Antiviral Research 76 (2007) 30–37 Treatment of hepatitis B virus-infected cells with -glucosidase inhibitors results in production of virions with altered molecular composition and infectivity Catalin Lazar a , David Durantel b , Alina Macovei a , Nicole Zitzmann c , Fabien Zoulim b , Raymond A. Dwek c , Norica Branza-Nichita a,c,∗ a Institute of Biochemistry, Splaiul Independentei, 296, Sector 6, Bucharest 77700, Romania b INSERM, U871, Universit´ e Lyon 1, IFR62 Laennec, et Hospices Civils de Lyon (HCL), Lyon, France c Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom Received 9 March 2007; accepted 30 April 2007 Abstract Trimming of the N-glycans attached to the envelope proteins of hepatitis B virus (HBV) is required in different steps of the viral life cycle. Inhibition of the host enzymes -glucosidases, involved in the endoplasmic reticulum (ER)-associated processing of the N-linked glycans, results in misfolding of the HBV envelope proteins, prevention of HBV secretion and accumulation of viral DNA within infected cells. However, the impact of these effects on HBV morphogenesis and infectivity of the viral particles that are still released from cells with inhibited -glucosidase has not been addressed so far. Using N-butyldeoxynojirimycin (NB-DNJ), a competitive inhibitor of the ER -glucosidases, we analyzed the role of these enzymes on HBV assembly and infectivity of the virions released from HepG2.2.2.15 cells. HBV secreted from drug-treated cells contained an envelope with altered composition of the disulfide-linked oligomers and no detectable middle (M) protein. These molecular changes had a significant effect on HBV infectivity, reducing it to 20% compared to controls, for the highest concentrations of NB-DNJ used. Our data show for the first time that an active -glucosidase activity is crucial for production of infectious HBV and provide new insights into the controversial role of the M protein in this process. © 2007 Elsevier B.V. All rights reserved. Keywords: HBV; Infectivity: HepaRG cells; Alpha-glucosidase inhibition; NB-DNJ 1. Introduction Hepatitis B virus (HBV) is an important human pathogen that causes chronic liver disease leading to cirrhosis, hepato- cellular carcinoma and death (Beasley, 1988; Hollinger, 1995). HBV is a small virus, member of the Hepadnaviridae family, which contains a partially double stranded DNA genome of approximately 3.5 kb (Leenders et al., 1992). The HBV enve- lope consists of a lipid membrane derived from the infected host cell in which multiple copies of the viral surface proteins are inserted. These proteins, designated large (L), middle (M) ∗ Corresponding author at: Institute of Biochemistry, Splaiul Independentei, 296, Sector 6, Bucharest 77700, Romania. Tel.: +40 1 2239069; fax: +40 1 2239068. E-mail addresses: nichita@biochim.ro, norica@glycob.ox.ac.uk (N. Branza-Nichita). and small (S) are translated from alternative start codons of the same open reading frame (ORF) on the HBV genome and share the same S domain at the C-terminal region (Nassal, 1996). In addition to the S domain, the M protein contains the pre-S2 region, also present in L. The N-terminal region of the L pro- tein further contains the pre-S1 domain, which is unique to this protein (Fig. 1). The virus-encoded envelope proteins are synthe- sized as glycosylated and nonglycosylated entities, depending on the occupancy of the N-glycosylation site at Asn-146 of the S domain. A second N-glycosylation site is located at Asn-4 of the pre-S2 domain; however, it is only utilized by the M protein, which is also O-glycosylated at Thr-37 in its pre-S2 domain (Heermann and Gerlich, 1992; Werr and Prange, 1998). The N-glycosylation sites are strictly conserved among all HBV genotypes, suggesting an important role of the glycans attached to the envelope proteins in the HBV life cycle (Norder et al., 1994). Indeed, inhibition of N-glycosylation or N-glycan 0166-3542/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.antiviral.2007.04.004