Woodchuck hepatitis virus (WHV) is a mammalian hepatitis virus and taxonomically and antigenically related to human hepatitis B virus (HBV). Previous report showed that the pre-S envelope protein of an avian hepatitis B virus, duck hepatitis B virus (DHBV), contains a region, DDPLL, that is necessary for virus assembly and secretion (Lenhoff R, Summers J. J Virol, 1994, 68: 4565~4571). It was found in the present study that amino acids 201 to 205 of the pre-S envelope protein of WHV form a conserved amino acid cluster, GDPAL, which shows a high similarity to the DDPLL sequence of DHBV. The GDPAL region was deleted or induced to be mutated at some amino acid sites so as to determine whether the region was functionally similar to the DDPLL region. The mutant DNA was trans feeted into human hepatoma cell line Huh7 and the medium was assayed according to virion concentration by use of immunoprecipitation and Southern blot analysis. It was found that an in-frame deletion of this small region resulted in inhibition of virion formation, suggesting that the GDPAL region of the pre-S envelope protein was necessary for virus assembly and/or secretion of WHV.Individual replacement of alamine 204, leucine 205 or serine 206 with other amino acid residues did not affect rims production. However, substitution of aspartic acid 202 with valine or substitution of proline 203 with leucine caused dramatical inhibition of WHV production. Furthermore, the GDPAL mutants were individually tested for their complementarity to a pre-S1 defective genome. The results showed that the GDPAL region functioned as part of the pre-S1 protein but was not necessary to function as part of the pre-S2 protein. Woodchuck hepatitis virus (WHV) is a mammalian hepatitis virus and taxonomically and antigenically related to human hepatitis B virus (HBV). Previous report showed that the pre-S envelope protein of an avian hepatitis B virus, duck hepatitis B virus (DHBV), contains a region, DDPLL, that is necessary for virus assembly and secretion (Lenhoff R, Summers J. J Virol, 1994, 68: 4565-4571). It was found in the present study that amino acids 201 to 205 of the pre- S envelope protein of WHV form a conserved amino acid cluster, GDPAL, which shows a high similarity to the DDPLL sequence of DHBV. The GDPAL region was deleted or induced to be mutated at some amino acid sites so as to determine whether the region was functionallyally The mutant DNA was transfected into human hepatoma cell line Huh7 and the medium was assayed according to virion concentration by use of immunoprecipitation and Southern blot analysis. It was found that an in-frame deletion of this small regio n resulted in inhibition of virion formation, suggesting that the GDPAL region of the pre-S envelope protein was necessary for virus assembly and / or secretion of WHV. Intravenous replacement of alamine 204, leucine 205 or serine 206 with other amino acid residues did not However, the substitution of aspartic acid 202 with valine or substitution of proline 203 with leucine caused dramatical inhibition of WHV production. Furthermore, the GDPAL mutants were singly tested for their complementarity to a pre-S1 defective genome. the GDPAL region functioned as part of the pre-S1 protein but was not necessary to function as part of the pre-S2 protein.