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The circular RNA genome of hepatitis delta virus (HDV) can fold into an unbranched rodlike structure. We mutagenized the two ends of this structure and assayed the effects on the ability of the genomes to replicate and accumulate processed RNA transcripts in transfected cells. The top end, defined as that nearest to the 5' end of the putative mRNA for delta antigen, was much more sensitive than the other end, defined as the bottom. Most of the 22 mutants made at the bottom were able to accumulate RNA as well as the wild type. For deletions extending as close as 2 nucleotides (nt) from the predicted domains needed for the two ribozymes, the accumulation levels dropped to &lt;0.1%. In one mutant, 13 nt of HDV was replaced with 57 nt of non-HDV sequences, and accumulation was at 20% of the wild-type level, consistent with the potential of HDV to act as a vector. However, after replacement with a second sequence, accumulation dropped to 1%. For most of the 14 mutants made at the top of the rod, we observed dramatic inhibitory effects. For example, after removal of 3 bp from the stem adjacent to the terminal loop, accumulation dropped to &lt;0.06% of the wild-type genome level. The top region that we considered was adjacent to both the 5' end of the putative mRNA and the domain that has been proposed to contain a promoter for RNA-directed RNA synthesis. The RNA accumulation abilities of certain mutants were tested under additional different experimental conditions. It was found that after longer times, some mutants began to catch up with the wild type. Also, it was found that certain top mutants gave much greater levels of accumulation when transfected into cells containing the small delta antigen. One interpretation of these data is that certain features at the top of the rod are needed for the accumulation of essential delta antigen mRNA species.

Effects of nucleotide changes on the ability of hepatitis delta virus to transcribe, process, and accumulate unit-length, circular RNA