In vitro-synthesized hepatitis delta virus RNA initiates genome replication in cultured cells.

Monomers of the genomic strand of hepatitis delta virus RNA were transcribed in vitro and then delivered to NIH 3T3 fibroblasts by using a liposome fusion technique. After 7 days, genome replication was detected, but only in fibroblasts that stably expressed the delta antigen. Sequence analysis of the replicated products identified them as faithful copies of the hepatitis delta virus genome found in virions.     

Initiation of RNA replication of cloned Taiwan-3 isolate of hepatitis delta virus genotype II in cultured cells

Hepatitis delta virus (HDV) genotype II is the predominant genotype in Taiwan and is associated with less progressive disease than genotype I. Although the Taiwan-3 (T3) clone was the first genotype II HDV isolated in Taiwan, its replication in cultured cells has not previously been established. Here, we demonstrate that cloned T3 HDV is capable of replicating in cultured cells. Furthermore, we show that: (1). the replication level of T3 clones is 100-fold lower than that of a genotype I HDV prototype of Italian origin; (2). both forms of the genotype II T3 delta antigen are expressed; and (3). T3 HDV undergoes RNA editing during replication, with 4.8% of the T3 genomes showing evidence of editing. The low level of RNA replication may be related to the milder clinical outcomes of genotype II HDV infections.     

Characterization of hepatitis delta antigen: specific binding to hepatitis delta virus RNA.

It has previously been shown that human hepatitis virus delta antigen has an RNA-binding activity (Chang et al., J. Virol. 62:2403-2410, 1988). In the present study, the specificity of such an RNA-protein interaction was demonstrated by expressing various domains of the delta antigen in Escherichia coli as TrpE fusion proteins and testing their RNA-binding activities in a Northwestern protein-RNA immunoblot assay and RNA gel mobility shift assay. Hepatitis delta virus (HDV) RNA bound specifically to the delta antigen in the presence of an excess amount of unrelated RNAs and a relatively high salt concentration. Both genome- and antigenome-sense HDV RNAs and at least two different regions of HDV genomic RNA bound to the delta antigen. Surprisingly, these two different regions of HDV genomic RNA could compete with each other for delta antigen binding, although they do not have common nucleotide sequences. In contrast, this binding could not be competed with by other viral or cellular RNA. Since both the genomic and antigenomic HDV RNAs had strong intramolecular complementary sequences, these results suggest that the binding of delta antigen is probably specific for a secondary structure unique to the HDV RNA. By expressing different subdomains of the delta antigen, we found that the middle one-third of delta antigen was responsible for binding HDV RNA. Neither the N-terminal nor the C-terminal domain bound HDV RNA. Binding between the delta antigen and HDV RNA was also demonstrated within the HDV particles isolated from the plasma of a human delta hepatitis patient. This in vivo binding resisted treatment with 0.1% sodium dodecyl sulfate and 0.5% Nonidet P-40. In addition, we showed that the antiserum from a human patient with delta hepatitis reacted with all three subdomains of the delta antigen, indicating that all of the domains are immunogenic in vivo. These studies demonstrated the specific interaction between delta antigen and HDV RNA.     

RNA conformational requirements of self-cleavage of hepatitis delta virus RNA.

Hepatitis delta virus (HDV) RNA subfragments undergo self-cleavage at varying efficiencies. We have developed a procedure of using repeated cycles of heat denaturation and renaturation of RNA to achieve a high efficiency of cleavage. This effect can also be achieved by gradual denaturation of RNA with heat or formamide. These results suggest that only a subpopulation of the catalytic RNA molecules assumes the active conformation required for self-cleavage. This procedure could be of general use for detecting catalytic RNA activities.     

RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur between replicating MHV RNAs and RNA fragments which do not replicate, suggesting the potential of RNA recombination for genetic engineering.     

Functional domains of delta antigens and viral RNA required for RNA packaging of hepatitis delta virus.

The functions of delta antigens (HDAgs) in the morphogenesis of hepatitis delta virus (HDV) have been studied previously. The C terminus of large HDAg has been shown to complex with the small surface antigen (HBsAg) of helper hepatitis B virus, whereas the assembly of small HDAg requires interaction with the N terminus of large HDAg (M.-F. Chang, C.-J. Chen, and S. C. Chang, J. Virol. 68:646-653, 1994). To further examine the molecular mechanisms by which HDAgs are involved in the assembly of HDV RNA, we have cotransfected Huh-7 cells with plasmids representing a longer than unit-length HDV and the small HBsAg cDNAs. We found that HDAg mRNA could be generated from an endogenous promoter within the HDV cDNA that was translated into large HDAg. Large HDAg is capable of complexing with monomeric HDV genomic RNA to form ribonucleoprotein particles (RNPs) and is capable of forming enveloped HDV-like particles in the presence of small HBsAg without undergoing HDV replication. In addition, the middle region from amino acid residues 89 to 145 of large HDAg is required for assembly of the RNPs but is dispensable for assembly of the enveloped particles. RNA assembly is also demonstrated with small HDAg when it is cotransfected with a packaging-defective large HDAg mutant and small HBsAg. Leu-115 within the putative helix-loop-helix structure of the small HDAg is important for the replication of HDV but is not essential for RNA assembly, suggesting that conformational requirements of small HDAg for replication and assembly of viral RNA may be different. Further studies indicate that a 312-nucleotide linear HDV RNA from one end of the HDV and structure is sufficient to form RNP complexes competent for assembly of virus-like particles with large HDAg and small HBsAg.     

Large hepatitis delta antigen is not a suppressor of hepatitis delta virus RNA synthesis once RNA replication is established

Moderation of hepatitis delta virus (HDV) replication is a likely prerequisite in the establishment of chronic infections and is thought to be mediated by the intracellular accumulation of large hepatitis delta antigen (L-HDAg). The regulatory role of this protein was suggested from several studies showing that cotransfection of plasmid cDNAs expressing both L-HDAg and HDV RNA results in a potent inhibition of HDV RNA replication. However, since this approach differs significantly from natural HDV infections, where HDV RNA replication is initiated from an RNA template, and L-HDAg appears only late in the replication cycle, it remains unclear whether L-HDAg can modulate HDV RNA replication in the natural HDV replication cycle. In this study, we investigated the effect of L-HDAg, produced as a result of the natural HDV RNA editing event, on HDV RNA replication. The results showed that following cDNA-free HDV RNA transfection, a steady-state level of RNA was established at 3 to 4 days posttransfection. The same level of HDV RNA was reached when a mutant HDV genome unable to make L-HDAg was used, suggesting that L-HDAg did not play a role. The rates of HDV RNA synthesis, as measured by metabolic labeling experiments, were identical at 4 and 8 days posttransfection and in the wild type and the L-HDAg-deficient mutant. We further examined the effect of overexpression of L-HDAg at various stages of the HDV replication cycle, showing that HDV RNA synthesis was resistant to L-HDAg when it was overexpressed 3 days after HDV RNA replication had initiated. Finally, we showed that, contrary to conventional thinking, L-HDAg alone, at a certain molar ratio with HDV RNA, can initiate HDV RNA replication. Thus, L-HDAg does not inherently inhibit HDV RNA synthesis. Taken together, these results indicated that L-HDAg affects neither the rate of HDV RNA synthesis nor the final steady-state level of HDV RNA and that L-HDAg is unlikely to act as an inhibitor of HDV RNA replication in the natural HDV replication cycle.     

RNA editing in the replication cycle of human hepatitis delta virus

For some time it has been known that the RNA genome of human hepatitis delta virus (HDV) undergoes a specific RNA editing event. This review describes the editing phenomenon and its potential biological significance, and evaluates the data regarding the mechanism involved, including the possible relationship to other RNA editing phenomena.     

RNA recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus A59 and fusion-negative mouse hepatitis virus 2.

It has previously been shown that the murine coronavirus mouse hepatitis virus (MHV) undergoes RNA recombination at a relatively high frequency in both tissue culture and infected animals. Thus far, all of the recombination sites had been localized at the 5' half of the RNA genome. We have now performed a cross between MHV-2, a fusion-negative murine coronavirus, and a temperature-sensitive mutant of the A59 strain of MHV, which is fusion positive at the permissive temperature. By selecting fusion-positive viruses at the nonpermissive temperature, we isolated several recombinants containing multiple crossovers in a single genome. Some of the recombinants became fusion negative during the plaque purification. The fusion ability of the recombinants parallels the presence or absence of the A59 genomic sequences encoding peplomers. Several of the recombinants have crossovers within 3' end genes which encode viral structural proteins, N and E1. These recombination sites were not specifically selected with the selection markers used. This finding, together with results of previous recombination studies, indicates that RNA recombination can occur almost anywhere from the 5' end to the 3' end along the entire genome. The data also show that the replacement of A59 genetic sequences at the 5' end of gene C, which encodes the peplomer protein, with the fusion-negative MHV-2 sequences do not affect the fusion ability of the recombinant viruses. Thus, the crucial determinant for the fusion-inducing capability appears to reside in the more carboxyl portion of the peplomer protein.     

Sequence and structure of the catalytic RNA of hepatitis delta virus genomic RNA.

Human hepatitis delta virus (HDV) RNA has been shown to contain a self-catalyzed cleavage activity. The sequence requirement for its catalytic activity appears to be different from that of other known ribozymes. In this paper, we define the minimum contiguous sequence and secondary structure of the HDV genomic RNA required for the catalytic activity. By using nested-set deletion mutants, we have determined that the essential sequence for the catalytic activity is contained within no more than 85 nucleotides of HDV RNA. These results are in close agreement with the previous determinations and confirmed the relative insignificance of the sequence at the 5' side of the cleavage site. The smallest catalytic RNA, representing HDV genomic RNA nucleotide positions 683 to 770, was used as the basis for studying the secondary structure requirements for catalytic activity. Analysis of the RNA structure, using RNase V1, nuclease S1 and diethylpyrocarbonate treatments showed that this RNA contains at least two stem-and-loop structures. Other larger HDV RNA subfragments containing the catalytic activity also have a very similar secondary structure. By performing site-specific mutagenesis studies, it was shown that one of the stem-and-loop structures could be deleted to half of its original size without affecting the catalytic activity. In addition, the other stem-and-loop contained a six base-pair helix, and the structure, rather than the sequence, of this helix was required for the catalytic activity. However, the structure of a portion of the stem-and-loop remains uncertain. We also report that this RNA can be divided into two separate molecules, which alone did not have cleavage activity but, when mixed, one of the RNAs could be cleaved in trans. This study thus reveals some features of the secondary structure of the HDV genomic RNA involved in self-catalyzed cleavage. A model of this RNA structure is presented.     

The nucleolar phosphoprotein B23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus RNA replication

Hepatitis delta virus (HDV) encodes two isoforms of delta antigens (HDAgs). The small form of HDAg is required for HDV RNA replication, while the large form of HDAg inhibits the viral replication and is required for virion assembly. In this study, we found that the expression of B23, a nucleolar phosphoprotein involved in disparate functions including nuclear transport, cellular proliferation, and ribosome biogenesis, is up-regulated by these two HDAgs. Using in vivo and in vitro experimental approaches, we have demonstrated that both isoforms of HDAg can interact with B23 and their interaction domains were identified as the NH(2)-terminal fragment of each molecule encompassing the nuclear localization signal but not the coiled-coil region of HDAg. Sucrose gradient centrifugation analysis indicated that the majority of small HDAg, but a lesser amount of the large HDAg, co-sedimented with B23 and nucleolin in the large nuclear complex. Transient transfection experiments also indicated that introducing exogenous full-length B23, but not a mutated B23 defective in HDAg binding, enhanced HDV RNA replication. All together, our results reveal that HDAg has two distinct effects on nucleolar B23, up-regulation of its gene expression and the complex formation, which in turn regulates HDV RNA replication. Therefore, this work demonstrates the important role of nucleolar protein in regulating the HDV RNA replication through the complex formation with the key positive regulator being small HDAg.