A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.

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The human norovirus (NoV) polyprotein is cleaved into mature non-structural proteins by both mature NoV protease (Pro, NS6) and its un-cleaved precursor (ProPol, NS6-7). Processing order is well-established with 'early' and 'late' cleavages, but the governing enzymatic mechanisms are unknown. Enzyme kinetics of a GII Pro and ProPol were analyzed using synthetic peptides representing the five natural polyprotein cleavage sites. The relative efficiency of cleavage of the individual peptides was consistent with established polyprotein processing order, and primarily correlated with enzyme turnover (kcat). Enzymatic efficiencies (kcat/Km) of ProPol at all five sites were equivalent to, or greater than, that of Pro. Binding affinities (Km) for the two least efficiently cleaved sites (p20/VPg, VPg/Pro) were 2-4-fold higher than the other sites. This work further defines the role of ProPol in NoV polyprotein cleavage, and demonstrates that human norovirus polyprotein processing order is primarily an inherent property of enzymatic activity.

ORI-9020, a novel dinucleotide, evaluated in transgenic mice expressing hepatitis B virus (HBV), significantly reduced liver HBV DNA (P < 0.001). Levels of HBeAg and HBsAg in serum and of HBcAg in liver were not affected by treatment. A minimal effective dosage was determined to be between 1.6 and 0.5 mg/kg of body weight/day, which was similar to that observed for adefovir dipivoxil.The discovery of safe and effective antiviral drugs continues to present considerable challenges for hepatitis B virus (HBV) therapies. The novel dinucleotides ORI-9020, ORI-7246, and ORI-7170 have been developed and identified to be active against HBV in cell culture (2a). The mode of antiviral action of ORI-9020 and ORI-7246 appears to be direct interference of HBV replication at the level of HBV reverse transcriptase and/or DNA polymerase at an early step, either at, or prior to, the production of the first strand of HBV DNA. These experiments show that these compounds have antiviral activities comparable with adefovir dipivoxil (ADV). Furthermore, when used in combination with lamivudine (3TC) possible synergistic antiviral effects were apparent.Given some of the favorable characteristics of ORI-9020, the anti-HBV activity was evaluated in a previously developed transgenic HBV mouse model (2). Experiments with transgenic mice expressing HBV have demonstrated the model's utility for evaluating potential anti-HBV compounds such as interleukin-12 (1), 3TC (5), alpha interferon (6), ADV (4), and entecavir (3). The results of the evaluation of ORI-9020 in the transgenic mouse model are reported here.Transgenic HBV mice were originally obtained from Frank Chisari (Scripps Research Institute, La Jolla, Calif.) (2). Animal use and care was in compliance with the Utah State University Institutional Animal Care and Use Committee.For the first animal experiment, ORI-9020 was prepared fresh daily at a dosage of 100 mg/kg of body weight /day, which was equal to 170 mol/kg/day, and was injected intraperitoneally (i.p.) using cremaphor-ethanol-saline (CES) (10:10:80) or physiological saline as vehicles. ADV (Gilead, Foster City, Calif.), the positive control, was prepared using the CES vehicle. A dosage of 10 mg/kg/day (19.9 mol/kg/day) was used. In the second experiment to determine the minimal effective concentration, ORI-9020 was prepared in sterile saline in onehalf-log dilutions from 50 to 0.05 mg/kg/day. The drug was delivered i.p. in a volume of 0.1 ml.Liver samples were analyzed for HBV DNA, HBV RNA, and HBcAg, and serum samples were processed for HBV DNA, HBeAg, and HBsAg according to previously published methods (4). HBV DNA and RNA were detected in liver by Southern and Northern blot analysis, respectively. A competitive quantitative PCR was used to detect HBV DNA in the serum. HBeAg and HBsAg were detected using an in-house assay.For the first experiment, male transgenic mice with HBeAg titers in the upper 85% of the range were block randomized across treatment groups according to HBeAg titers. Mice received i.p. injections once da...

The VPg protein of human Norovirus (hNoV) is a multi-functional protein essential for virus replication. The un-cleaved viral precursor protein, ProPol (NS5-6) was 100-fold more efficient in catalyzing VPg nucleotidylylation than the mature polymerase (Pol, NS6), suggesting a specific intracellular role for ProPol. Sequential and single-point alanine substitutions revealed that several positively charged amino acids in the N-terminal region of VPg regulate its nucleotidylylation by ProPol. We provide evidence that VPg directly binds NTPs, inhibition of binding inhibits nucleotidylylation, and NTP binding appears to involve the first 13 amino acids of the protein. Substitution of multiple positively charged amino acids within the first 12 amino acids of the N-terminal region inhibits nucleotidylylation without affecting binding. Substitution of only Lys20 abolishes nucleotidylylation, but not NTP binding. These studies indicate that positively charged amino acids in the first 20 amino acids of hNoV VPg regulate its nucleotidylylation though several potential mechanisms.

BackgroundEgypt has the world’s highest prevalence of infection with hepatitis C virus (HCV), which is a major cause of hepatocellular carcinoma. The high HCV prevalence is largely attributed to the parenteral antischistosomal therapy (PAT) campaigns conducted from the 1950s through the 1980s; however, the primary modes of transmission in the post-PAT period are not well known. In this study we examined the associations between HCV prevalence and exposures to risk factors, including PAT, in a high HCV prevalence population.MethodsUsing a cross-sectional design, we examined the associations between demographic characteristics and risk factors for HCV transmission and HCV positivity prevalence among a sample of Egyptian residents. Data were collected through an interview-administered survey, and the association estimates were determined using χ2 and logistic regression.ResultsThe highest HCV positivity prevalence was observed in cohorts born before 1960, and declined precipitously thereafter; whereas the proportion of subjects reporting PAT remained relatively stable. Being male, having a rural residence, and having received PAT were all associated with HCV positivity; however, PAT alone could not account for the high prevalence of HCV.ConclusionsIn Egypt, PAT and other transmission factors yet to be identified, as well as cohorts born before the 1960s and infected with HCV, are most likely the main contributors to the current HCV endemic.

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