Recombination between sequences of hepatitis B virus from different genotypes

A comparison of 25 hepatitis B virus (HBV) isolates for which complete genome sequences are available revealed two that occupied different positions in phylogenetic trees reconstructed from different open reading frames. Further analysis indicated that this incongruence was the result of recombination between viruses of different genomic and antigenic types. Both putative recombinants originated from geographic regions where multiple genotypes are known to cocirculate. A search of the sequence databases showed evidence of similar intergenotypic recombinants. These observations indicate that recombination between divergent strains may represent an important source of genetic variation in HBV.     

Self-ligating RNA sequences on the antigenome of human hepatitis delta virus.

A 179-base fragment of RNA from the 1,679-base antigenome of hepatitis delta virus can not only self-cleave but, when the ends of the resultant fragments are brought into apposition by base pairing to another RNA, also self-ligate. Thus, processing events needed for genome replication in vivo may be strictly RNA mediated.     

Heterogeneity and evolution rates of delta virus RNA sequences.

To investigate the geographical divergence of delta virus RNA sequences, 868 nucleotides (nt), including the delta antigen-coding region, were determined in isolates from two Japanese patients, M and S, by polymerase chain reaction and direct sequencing and compared with three previously reported nucleotide sequences. The sequence obtained for hepatitis delta virus RNA from patient M was approximately 92% identical to sequences previously obtained for two other strains of hepatitis delta virus, whereas the sequence of hepatitis delta virus RNA obtained from patient S was approximately 81% identical to the previously sequenced strains. This suggests that delta agent in Japan has a heterogeneous origin and the delta virus RNA sequence from Japanese patient S is the most divergent delta virus isolate yet analyzed. To study the evolution rate of delta virus RNA, viral isolates obtained 3 and 4 years apart from each of two patients were also sequenced. It was estimated that the substitution rate of viral RNA was 0.57 x 10(-3) nt per site per year in patient M and 0.64 x 10(-3) nt per site per year in patient S for the delta antigen gene.     

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.     

The topology of hepatitis B virus pregenomic RNA promotes its replication

Previous analysis of hepatitis B virus (HBV) indicated base pairing between two cis-acting sequences, the 5' half of the upper stem of epsilon and phi, contributes to the synthesis of minus-strand DNA. Our goal was to identify other cis-acting sequences on the pregenomic RNA (pgRNA) involved in the synthesis of minus-strand DNA. We found that large portions of the pgRNA could be deleted or substituted without an appreciable decrease in the level of minus-strand DNA synthesized, indicating that most of the pgRNA is dispensable and that a specific size of the pgRNA is not required for this process. Our results indicated that the cis-acting sequences for the synthesis of minus-strand DNA are present near the 5' and 3' ends of the pgRNA. In addition, we found that the first-strand template switch could be directed to a new location when a 72-nucleotide (nt) fragment, which contained the cis-acting sequences present near the 3' end of the pgRNA, was introduced at that location. Within this 72-nt region, we uncovered two new cis-acting sequences, which flank the acceptor site. We show that one of these sequences, named omega and located 3' of the acceptor site, base pairs with phi to contribute to the synthesis of minus-strand DNA. Thus, base pairing between three cis-acting elements (5' half of the upper stem of epsilon, phi, and omega) are necessary for the synthesis of HBV minus-strand DNA. We propose that this topology of pgRNA facilitates first-strand template switch and/or the initiation of synthesis of minus-strand DNA.     

Recurring features of local tertiary structural elements in RNA molecules exemplified by hepatitis D virus RNA

Elements of local tertiary structure in RNA molecules are important in understanding structure-function relationships. The loop E motif, first identified in several eukaryotic RNAs at functional sites which share an exceptional propensity for UV crosslinking between specific bases, was subsequently shown to have a characteristic tertiary structure. Common sequences and secondary structures have allowed other examples of the E-loop motif to be recognized in a number of RNAs at sites of protein binding or other biological function. We would like to know if more elements of local tertiary structure, in addition to the E-loop, can be identified by such common features. The highly structured circular RNA genome of the hepatitis D virus (HDV) provides an ideal test molecule because it has extensive internal structure, a UV-crosslinkable tertiary element, and specific sites for functional interactions with proteins including host PKR. We have now found a UV-crosslinkable element of local tertiary structure in antigenomic HDV RNA which, although differing from the E-loop, has a very similar pattern of sequence and secondary structure to the UV-crosslinkable element found in the genomic strand. Despite the fact that the two structures map close to one another, the sequences comprising them are not the templates for each other. Instead, the template regions for each element are additional sites for potential higher order structure on their respective complementary strands. This wealth of recurring sequences interspersed with base-paired stems provides a context to examine other RNA species for such features and their correlations with biological function.     

Recombination in the genesis and evolution of hepatitis B virus genotypes

Hepatitis B virus (HBV) infection is widely distributed in both human and ape populations throughout the world and is a major cause of human morbidity and mortality. HBV variants are currently classified into the human genotypes A to H and species-associated chimpanzee and gibbon/orangutan groups. To examine the role of recombination in the evolution of HBV, large-scale data retrieval and automated phylogenetic analysis (TreeOrder scanning) were carried out on all available published complete genome sequences of HBV. We detected a total of 24 phylogenetically independent potential recombinants (different genotype combinations or distinct breakpoints), eight of which were previously undescribed. Instances of intergenotype recombination were observed in all human and ape HBV variants, including evidence for a novel gibbon/genotype C recombinant among HBV variants from Vietnam. By recording sequence positions in trees generated from sequential fragments across the genome, violations of phylogeny between trees also provided evidence for frequent intragenotype recombination between members of genotypes A, D, F/H, and gibbon variants but not in B, C, or the Asian B/C recombinant group. In many cases, favored positions for both inter- and intragenotype recombination matched positions of phylogenetic reorganization between the human and ape genotypes, such as the end of the surface gene and the core gene, where sequence relationships between genotypes changed in the TreeOrder scan. These findings provide evidence for the occurrence of past, extensive recombination events in the evolutionary history of the currently classified genotypes of HBV and potentially in changes in its global epidemiology and associations with human disease.     

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.     

Adenylic acid - rich sequences in influenza virus RNA.

Some influenza virus complementary RNA (cRNA) from infected chick cells is polyadenylated as judged by oligo(dT)-cellulose chromatography. However, none of the virion RNA or the vRNA synthesised in infected cells contain poly(A) sequences. cRNA containing poly(A) sequences was further characterised by polyacrylamide gel electrophoresis and under the conditions used only some size classes of cRNA were polyadenylated.     

The 30S Moloney sarcoma virus RNA contains leukemia virus nucleotide sequences.

The 50S-70S RNA of a Moloney sarcoma-leukemia virus [Mo-MSV(MLV)] complex produced by a particular mouse cell line was shown by gel electrophoresis to contain a major (97%) 30S sarcoma-specific subunit species and a minor (3%) 38S leukemia virus-specific subunit. On the basis of its sedimentation coefficient and known complexity, the 30S Mo-MSV RNA was estimated to be a unique RNA molecule of about 6000 nucleotides. Hybridization experiments using viral RNA and DNA complementary to viral RNA (cDNA) made by viral DNA polymerase indicated that the 30S Mo-MSV RNA shared 70% of its sequences with Mo-MLV, 30% with another MLV derived from Mo-MLV, and 30% with Kirsten sarcoma-xenotropic leukemia virus. The 30S Mo-MSV RNA sequences shared with these viruses were not additive. The Tm of a Mo-MSV RNA-MLV cDNA hybrid was 83 degrees C, indicating that large contiguous nucleotide sequences were shared between the two nucleic acids. Mo-MSV RNA and Mo-MLV RNA shared possibly seven of 20-30 RNAase T1-resistant oligonucleotides, while Mo-MSV RNA contained three, and Mo-MLV RNA contained at least five specific oligonucleotides. We conclude that the 30S Mo-MSV RNA molecule consists of approximately 70% (about 4200 nucleotides) Mo-MLV-specific sequences and of 30% (1800 nucleotides) Mo-MSV-specific sequences covalently linked. Our results favor the hypothesis that 30S Mo-MSV RNA was generated by recombination between Mo-MLV and other genetic elements. We discuss whether all or only the MSV-specific sequences of the 30S Mo-MSV RNA function as sarcoma genes. Mo-MLV cDNA was hybridized about 45% by unfractionated Mo-MSV (MLV) RNA at RNA/DNA ratios of up to 10, about 50% by electrophoretically purified 30S Mo-MSV RNA at RNA/DNA ratios up to 500, but close to 100% by unfractionated Mo-MSV(MLV) RNA at RNA/DNA ratios over 900. This indicated that unfractionated RNA of our Mo-MSV(MLV) contained a complete complement of Mo-MLV, albeit at a low ratio.     

Integration of hepatitis B virus DNA in chromosome-specific satellite sequences.

We previously reported the cloning and detailed analysis of the integrated hepatitis B virus sequences in a human hepatoma cell line. We report here the integration of at least one of hepatitis B virus at human satellite DNA sequences. The majority of the cellular sequences identified by this satellite DNA were organized as a multimeric composition of a 0.6-kilobase EcoRI fragment. This clone hybridized in situ almost exclusively to the centromeric heterochromatin of chromosomes 1 and 16 and to a lower extent to chromosome 2 and to the heterochromatic region of the Y chromosome. The immediate flanking host sequence appeared as a hierarchy of repeating units which were almost identical to a previously reported human satellite III DNA sequence.