral genome revealed that 43% of inserts were in the preC/C region and 57% were in the HBV X gene. Strikingly, approximately 24% of the integrations examined had a breakpoint in a short 15 nt viral genome region (1820-1834 nt). As a consequence, all of the confirmed X gene insertions were C-terminal truncated, losing their growth-suppressive domain. However, the same pattern of X gene C-terminal truncation was found in both tumor and non-tumor derived samples. Furthermore, the integrated viral sequences in both groups had a similar low frequency of C1653T, T1753V and A1762T/G1764A mutations. The frequency and patterns of HBV insertions were similar between tumor and their adjacent non-tumor samples indicating that the majority of HBV DNA integration events are not associated with hepatocarcinogenesis.

The molecular biology of rotaviruses X: intercellular dissemination of rotavirus NSP4 requires glycosylation and is mediated by direct cell-cell contact through cytoplasmic extrusions.

The effect of expressing the NSP4 protein of group A rotaviruses in cells has been studied. It led to the rapid appearance of long cytoplasmic extrusions, which, through site-directed mutagenesis of the N-linked glycosylation sites near the amino terminus of the protein, were shown to be dependent on its ability to become fully glycosylated. Real-time confocal microscopy was used to follow the appearance of similar cytoplasmic extrusions in virus-infected cells and revealed them to grow at a rate of ~2 microns/min to more than three cell diameters and to have a lifespan of 30-60 minutes. CellTracker dyes were used to label cell populations and facilitate the monitoring of the transfer of cytoplasm from virus-infected to surrounding uninfected cells through cytoplasmic extrusions that broke down to vesicles, seen both on the surface of and within uninfected cells in mixed cell populations. Staining of these tagged cell mixtures with monospecific antibody to NSP4 revealed the presence of the protein on uninfected cells, suggesting that the cytoplasmic extrusions formed by virus-infected cells facilitates the direct cell-cell spread of NSP4. This direct cell-cell transfer of infected cell material triggered by expression of glycosylated NSP4 in virus-infected cells may contribute to viral pathogenesis and facilitate host invasion by rotaviruses.

The rotavirus enterotoxin (NSP4) promotes re-modeling of the intracellular microtubule network.

Expression of the rotavirus enterotoxin (NSP4) in transfected monkey kidney cells was found to result in a dramatic re-modeling of the microtubule (MT) network. This important centrosome organized cytoskeletal element was dissolved by expression of NSP4 and re-formed in a ring array at the periphery of the cell, similar to that seen following normal virus infection. Site directed mutagenesis of the N-linked glycosylation sites in NSP4 was employed to show that glycosylation of NSP4 was not required for it to promote changes in the MT network. This result together with experiments using conventional inhibitors indicated that NSP4's ability to cause elevation of intracellular calcium levels was also not necessary to effect the changes in the MT network. Use of the centrosome function inhibitor nocodazole demonstrated that NSP4 based remodeling of the MT network was dominant over the normal organizational role of the centrosome. Finally the remodeling of the MT network was shown not to be linked to cellular apoptosis or necrosis. The potential importance of this newly recognised role for NSP4 in the overall process of intracellular pathogenesis by rotaviruses is discussed.

Aberrant expression of microRNA 155 may accelerate cell proliferation by targeting sex-determining region Y box 6 in hepatocellular carcinoma.

BACKGROUND: Recent research has suggested that the oncomir microRNA 155 (miR-155) is up-regulated in hepatocellular carcinoma (HCC). In this study, the authors investigated the tumorigenic mechanism of this oncomir in the development of HCC. METHODS: Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted to analyze the expressions of miR-155 and its potential target genes in paired tumor tissues and adjacent tumor-free tissues and in disease-free liver tissue samples. The in silico predicted target genes of miR-155 were assessed by dual-luciferase reporting assay, real-time RT-PCR, and Western blot analyses. U6 promoters that drive miR-155 precursor overexpression and miR-155 tough decoy knock-down constructs were used to study its affects on cell proliferation in vitro and on tumor formation in nude mice. RESULTS: Quantitative RT-PCR demonstrated a gradual ascension of miR-155 expression in cirrhotic liver tissues and in HCC tumor tissues compared with low expression levels in normal liver tissues. Ectopic expression of miR-155 in HepG2 cells enhanced its tumorigenesis, whereas depletion of the endogenous miR-155 reversed these tumorigenic properties. Ectopic expression of sex-determining region Y box 6 (SOX6) was able to reverse the growth-promoting property of miR-155. Concordantly, the results demonstrated for the first time that SOX6 is a direct target of miR-155. Further analysis revealed that SOX6 reduced cell growth by up-regulating p21waf1/cip1 expression in a p53-dependent manner. In addition, a decline in p21waf1/cip1 expression caused by miR-155 could be reversed by SOX6 expression. CONCLUSIONS: The current data indicated that SOX6 is a novel target of miR-155 and that miR-155 enhances liver cell tumorigenesis at least in part through the novel miR-155/SOX6/p21waf1/cip1 axis. These findings suggest that miR-155 may be a potential target for HCC treatment.

Interactions in vivo between the Vif protein of HIV-1 and the precursor (Pr55(GAG)) of the virion nucleocapsid proteins.

The abnormality of viral core structure seen in vif-defective HIV-1 grown in PBMCs has suggested a role for Vif in viral morphogenesis. Using an in vivo mammalian two-hybrid assay, the interaction between Vif and the precursor (Pr55(GAG)) of the virion nucleocapsid proteins has been analysed. This revealed the amino-terminal (aa 1-22) and central (aa 70-100) regions of Vif to be essential for its interaction with Pr55(GAG), but deletion of the carboxy-terminal (aa 158-192) region of the protein had only a minor effect on its interaction. Initial deletion studies carried out on Pr55(GAG) showed that a 35-amino-acid region of the protein bridging the MA(p17)-CA(p24) junction was essential for its ability to interact with Vif. Site-directed mutagenesis of a conserved tryptophan (Trp(21)) near the amino terminus of Vif showed it to be important for the interaction with Pr55(GAG). By contrast, mutagenesis of the highly conserved YLAL residues forming part of the BC-box motif, shown to be important in Vif promoting degradation of APOBEC3G/3F, had little or no effect on the Vif-Pr55(GAG) interaction.

Characterization of the NSP6 protein product of rotavirus gene 11.

The 12kDa non-structural protein 6 (NSP6) is the least studied of the rotavirus proteins. In an attempt to further characterize this protein mono-specific antisera was generated using purified protein expressed in E. coli. Pulse/chase radio-labeling of virus infected cells was used to show that it is expressed at a steady but low rate throughout the virus replication cycle. In contrast to the other rotavirus non-structural proteins, NSP6 was found to have a high rate of turnover, being completely degraded within 2h of synthesis. NSP6 tagged with GFP was used to probe the intracellular distribution of the protein, perinuclear aggregates were observed in the cytoplasm of transfected cells. Following virus infection of these transfected cells the aggregates were seen to redistribute to the viroplasms. Consistent with its localization to the site of viral genome replication and packaging, NSP6 was found to be a sequence independent nucleic acid binding protein, with similar affinities for ssRNA and dsRNA.

Changes in the Vif protein of HIV-1 associated with the development of resistance to inhibitors of viral protease.

The protease (PR) and virus infectivity factor (vif) gene sequences of a cohort of HIV-1 infected patients showing evidence of developing protease inhibitor (PI) resistance whilst undergoing highly active antiretroviral therapy (HAART) have been determined. The PR sequences showed the presence of the classical mutations associated with resistance to PIs. The sequence of the Vif protein showed less variation in samples from PI treated patients than in specimens prepared from treatment-naive patients. In addition a number of amino acid positions within Vif showed highly significant preferences for a particular amino acid in the PI-treated cohort compared to the untreated control cohort.

Sequence analysis of the guanylyltransferase (VP3) of group A rotaviruses.

The RNA segment encoding the guanylyltransferase (VP3) from 12 group A rotavirus isolates has been sequenced following RT-PCR and molecular cloning of the full-length amplicons produced. Alignment of the derived amino acid sequences including those of the four VP3 sequences available from GenBank revealed two levels of sequence divergence. Virus isolates from humans showed greater than 94% sequence identity, whereas those isolated from different mammalian species showed as low as 79% sequence identity. The exceptions were avian virus isolates, which diverged approximately 45% from those of mammalian origin, and the human virus isolates DS1 and 69M, which showed much closer (over 90%) identity to viruses of bovine origin, suggesting that these human isolates may have undergone recent reassortment events with a bovine virus. Analysis of the sequences for a putative enzymic active site has revealed that the KXTAMDXEXP and KXXGNNH motifs around amino acids 385 and 545, respectively, are conserved across both group A and C rotaviruses.