Hepatitis delta virus (HDV) is a defective RNA virus that depends on the presence of hepatitis B virus (HBV) for the creation of new virions and propagation of the infection to hepatocytes. Chronic infection with HDV is usually associated with a worsening of HBV infection, leading more frequently to cirrhosis, increased risk of liver decompensation and hepatocellular carcinoma (HCC) occurrence. In spite of a progressive declining prevalence of both acute and chronic HDV infection observed over several years, mainly due to increased global health policies and mass vaccination against HBV, several European countries have more recently observed stable HDV prevalence mainly due to migrants from non-European countries. Persistent HDV replication has been widely demonstrated as associated with cirrhosis development and, as a consequence, development of liver decompensation and occurrence of HCC. Several treatment options have been attempted with poor results in terms of HDV eradication and improvement of long-term prognosis. A global effort is deemed urgent to enhance the models already existing as well as to learn more about HDV infection and correlated tumourigenesis mechanisms.

The natural substrate cleaved by the hepatitis delta virus (HDV) ribozyme contains a 3′,5′-phosphodiester linkage at the cleavage site; however, a 2′,5′-linked ribose-phosphate backbone can also be cleaved by both trans-acting and self-cleaving forms of the HDV ribozyme. With substrates containing either linkage, the HDV ribozyme generated 2′,3′-cyclic phosphate and 5′-hydroxyl groups suggesting that the mechanisms of cleavage in both cases were by a nucleophilic attack on the phosphorus center by the adjacent hydroxyl group. Divalent metal ion was required for cleavage of either linkage. However, although the 3′,5′-linkage was cleaved slightly faster in Ca2+ than in Mg2+, the 2′,5′-linkage was cleaved in Mg2+ (or Mn2+) but not Ca2+. This dramatic difference in metal-ion specificity is strongly suggestive of a crucial metal-ion interaction at the active site. In contrast to the HDV ribozymes, cleavage at a 2′,5′-phosphodiester bond was not efficiently catalyzed by the hammerhead ribozyme. The relaxed linkage specificity of the HDV ribozymes may be due in part to lack of a rigid binding site for sequences 5′ to the cleavage site.

The crystal structure of a genomic hepatitis delta virus (HDV) ribozyme 3′ cleavage product predicts the existence of a 2 bp duplex, P1.1, that had not been previously identified in the HDV ribozymes. P1.1 consists of two canonical C-G base pairs stacked beneath the G[bull ]U wobble pair at the cleavage site and would appear to pull together critical structural elements of the ribozyme. P1.1 is the second stem of a second pseudoknot in the ribozyme, making the overall fold of the ribozyme a nested double pseudoknot. Sequence comparison suggests the potential for P1.1 and a similar fold in the antigenomic ribozyme. In this study, the base pairing requirements of P1.1 for cleavage activity were tested in both the genomic and antigenomic HDV ribozymes by mutagenesis. In both sequences, cleavage activity was severely reduced when mismatches were introduced into P1.1, but restored when alternative base pairing combinations were incorporated. Thus, P1.1 is an essential structural element required for cleavage of both the genomic and antigenomic HDV ribozymes and the model for the antigenomic ribozyme secondary structure should also be modified to include P1.1.

Hepatitis delta virus (HDV) is a human pathogen that can greatly increase the severity of liver damage caused by an hepatitis B infection. HDV contains a circular, single-stranded RNA genome that encodes a unique protein, the delta antigen. Two forms of the delta antigen, δAg-S and δAg-L, are derived from a single open reading frame by RNA editing. Here we analyze the subcellular distribution of HDV RNA and its spatial relationship to known intranuclear structures. The human hepatoma cell line Huh7 was stably transfected with wild-type HDV cDNA and the viral RNAs were localized by in situ hybridization and fluorescence confocal microscopy. HDV RNA is detected throughout the nucleoplasm, with additional concentration in focal structures closely associated with nuclear speckles or clusters of interchromatin granules. Both the smaller form of the delta antigen (δAg-S), which is required for HDV genomic replication, and the larger form of the delta antigen (δAg-L), which represses replication, co-localize with delta RNA throughout the nucleoplasm and in the foci. However, the foci do not incorporate bromo-UTP and do not concentrate either RNA polymerase II or cleavage and polyadenylation factors required for viral RNA synthesis and 3′ end processing, respectively. Thus, it is unlikely that the delta foci represent major sites of viral transcription or replication. In conclusion, the data show that viral RNA–protein complexes accumulate in structures closely associated with interchromatin granules, a subnuclear domain highly enriched in small nuclear ribonucleoproteins, poly(A+) RNA, and RNA splicing protein factors. This implies a specific compartmentalization of ribonucleoproteins in the nucleus.