HEPATITIS DELTA VIRUS

Human hepatitis delta virus (HDV) is an infectious agent found in nature only in association with hepatitis B virus (HBV). In fact, HDV is less than a virus, in that it needs help from HBV to carry out a cycle of replication. Our efforts are directed towards understanding the structure and replication of HDV, and the nature of its interactions with HBV.

Transfection of cells with HDV RNA does not lead to genome replication; however, if the delta antigen is already present either within the cell or in a virus-like complex, replication occurs. As part of a collaborative study, we have obtained large amounts of pure delta antigen following expression in bacteria. This antigen made ribonucleoprotein (RNP) complexes with HDV RNA as transcribed in vitro, which when transfected into animal cells, initiated genome replication. Furthermore, although the delta antigen could not be replaced by several of the other RNA binding proteins tested, it could be replaced by a mutant form of the delta antigen. Surprisingly, other studies show that the mutant protein, per se, was not sufficient to support genome replication. Further studies are needed to determine how it is that this mutant protein was seemingly able to facilitate only the early events in genome replication.

A sensitive procedure (rapid amplification of cDNA ends, followed by cloning and nucleotide sequencing) to determine the 5'ends of polyadenylated mRNA was applied to mRNA species isolated from various sources, including infected liver and cultured cells transfected with wild type and various mutants of the HDV genome. We found that nt 1630 was normally the preferred 5'-end. Moreover, for various genomes mutated in this region the 5'-ends were either unchanged, additional novel sites were found, or the 1630 site was totally absent and only a novel site(s) was found. Overall, these data support the significance of this region on the genomic RNA in directing the initiation of RNA-directed transcription. Furthermore, the site at nt 1630 and the additional sites detected with the mutant genomes were all consistent with initiation by RNA polymerase II.

The delta antigen is an RNA binding protein that demonstrates specificity in terms of its interaction with HDV RNAs. However, it also demonstrates some non-specific nucleic acid binding ability. We found that like some other nucleic acid binding proteins, it has activities that are described as chaperone-like. For example, at 30 nM it promoted a 600-fold enhancement in the rate of reassociation of denatured double stranded DNA. Further studies are underway to test whether such chaperone-like activities contribute to the ways in which this antigen provides essential support for HDV genome replication.

This study has focused on the isoprenylation of a cysteine 4 amino acids from the Cterminus of the large delta antigen. Others have previously reported that this modification occurs and have interpreted it as being essential for the large delta antigen to act in supporting particle assembly. We used gel electrophoresis to separate and hence quantitate the modified from unmodified forms of the protein. In addition, we have applied mass spectrometry to protease fragments of purified protein to more specifically characterize the isoprenylation event.

We have previously discovered RNA-editing as it applies to HDV. During replication of the HDV genome, there occurs for some antigenomic RNAs a specific change of adenosine to inosine. It is because of this specific change that translation can switch from the small delta antigen, which is needed for genome replication, to the large delta antigen, which is needed for particle assembly. Now we have begun to ask some additional questions--when does editing occur and what is the actual substrate? Also, we are investigating an unusual situation in which we have detected what we call "hyper-editing."

Previously, others and we have studied the transcription by RNA polymerase II, in vitro, of HDV RNA by extracts of mammalian cells. Chiang and coworkers have recently developed conditions for the purification of protein pol II holoenzyme complexes from disrupted human cells that are capable of DNA-directed RNA transcription. As a collaboration, we are now attempting to define conditions in which this same holoenzyme will transcribe HDV RNA templates. To date, our studies have detected 3'-end addition of ribonucleotides to the RNA templates. The system will be modified to determine whether accessory factors lead to actual initiation of transcription.

Recent studies by zuccola and coworkers have determined the crystal structure of the domain on the delta protein that is involved in dimerization and multimerization. this information has been used to design point mutations predicted to be critical for multimerization and/or dimerization. as a collaborative study, we have constructed such mutants and are testing them in the context of hdv genome replication. Small peptides that should interfere with multimerization and/or dimerization of the wild type delta protein have also been designed and constructed. These peptides are being tested both in vivo and in vitro. These studies will not only provide information on the requirements of protein-protein interactions in HDV replication but will also provide insights into antiviral strategies.

BICHKO, V.V. Cationic liposomes. In DNA Transfer to Cultured Cells, edited by K. Ravid, I. Freshney. Wiley-Liss, pp. 193-212, 1998.

DINGLE, K., MORALEDA, G., BICHKO, V., TAYLOR, J. Electrophoretic analysis of the ribonucleoproteins of hepatitis delta virus. J. Virol. Meth. 75:199-204, 1998.

TAYLOR, J.M. Replication of human hepatitis delta virus: influence of studies of subviral plant pathogens. Adv. Virus Res. (in press).

TAYLOR, J.M., WU, T.-T., BICHKO, V.V. Variations on the HDV RNA genome. In Viral Hepatitis and Liver Disease, edited by M. Rizzetto, R.H. Purcell, J.L. Gerin, and G. Verme. Edizioni Minerva Medica, Turin, pp. 295-297, 1998.

DINGLE, K., BICHKO, V., ZUCCOLA, H., HOGLE, J., TAYLOR, J. Initiation of hepatitis delta virus genome replication. J. Virol. 72:4783-4788, 1998.

TAYLOR, J.M. Hepatitis delta virus. In Microbiology and Microbial Infection, 9th edition. Topley and Wilson, Chapter 37, pp. 775-779, 1998.

TAYLOR, J.M. Human hepatitis delta virus: structure and replication of the genome. Curr. Top. Microbiol. Immunol. (in press).

WU, T.-T., SU, Y.-H., BLOCK, T.M., TAYLOR, J.M. Atypical splicing of the latency associated transcripts of herpes simplex type I. Virology 243:140-149, 1998.

^§   Fox Chase researcher

^a   H.J. Netter: Royal Children's Hospital, Hurston, Australia, 4029

^b   K. Dingle: Present address--Department of Biochemistry, Oxford University, United Kingdom, OX1 3QU

^c   T.-T. Wu: Present address--Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095

^d   V.V. Bichko: Scriptgen Pharmaceuticals, Medford, MA 02115

^e   H. Zuccola, C. Elkin, J. Hogle: Harvard Medical School, Boston, MA 02115

^f   J. Otto: Duke University Medical Center, Durham, NC 27710

^g   C.-M. Chiang: Department of Biochemistry, University of Illinois, Urbana, IL 61801

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998