The pre-S region determines the intracellular localization and appearance of hepatitis B virus

The functional role of the hepatitis B virus (HBV) pre-S region for assembl y and appearance of the virus is not completely understood. In this study, 3 natural-occurring mutants were investigated. Three mutants of the pre-S r egion-a point mutation in the CCAAT box (MUT1), a 6-bp deletion (MUT2) 3' o f the CCAAT box, and a 153-bp deletion (MUT3) in the preS2 domain-were clon ed alone or in combinations in replication-competent HBV plasmids and trans fected in hepatoma cells. The impact on HBV assembly and appearance was stu died by Northern Blot, primer extension analysis, immunofluorescence studie s, enzyme-linked immunosorbent assay, and electron microscopy. An inversed ratio of pre-S/S mRNA transcripts compared with wild-type (wt) HBV was foun d when either MUT1 or -2 were included into the plasmid. Intracellular loca lization with both mutants showed retention of large S-protein in the endop lasmic reticulum and nuclear accumulation of core protein. The extracellula r amount of S-protein was reduced with MUT1 and -2 or combinations in which 1 of the mutants was included. However, the extracellular appearance of vi ral products was comparable with wtHBV. In contrast, MUT3 showed major chan ges. Virion-like particles had a fried-egg, and filaments a screw-like appe arance. The S-promoter mutations MUT1 and MUT2 correlated with viral retent ion. MUT3 leads to malformed viral particles. Therefore, different regions in the pre-S domain are essential to determine the intracellular localizati on and extracellular appearance of HBV, and might contribute to the prognos is of chronic HBV infection.