THE LARGE SURFACE PROTEIN OF DUCK HEPATITIS-B VIRUS IS PHOSPHORYLATED IN THE PRE-S DOMAIN

The two major envelope proteins (large [L] and small [S]) of duck hepatitis B virus are encoded by the pre-S/S open reading frame. The L protein is initiated fro the AUG at position 801 in the pre-S region of the pre-S/S coding sequence, yielding an N-terminal consensus sequence for myristylation. Western immunoblots of the L protein often reveal a doublet at 36 and 34 kDa, with the latter attributed to the use of one of the three internal initiation codons. However, metabolic labelling with [H-3]myristic acid results in labelling of both P35 and P36, indicating that both species must be initiated fro the same start codon. Using metabolic labeling with P-32 and digestion with residue-specific phosphatases, we demonstrate that L protein heterogeneity is due to phosphorylation of threonine and/or serine residues within the pre-S domain. We propose that at lest one possible phosphorylation site is located at a novel (S/T)PPL motif which is conserved near the carboxyl end of the pre-S1 domain in all hepadnavirus sequences. Two to three additional (S/Ta)P motifs are also present in the carboxyl half of the pre-S1 (but not pre-S2 or S) domain of all hepadnaviruses. L protein in serum-derived particles is resistant to phosphatase digestion in the absence of detergent, reflecting an internal disposition of the phosphorylated pre-S domain and suggesting a role for dephosphorylation in the topological shift within L during morphogenesis (P. Ostapchuk, P. Hearing, and D. Ganem, EMBO J. 13:1048-1057, 1994). Furthermore, we observe that the relative amount of the phosphorylated form of L increases with time in the viral growth cycle. These findings imply that phosphorylation-desphosphorylation of the L protein is an important, regulated mechanism necessary for correct virion morphogenesis.