Systemic but not preS-specific inhibition of N-glycosylation reduces virus production. (A) To investigate the impacts of glycosylation inhibitors on HBV formation in the absence of the M protein, HuH-7 cells were (co)transfected with a plasmid-based HBV genome defective in envelope protein synthesis (pHBV.env-), a vector encoding the L gene with inactivated start codons for M and S (Lo), plus an S expression vector. Upon transient expression, cells were treated with the indicated drugs for 70 h. Secretion of enveloped virions in the culture medium was detected by immunoprecipitation and radioactive labelling of the viral genome by the endogenous viral polymerase. The migration of the genome was visualized by agarose electrophoresis and PhosphorImaging (Bottom). Nonenveloped cytosolic nucleocapsids were immunoprecipitated from cell lysates and processed as above (Top). The amounts of virions released into the media were quantitated by measurement of the corresponding band intensities and demonstrated in % amount relative to corresponding mock-treated cells (Virus production). The amounts of subviral envelope particles harvested from the media were measured by an S-specific ELISA and depicted as above (S secretion). (B) Complementation of the env-negative HBV genome with Lo mutants carrying single or double mutations in the preS-specific N-glycosylation sequons. HuH-7 cells were cotransfected with pHBV.env-, S, and the indicated wild-type or mutant Lo constructs, and intracellular nucleocapsid assembly (Top) and virus release (Bottom) were analyzed essentially as in A.