Inhibitors of alpha glucosidases prevent the trimming of oligosaccharides on certain nascent glycoproteins, including the hepatitis B virus MHBs envelope glycoprotein. MHBs proteins with untrimmed oligosaccharides do not interact with calnexin, increasing protein misfolding and subsequent degradation by proteasomes. As peptides loaded onto newly synthesized MHC class I complexes are predominantly derived from proteasomes, the possibility that glucosidase inhibition could increase presentation by MHC class I was determined. Using either a model epitope, or a natural MHBs epitope, it was demonstrated that glucosidase inhibitors enhanced presentation by MHC class I and promoted activation of antigen-specific CTLs, suggesting a pharmacologic approach to immune modulation.

The signal sequences that mediate entry of the hepatitis B virus (HBV) envelope proteins into the endoplasmic reticulum (ER) are located within the S domain at positions 11–32 and at positions 80–98 (from the start of the S domain). In addition, hydrophobic patches at positions 160–184 and 189–210 of the S domain may also be involved in entry into the ER. The role of each of these domains in the entry of the HBV M glycoprotein into the ER was studied by deletion mutations of each of the signal sequences. Glycosylation of proteins was used as a marker of entry into the ER. Our results indicate that association with the ER could not be prevented by the deletion of either individual or combinations of the HBV signal sequences. M protein lacking signal sequence I was able to enter the ER and had limited secretion. In contrast, M protein lacking signal sequence II could not be secreted but still entered the ER. M protein lacking signal sequences I and II, while still associated with the ER, was rapidly degraded by the cytosolic proteasome. The potential use of such a vector as a CTL vaccine was tested through an in vitro antigen presentation assay. In this assay, a DNA vaccine candidate lacking signal sequences I and II lead to a > 6-fold increase in CTL activation, as compared to the vector expressing wild type M protein. These results suggest that increased degradation of the HBV envelope proteins can lead to enhanced antigen presentation.

ABSTRACT The high levels of hepatitis B virus (HBV) surface antigen (HBsAg)-bearing subviral particles in the serum of chronically infected individuals are thought to play a role in suppressing the HBV-specific immune response. Current therapeutics are not directed at reducing this viral antigenemia; thus, our group has focused on identifying inhibitors of HBsAg secretion. By using the HBV-expressing cell line HepG2.2.15, high-throughput screening of an 80,288-compound synthetic small-molecule library identified HBF-0259, an aromatically substituted tetrahydro-tetrazolo-(1, 5- a )-pyrimidine. Following resynthesis, HBF-0259 had a 50% effective concentration of approximately 1.5 μM in a secondary, HBV-expressing cell line, with a concentration that exhibited 50% cytotoxicity of >50 μM. The equilibrium concentration of HBF-0259 in aqueous solution at physiological pH was 15 to 16 μM; the selective index was thus >9. As intended by our screening paradigm, HBF-0259 is a selective, potent inhibitor of secretion of both subviral and DNA-containing viral particles, while the secretion of α-1-acid glycoprotein and α-1-antitrypsin was unaffected. The HBV e antigen, which is not a constituent of HBV particles, was also unaffected, suggesting that the secretion of particles bearing HBV structural glycoproteins is targeted directly. Inhibitory activity was also confirmed by transfection of HBsAg, indicating that the action of the compound is independent of those of other viral proteins. HBF-0259 had no effect on HBV DNA synthesis, demonstrating that inhibition is independent of viral genomic replication. Finally, HBF-0259 had little or no effect on the cell-to-cell spread of two unrelated viruses, suggesting that it is a specific inhibitor of secretion of HBsAg. Possible mechanisms of action and the implications for its development are discussed.

To study the pathway of degradation of the hepatitis B virus (HBV) middle envelope protein (M), human hepatoblastoma cells were transfected with a plasmid that specifies production of M in the absence of other viral proteins. When expressed in HepG2 cells, 90% of M protein was secreted into the culture media within a 24-h period. However, quite surprisingly, 10% of this protein remained cell associated and was only slowly degraded over a 24-48-h period. Treatment with inhibitors of the cytosolic proteasome complex resulted in the accumulation of full-length HBV M protein and M derived HBV-specific polypeptides of 20 and 17 kDa. Treatment with the endoglycosidases PNGase F and Endo H, confirmed that the two species were derived from a similar polypeptide with a N-linked glycan modification. Evidence that this peptide was derived from a proteolytic processing event was determined through the detection of the C-terminal fragment using a C-terminal tagged HA tagged construct. The hypothesis that the 20 and 17 kDa polypeptide species are intermediates of M degradation was reinforced by their detection in cells transfected with vectors specifying M secretion defective mutants that accumulate intracellular M. Moreover, deletion of a putative cleavage sites prevented the detection of the 20 and 17 kDa species, consistent with the notion that they are generated by the action of a cellular protease prior to proteasomal degradation. Thus, these results highlight an important way in which large protein aggregates, such as the HBsAg can be processed for efficient degradation via the proteasomes and allow for proper antigen presentation via the MHC I pathway. (C) 2007 Elsevier Inc. All rights reserved.

Previous work has shown that the secretion of enveloped hepatitis B virus (HBV) DNA and the HBV middle envelope protein (MHBs) are sensitive to glucosidase inhibition. Here, it is shown that HBV DNA secretion remains depressed after the removal of the glucosidase inhibitor and long after glucosidase function returns to normal. For example, glycoprocessing and the secretion of α-1 anti-trypsin returned to normal within 3 h of the removal of the glucosidase inhibitor. In contrast, the secretion of HBV did not return to normal for more than 7 days after the removal of the inhibitor. Consistent with the inhibition of HBV virion secretion, the levels of HBV L and HBV M proteins were also reduced by treatment with the glucosidase inhibitor and remained reduced for 7 days after compound withdrawal. The implications of the prolonged antiviral effect against HBV and the use of glucosidase inhibitors as antiviral agents are discussed.

Imino sugar glucosidase inhibitors have selective antiviral activity against certain enveloped, mammalian viruses. Deoxynojirimycins (DNJs) modified by N-alkylation to contain a nine carbon atom side chain (N-n-nonyl-deoxynojirimycin; N-nonyl-DNJ, NN-DNJ) were shown to be, for example, at least 20 times more potent in inhibiting hepatitis B virus (HBV) and bovine viral diarrhoea virus (BVDV) in cell based assays than the non-alkylated DNJ. These data suggested that modification of the alkyl side chain could influence antiviral activity. Previous work has focused on varying side chain length. In this report, the influence of side chain branching and cyclization upon toxicity and antiviral activity was explored. Briefly, using a virus secretion assay for HBV and a single step growth (yield reduction) assay for BVDV, 14 different DNJ-based sugars, possessing various N-alkyl substitutions, were tested for antiviral activity. Of the series, N-methoxy-nonyl-DNJ and N-butyl-cyclohexyl DNJ were determined to have the best selectivity index against BVDV and HBV, with the N-methoxy analogue being the most potent with micromolar antiviral activity. The results of this antiviral survey and the implications for the mechanism of action and ultimate therapeutic potential of the DNJ-based imino sugars is provided and discussed.