Conserved features of an RNA promoter for RNA polymerase II determined from sequence heterogeneity of a hepatitis delta virus population

The right terminal domain of genomic hepatitis delta virus (HDV) RNA is involved in viral replication by recruiting host RNA polymerase II. To identify conserved features of this region, we performed high-throughput 454 sequencing of an HDV population actively replicating in cells. We generated 473,139 sequences representing 2351 new HDV variants of this region and investigated nucleotide conservation and positions of covariation in the population. We found that the sequence is heterogeneous and the rod-like conformation is conserved for both polarities of the HDV RNA genome at this location. Additionally, we identified conserved nucleotides near the previously reported initiation site of transcription, and corroborated our finding with sequences from HDV variants isolated in various hosts. Our analysis highlights the importance of both a conserved sequence at the tip of the rod-like structure and the RNA secondary structure upstream of the tip, which are likely important for HDV replication.