Duck hepatitis B virus can tolerate insertion, deletion, and partial frameshift mutation in the distal pre-S region

Identification of cell surface viral binding proteins is important for understanding viral attachment and internalization. We have fused the pre-S domain of the duck hepatitis B virus (DHBV) large envelope protein to glutathione S-transferase and demonstrated a 170-kDa binding protein (p170) in [ 35 S]methionine-labeled duck hepatocyte lysates. This glycoprotein was found abundantly in all extrahepatic tissues infectible withDHBV and in some noninfectible tissues, though it is not secreted into the blood. The interaction of pre-S fusion protein with p170 was competitively inhibited by wild-type DHBV in a dose-dependent manner. In addition, infection of hepatocytes with DHBV blocked the binding of pre-S fusion protein to p170, which suggests a biological role for p170 during natural infection. The p170 binding site was mapped to a conserved sequence of 16 amino acid residues (positions 87 to 102) by using 24 pre-S deletion mutants; this binding domain coincides with a major virus-neutralizing antibody epitope. Furthermore, site-directed mutagenesis revealed that an arginine residue at position 97 is critical for p170 binding. p170 was purified by a combination of ion-exchange and affinity chromatographies, and four peptide sequences were obtained. Two peptides showed significant similarities to human and animal carboxypeptidases H, M, and N. Taken together, these results raise the possibility that the p170 binding protein is important during the replication cycle of DHBV.

Section: Discussionmentioning

confidence: 99%

“…The locations of the type II and IV epitopes are those of Yuasa et al(33), and the locations of the M900 and SD20 epitopes are those of Chassot et al(3). The location of a sequence nonessential for viral infectivity is that of Li et al(16).VOL. 69, 1995DHBV Pre-S-BINDING PROTEIN 7111…”

mentioning

confidence: 92%

Interaction between duck hepatitis B virus and a 170-kilodalton cellular protein is mediated through a neutralizing epitope of the pre-S region and occurs during viral infection

Tong

Wands

1995

“…As a control, a mutant with triple amino acid substitutions, K95S-R97L-E98A, was used (the mutations cover the p170 binding site but do not abolish p170 binding). Although some of the mutations caused amino acid changes in the overlapping polymerase gene (R101I-R012D: S311Y-P312R; F100V-R101L: F310C; Y103C-Q104F: S314F; and K95S-R97L-E98A: S306C), this portion of the polymerase is a spacer region tolerant of substantial sequence alterations (1,16). After the mutants were transfected into LMH cells, the secretion of pelletable particles into culture medium at different time points was measured.…”

Section: Retention Of Low-level P120 By Intact Pre-s Constructmentioning

confidence: 99%

“…The epitopes for amino acids 58 to 66, 91 to 99, 127 to 138, and 139 to 145 are according toYuasa et al (30); those for amino acids 83 to 90 and 100 to 107 are according to Chassot et al(2); and those for amino acids 98 to 104 and 112 to 126 are according toLi et al (16a). A nonessential region for viral infectivity is shown (open bar with dots)(16).VOL. 70, 1996 TISSUE-SPECIFIC DHBV-BINDING PROTEIN 6031…”

mentioning

confidence: 99%

Characterization of a 120-Kilodalton pre-S-binding protein as a candidate duck hepatitis B virus receptor

Tong

Wands

1996

Self Cite

Infection by human and animal hepadnaviruses displays remarkable host and tissue tropism. The infection cycle probably initiates with binding of the pre-S domain of viral envelope protein to surface receptors present on the hepatocyte. Three types of neutralizing monoclonal antibodies against duck hepatitis B virus (DHBV) have their binding sites clustered within residues 83 to 107 of the pre-S protein, suggesting that this region may constitute a major receptor binding site. A 170-or 180-kDa duck protein (p170 or gp180) which binds DHBV particles through this part of the pre-S sequence has been identified recently. Although the p170 binding protein is host (duck) specific, its distribution is not restricted to DHBV-infectible tissues. Using the pre-S protein fused to glutathione S-transferase and immobilized on Sepharose beads, we have now identified an additional binding protein with a size of 120 kDa (p120). p120 expression is restricted to the liver, kidney, and pancreas, the three major organs of DHBV replication. While optimal p170 binding requires an intact pre-S protein, binding to p120 occurs much more efficiently with a few N-or C-terminally truncated forms. The p120 binding site was mapped to residues 98 to 102 of the pre-S region, which overlaps with a cluster of known virus-neutralizing epitopes. Site-directed mutagenesis revealed residues 100 to 102 (Phe-Arg-Arg) as the critical p120 contact site; nonconservative substitution in any of the three positions abolished p120 binding. Double mutations at positions 100 to 102 markedly reduced DHBV infectivity in cell culture. Short pre-S peptides covering the clustered neutralizing epitopes (also p170 and p120 binding sites) reduced DHBV infectivity in primary duck hepatocyte cultures. Thus, p120 represents a candidate component of the DHBV receptor complex.Interaction between a viral envelope protein and its cell surface receptor(s) initiates the virus infection cycle and often determines the host range and tissue tropism (for a recent review, see reference 6). Virus-receptor interactions may be complex processes. For example, binding of human immunodeficiency virus to CD4 molecules is insufficient to allow viral infection of mouse T lymphocytes (18), and adenovirus attachment to cell surface and subsequent internalization require two separate steps involving distinct cellular proteins (29). We are interested in identifying the cellular protein(s) required for attachment and penetration of duck hepatitis B virus (DHBV), an avian relative of the human hepatitis B virus. Accumulating evidence supports the idea that the pre-S region of the DHBV large envelope protein mediates receptor binding and determines species specificity (7, 10, 26). Binding of DHBV particles to Pekin duck hepatocytes has recently been demonstrated (22), but the exact receptor contact sites have not been identified. Binding sites for pre-S-neutralizing antibodies are located in residues 58 to 66, 83 to 90, 91 to 99, 100 to 107, and 139 to 145 (2, 30), with three neutralizing epitopes b...

“…The similarities between hepadnaviral and retroviral pol genes and evidence that replication of the hepadnaviral DNA genome involves reverse transcription have indicated the existence of a virally encoded, multifunctional polymerase. In vitro transfection experiments which examined the effects of mutations in the pol gene on production of specific replicative intermediates suggested four domains within the polymerase: an amino-terminal primer, a spacer region, an RT, and a carboxy-terminal RNase H (8,10,31,43).…”

mentioning

confidence: 99%

Detection of an RNase H activity associated with hepadnaviruses

Oberhaus

Newbold

1995

Replication of the hepadnavirus DNA genome is accomplished via reverse transcription of an intermediate, pregenomic RNA molecule. This process is likely to be carried out by a virally encoded, multifunctional polymerase which possesses DNA-and RNA-dependent DNA polymerase and RNase H activities. However, the nature of the product(s) of the polymerase gene predicted to mediate these functions is unclear. Biochemical studies of the polymerase protein(s) have been limited by its apparent low abundance in virus particles and, until recently, the inability to express active polymerase protein(s) heterologously. We have used activity gel assays to detect DNA-and RNA-dependent DNA polymerase activities associated with highly purified duck hepatitis B virus (DHBV) core particles (S. M. Oberhaus and J. E. Newbold, J. Virol. 67:6558-6566, 1993). Now we report that the same approach identifies a 35-kDa RNase H activity in association with highly purified DHBV core particles and crude preparations of virions from DHBV-infected ducks and woodchuck hepatitis virus-infected woodchucks. This is the first report of the detection of an hepadnavirus-associated RNase H activity. Its apparent size is smaller than any of the DNA polymerase activities that we detected previously and significantly smaller than the full-length protein predicted from the polymerase open reading frame (p85 for DHBV). These data suggest that the viral polymerase and RNase H activities are separable and that these enzymes may coordinate their activities in vivo by forming a complex.