PHOSPHORYLATION OF DHBV PRE-S - IDENTIFICATION OF THE MAJOR SITE OF PHOSPHORYLATION AND EFFECTS OF MUTATIONS ON THE VIRUS LIFE-CYCLE

Four potential serine/threonine phosphorylation sites [(S/T)-P motif], designated P1-P4, on the pre-S protein of duck hepatitis B virus (DHB V) have been mutated. Mutants include single (P2, P3, P4) and double a mino acid substitutions (P1+P2, P3+P4) and one with all four sites mut ated (4P). Serine at position 118 (P3) was identified as the major sit e of phosphorylation by Western blotting and radioimmunoprecipitation after in vitro cell labeling with [S-35]methionine or [P-33]orthophosp hate. Mutant virions generated by transfection of LMH cells were infec tious both in vitro in duck hepatocyte primary cultures and in vivo in Pekin ducks. Intracellular relaxed circular (RC) and covalently close d circular (ccc) DNA syntheses were not affected by the P3 mutation or even the quadruple mutant. Extracellular virus production was slightl y increased when the P3 site was mutated. CsCl gradient centrifugation showed no clear difference between mutant and wild-type virus with re spect to the ratios of enveloped virus and nucleocapsid particles in h epatocyte culture supernatants. Trypsin or Vs protease digestion with or without NP-40 indicated that phosphorylation of the pre-S domain is nor involved in determining the transmembrane topology of DHBV large protein. This phenotypic analysis indicates that DHBV pre-S phosphoryl ation has no apparent effect on DHBV replication and formation of matu re viral particles in duck hepatocyte primary culture and does not aff ect infectivity in ducklings. (C) 1998 Academic Press.