Hepatitis B virus core gene deletions

Summary: The hepatitis B virus nucleocapsid, or core particle, forms the major internal structural component of the infectious viral Dane particle. Core particles play essential roles in the morphogenesis of the virion and in reverse transcription of pregenomic RNA, one of the initial steps in genome replication. The 183 amino acid core protein is the sole component of the core particle. In a disulphide-bonded dimeric form, the core protein accumulates to reach a critical threshold concentration leading to the co-operative core particle assembly process. The core protein consists of two domains: the N-terminal 140 amino acids and the C-terminal 43 amino acids. The latter region can be removed without affecting particle assembly. This region also contains a high proportion of basic residues and is tethered in the particle interior, where it interacts with the viral genome. The core protein is also an important immunological target during HBV infection. It contains B- and T-cell epitopes throughout its length and the core-specific helper T-cell response is thought to be an important factor in resolution of infection. During HBV infection subpopulations of viral variants arise with mutations in their nucleotide sequences. Variants with deletions in genomic regions are also detected, with deletions within the core gene being the most common. Determination of the functional ability of these variants was important as they are proposed to represent immune escape variants in patients and, as such, may be responsible for the chronic hepatitis observed. To allow detection of core deletion proteins with the immunodominant B-cell epitope (amino acids 74-89) removed, a ten amino acid epitope tag was inserted at the C- terminus of all proteins. The tag sequence did not affect particle assembly by full-length protein and allowed detection of bacterially-expressed core deletion proteins A84-109, A81-121, A60-117 and A79-125 (numbers indicate the residues deleted). Although, after sucrose gradient centrifugation, core deletion proteins were present in regions of the gradient where particles were expected, no particulate structures were detectable by electron microscopy. It was concluded that these deletion proteins were incapable of stable particle assembly and, instead, only formed large non-specific aggregates. Similar results were obtained when samples used for electron microscopy were tested in an agarose gel assay for core particles. The inability of the four core deletion proteins to assemble into core particles was not due to a failure of the proteins to dimerise, which is the first step of the particle assembly pathway. The deleted region of the A81-121 gene was replaced by an identical-sized region from the HBV surface gene and the resulting Acore-surface hybrid protein purified by density gradient centrifugation. This protein was also unable to form stable particles, indicating that the central region of the core protein is important for the formation of core particles. Step-wise replacement of the original core gene sequence to the A81-121 core gene deletion, from both the 5' and 3' ends of the deleted sequence, was carried out. This gave two sets of genes encoding three 'fill-in' proteins (5'FIO, 20, 30 and 3'FIO, 20, 30) with decreasing deletions (31 amino acids to 11 amino acids). However, all proteins were unable to form stable particles when purified as before. Results published during this project showed that central deletions of the core protein disrupted a motif consisting of a heptad repeat of hydrophobic residues. These results showed that even small disturbances of, or within, this motif prevented core particle assembly. As viral variants with core gene deletions are found co-existing with wild-type virus the possibility that both forms of protein could co-assemble to form novel mixed particles was examined. Density gradient purification of extracts from cells co-expressing full- length protein and the A84-109 or A81-121 proteins showed that both proteins were present in the same fractions, with a gradient profile similar to that of particles formed from full-length protein. These profiles were also different to those obtained for A84-109 and A81-121 proteins expressed in isolation. Therefore it seemed that mixed particles were able to assemble. In experiments where full-length and 5'F30 proteins were coexpressed similar results were not obtained, suggesting that mixed particles were unable to form, despite the smaller size of the deletion. In mammalian cells, core protein shows a cell-cycle dependent cellular localisation. The protein is present in the nucleus during the G, and Gj phases of the cell cycle and in the cytoplasm during S phase. Although the mechanism and function of this cell cycle-dependent localisation are unknown, a decision was made to investigate whether or not the core deletion proteins were subject to the same regulation. Using aphidicolin to arrest cells at the G,/S boundary, the location of the core deletion proteins in transfected HepG2 cells was examined by immunofluorescence. All four core deletion proteins displayed a localisation which was both nuclear and cytoplasmic within the same cell. This altered pattern was probably due to the presence of the deletion upstream to the nuclear localisation signal in the C-terminus of the core protein. The significance of this result is unknown at present, but may be relevant to the disease state observed in these patients. Sequencing of full-length core genes from viral DNA extracted from the serum of a chronic active hepatitis patient with core gene deletions (supplied by Dr. N. Naoumov) was carried out to examine whether or not the full-length viral subpopulation was responsible for the nature of the infection. The ten clones isolated showed a number of mutations throughout the pre-core and core regions, but these did not cluster in immunologically important epitopes. The results from this project have shown that HBV variants with core gene deletions are not functionally viable by themselves. However, the interactions between core deletion proteins and full-length protein require further study, as does the significance of the altered core deletion protein localisation. Results from these studies may explain the disease severity observed in the affected patients. Altematively, further studies on the functional differences displayed by full-length core protein variants will yield more information on the mechanism of chronic hepatitis development. Studies on the structures of the core particle and the core protein published recently should also allow a more rational and directed approach to identifying regions of the core protein required for core particle assembly