Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular carcinoma (HCC).The diagnosis of HCC is usually made in the late stages of the disease, when treatment options are limited and prognosis is poor. We therefore have developed a method of glycoproteomic analysis in an attempt to discover serum markers that can assist in the early detection of HBV-induced liver cancer. Briefly, a comparative method for analysis of oligosaccharides released from serum glycoproteins and for recovery and identification of proteins with aberrant glycosylation, as a function of cancer diagnosis, is described. The model we have used is the woodchuck (Marmota monax), which shares similarities in the glycosylation pattern associated with liver proteins in human HCC. In this report, we show that woodchucks diagnosed with HCC have dramatically higher levels of serumassociated core ␣-1,6-linked fucose, as compared with woodchucks without a diagnosis of HCC. The coupling of this methodology with 2D gel proteomics has permitted the identification of several glycoproteins with altered glycosylation as a function of cancer. One such glycoprotein, Golgi Protein 73 (GP73), was found to be elevated and hyperfucosylated in animals with HCC. Further, the study showed GP73 to be elevated in the serum of people with a diagnosis of HCC, providing a validation of our approach. The potential of this technology for biomarker discovery and the implications of increased levels of GP73 in liver cancer are discussed.glycomics ͉ hepatitis B virus ͉ hepatocellular carcinoma ͉ proteomics

We show that hepatitis C virus (HCV) p7 protein forms ion channels in black lipid membranes. HCV p7 ion channels are inhibited by long-alkyl-chain iminosugar derivatives, which have antiviral activity against the HCV surrogate bovine viral diarrhea virus. HCV p7 presents a potential target for antiviral therapy.H epatitis C virus (HCV) is the major cause of chronic hepatitis with a significant risk of end-stage liver cirrhosis and hepatocellular carcinoma (1). HCV belongs to the family Flaviviridae, which consists of three genera: flaviviruses, pestiviruses, and hepaciviruses. In the absence of both a suitable small animal model and a reliable in vitro infectivity assay for HCV, potential antiviral drugs initially have been tested by using a related pestivirus, bovine viral diarrhea virus (BVDV) (2). BVDV in vitro infectivity assays were used to demonstrate that long-alkyl-chain iminosugar derivatives containing either the glucose analogue deoxynojirimycin (DNJ) or the galactose analogue deoxygalactonojirimycin (DGJ) are potent antiviral inhibitors (3).DNJ derivatives inhibit endoplasmic reticulum (ER) ␣-glucosidases I and II (4, 5), and this inhibition leads to the misfolding of many host-and virus-encoded glycoproteins, including the envelope glycoproteins of BVDV (6) and HCV (7). Previous experiments have shown that the antiviral effect of the longalkyl-chain derivative N-nonyl-DNJ (NN-DNJ) is more pronounced than that of the short-alkyl-chain derivative N-butyl-DNJ (NB-DNJ), although the latter achieves a more effective ER ␣-glucosidase inhibition in cellulo. In addition, long-alkylchain DGJ derivatives that are not recognized by and do not inhibit ER ␣-glucosidases also show potent antiviral activity (3). Therefore, ER ␣-glucosidase inhibition does not correlate directly with the observed antiviral effect and is ruled out as the sole antiviral mechanism.The additional mechanism of action apparently is associated with the length of the alkyl side chain, because the short-chain N-butyl-DGJ (NB-DGJ) shows no antiviral activity, whereas the long-alkyl-chain derivative NN-DGJ is a potent inhibitor (3).The predominant antiviral mechanism is proposed to be mediated directly or indirectly by an effect of the long-alkyl side chains on the membrane and͞or membrane proteins, because treatment with long-alkyl-chain iminosugars affects the dimerization of viral membrane glycoproteins and alters the membrane glycoprotein composition of secreted BVDV virions but does not influence either viral RNA replication or protein synthesis (3).We decided to investigate the small membrane-spanning protein p7 as a potential target of long-alkyl-chain iminosugar derivatives, because flaviviruses such as dengue virus and Japanese encephalitis virus (8), which do not contain p7, are not inhibited by long-alkyl-chain DGJ derivatives, whereas pestiviruses are (3). Pesti-and hepaciviruses both contain the p7 protein.Most functional data about p7 are derived from the pestivirus p7, a 70-aa protein very similar to HCV p7. Functional data hav...

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One function of N-linked glycans is to assist in the folding of glycoproteins by mediating interactions of the lectin-like chaperone proteins calnexin and calreticulin with nascent glycoproteins. These interactions can be prevented by inhibitors of the ␣-glucosidases, such as N-butyl-deoxynojirimycin (NB-DNJ) and N-nonyl-DNJ (NN-DNJ), and this causes some proteins to be misfolded and retained within the endoplasmic reticulum (ER). We have shown previously that the NN-DNJ-induced misfolding of one of the hepatitis B virus (HBV) envelope glycoproteins prevents the formation and secretion of virus in vitro and that this inhibitor alters glycosylation and reduces the viral levels in an animal model of chronic HBV infection. This led us to investigate the effect of glucosidase inhibitors on another ER-budding virus, bovine viral diarrhea virus, a tissue culture surrogate of human hepatitis C virus (HCV). Here we show that in MDBK cells ␣-glucosidase inhibitors prevented the formation and secretion of infectious bovine viral diarrhea virus. Data also are presented showing that NN-DNJ, compared with NB-DNJ, exhibits a prolonged retention in liver in vivo. Because viral secretion is selectively hypersensitive to glucosidase inhibition relative to the secretion of cellular proteins, the possibility that glucosidase inhibitors could be used as broadbased antiviral hepatitis agents is discussed. A single drug against HBV, HCV, and, possibly, HDV, which together chronically infect more than 400 million people worldwide, would be of great therapeutic value.

An aqueous extract of the plant PhyUanthus niruri inhibits endogenous DNA polymerase of hepatitis B virus and binds to the surface antigen of hepatitis B virus in vitro. The extract also inhibits woodchuck hepatitis virus (WHV) DNA polymerase and binds to the surface antigen of WHV in vitro. The extract, nontoxic to mice, was tested for antiviral activity in woodchucks (Marmota monax). In a trial using six long-term WHV-carrier woodchucks, five treated animals showed a faster decrease in woodchuck hepatitis virus surface antigen titer compared to one untreated control. In animals recently infected with WHV, the extract was effective when administered i.p. in three out of four animals in reducing and within 3-6 weeks eliminating both the surface antigen titer and DNA polymerase activity in serum. The treatment was discontinued after 10 weeks, and the treated animals have remained free of detectable markers of WHV for more than 45 weeks. In contrast, three untreated controls remained positive for both markers for WHV. One of the controls died after 8 weeks; the other two controls have remained positive for WHV markers for more than 45 weeks. In a third trial with long-term carriers, test animals treated subcutaneously with the extract for 12 weeks did not respond; but on switching the mode of administration to i.p., two out of the five animals showed a significant decrease in woodchuck hepatitis virus surface antigen titer compared to controls.Chronic carriers of the hepatitis B virus (HBV) may remain asymptomatic for long periods, but many are at high risk of eventually developing post-hepatitic cirrhosis and primary hepatocellular carcinoma. Carriers are often infected within the first few years of life, but symptoms of chronic liver disease and primary hepatocellular carcinoma may not be perceived until the third, fourth, or later decades; pathogenesis, even though relentless, is slow (1, 2).Materials of animal, bacterial, and plant origin (3) have been described that appeared to interfere with the binding of the HBV surface antigen (HBsAg) to the HBsAg antibody (anti-HBs). Subsequently, about 1200 species of plant were tested, and about one-third were found to inhibit antiHBs-HBsAg binding. To obtain more specificity and increase the probability of obtaining an effective therapeutic agent, in addition to the inhibition of HBsAg-anti-HBs binding, we examined plant extracts in vitro to determine if they inhibited the endogenous DNA polymerase (DNAp) of HBV, which is necessary for its replication. The first plant tested was Phyllanthus niruri, which has been and is used widely (4) To assess the effects of P. niruri on the replication of HBV-like viruses in vivo, we used the woodchuck (Marmota monax) as an animal model. The carrier state in woodchucks and humans is similar. Liver diseases including primary hepatocellular carcinoma induced by woodchuck hepatitis virus (WHV) in woodchucks are very similar to those induced by HBV in humans. WHV is similar to HBV (6, 7) with substantial immunological cross-re...