IDENTIFICATION AND CHARACTERIZATION OF HEPADNA VIRUS ENVELOPE PROTEINS (HEPATITIS B VIRUS, WOODCHUCK, PRE-S, DUCK, OPEN READING FRAMES)

Abstract

The hepadna viruses are a family of hepatotropic, DNA-containing viruses for which the human hepatitis B virus (HBV) serves as a prototype. The association of HBV infection with acute and chronic liver disease has been extensively studied. However, the inability until recently to propagate these viruses in tissue culture or convenient laboratory animals has slowed progress towards understanding their basic biology. Our laboratory has focused on the identification and characterization of hepadnaviral proteins.;The open reading frame (ORF) which encodes the major envelope protein (surface antigen) of HBV has the capacity to encode an additional 164 to 175 amino acids (pre-s) upstream of and in frame with the surface antigen ATG. All members of the hepadna virus family have a similarly arranged envelope protein ORF. Studies to determine whether pre-s encoded amino acids are found in viral polypeptides, and what their function may be, have been carried out with two of the animal model viruses, the woodchuck hepatitis virus (WHV) and the duck hepatitis B virus (DHBV). Initial studies utilized computer algorithms to compare the predicted amino acid sequences of viral ORFs from HBV and WHV, at the levels of primary sequence homology, hydropathicity, and secondary structural propensity. Further studies involved construction of a fusion protein containing WHV pre-s sequences, which was overproduced in and purified from E.coli. Antiserum generated to this fusion protein was utilized in Western blot analyses to identify WHV polypeptides having molecular weights consistent with their translation from the first and second ATGs of the pre-s open reading frame. Each of the primary translation products from the pre-s:S ORF exists as two differentially glycosylated derivatives. In addition, the fusion protein antiserum has been used to immunoprecipitate polymerase-containing virions, indicating the presence of pre-s determinants on the viral surface. A similar approach, involving expression of a fusion protein containing duck hepatitis B virus pre-s sequences has been used to identify pre-s encoded polypeptides from DHBV. In contrast to the mammalian viruses, N-linked carbohydrates were not identified on DHBV pre-s molecules. In a collaborative study it has been shown that antiserum to a duck virus pre-s fusion protein has the ability to block viral infection of cultured duck hepatocytes. In addition, preliminary studies indicate that the fusion protein itself will also inhibit viral replication in tissue culture.