Susceptibility of Human Hepatitis Delta Virus RNAs to Small Interfering RNA Action

Abstract

In animal cells, small interfering RNAs (siRNA), when exogenously provided, have been reported to be capable of inhibiting replication of several different viruses. In preliminary studies, siRNA species were designed and tested for their ability to act on the protein expressed in Huh7 cells transfected with DNAdirected mRNA constructs containing hepatitis delta virus (HDV) target sequences. The aim was to achieve siRNA specific for each of the three RNAs of HDV replication: (i) the 1,679-nucleotide circular RNA genome, (ii) its exact complement, the antigenome, and (iii) the less abundant polyadenylated mRNA for the small delta protein.Many of the 16 siRNA tested gave >80% inhibition in this assay. Next, these three classes of siRNA were tested for their ability to act during HDV genome replication. It was found that only siRNA targeted against HDV mRNA sequences could interfere with HDV genome replication. In contrast, siRNA targeted against genomic and antigenomic RNA sequences had no detectable effect on the accumulation of these RNAs. Reconstruction experiments with nonreplicating HDV RNA sequences support the interpretation that neither the potential for intramolecular rod-like RNA folding nor the presence of the delta protein conferred resistance to siRNA. In terms of replicating HDV RNAs, it is considered more likely that the genomic and antigenomic RNAs are resistant because their location within the nucleus makes them inaccessible to siRNA-mediated degradation.Human hepatitis delta virus (HDV) has a 1,679-nucleotide (nt) single-stranded circular RNA genome that is replicated by RNA-directed RNA synthesis, most probably involving host RNA polymerase II (25). During this replication, three RNA species accumulate (5), as represented in Fig. 1. The genome and its exact complement, the antigenome, are considered unit length. They exist primarily in a circular conformation but also in a linear conformation; these two conformations can be resolved using appropriate conditions of gel electrophoresis (2). The third RNA species consists of relatively lower amounts of an 800-nt polyadenylated RNA (of the same polarity as the antigenome), which is translated to produce a 195-amino-acid protein, known as the delta antigen (␦Ag-S), and is essential for HDV genome replication (14).An increasing number of reports have shown that small interfering RNAs (siRNA) can be exogenously provided to cells undergoing animal virus replication and achieve inhibition (6,8,9,12,13,17,23). For the following reasons, we were specifically interested in the possible susceptibility to siRNA of HDV RNAs. (i) The HDV genomic and antigenomic RNAs can fold into an unbranched rod-like structure with 74% of the bases paired (15), and this folding might interfere with siRNA action. (ii) The delta protein has the ability to bind doublestranded RNA (4) and thus might also interfere. (iii) While several reports indicate that HDV genomic and antigenomic RNAs are predominantly located in the cell nucleus (7,21,24), two recent studies using cell fract...

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