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

Abstract

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 <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 <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.

Virology of Hepatitis D Virus

“…Certainly, predicted sequence and structure features near this end of the rod are essential for RNA transcription and accumulation. 53,54 Also, the ends of the rod, whether genomic or antigenomic, will bind RNA pol II and even act as templates for short transcripts. 55 However, the biologic relevance of these latter findings is questioned, especially because the transcripts are not primer-independent.…”

Genotype-Specific Complementation of Hepatitis Delta Virus RNA Replication by Hepatitis Delta Antigen

“…In cultured cells, HDV RNA replication requires expression of the short form of HDAg (16). Thus, transfection of HDV cDNA constructs deficient in HDAg expression yields replicating HDV RNA only if an expression construct for HDAg is also cotransfected and if the defect which abrogates HDAg expression leaves intact structural elements required for RNA replication (1,15,17,35). To examine the ability of HDAg to support replication of genotype I and genotype III HDV RNAs, constructs for intracellular expression of RNAs defective for HDAg synthesis were created (Fig.…”

“…This insertion created a stop codon as well as a frameshift. A single-base insertion was used in order to minimize potential effects on the RNA secondary structure that could also affect the ability of the RNA to replicate (35).…”

Restoration in vivo of defective hepatitis delta virus RNA genomes

“…For example, even after maintenance of the ribozyme domains and an attempt to conserve the rodlike folding, along with providing a separate source of dAg, such modified RNAs failed to replicate in transfected cells (Lazinski and Taylor 1994). In contrast, when relatively small changes, insertions, deletions, or even sequence replacements were made at either end of the rodlike structure, some of these RNAs were able to replicate (Netter et al , 1995aWu et al 1997;Gudima et al 1999). In another study, an 8-nt palindromic sequence was inserted at a series of sites around the genome.…”