Reconstitution in cultured cells of replicating HDV RNA from pairs of less than full-length RNAs.
The genome of hepatitis delta virus (HDV) is a small single-stranded circular RNA that is replicated via RNA-directed RNA synthesis. This makes use of a host RNA polymerase, probably pol II, that normally transcribes DNA templates. In vivo, the host polymerase can initiate replication from transfected linear RNAs using intramolecular template-switching. The present studies report that the polymerase could also achieve intermolecular switching leading to "reconstitution" of full-length HDV RNAs following transfection with two linear RNAs that were less than full length and yet lacking different regions of the genome. These two RNAs were synthesized in vitro, gel purified, pre-annealed, and then transfected into delta293, a cell line conditionally expressing the small delta antigen that is essential for HDV replication. Northern analyses of total RNA harvested from transfected cells detected the accumulation of full-length HDV genomic and antigenomic RNAs. Such reconstitution of full-length replicating HDV RNA was also achieved using nine other pairs of antigenomic RNAs and three pairs of genomic RNAs. Annealing of the RNAs prior to transfection was required for detectable HDV reconstitution. A second cell line, Huh7, also supported reconstitution when a pair of RNAs was cotransfected together with mRNA for the small delta protein. Taken together, these results support a model that observed genome reconstitution is a special form of recombination involving intermolecular template switches and they provide insights into the mechanism of RNA-directed RNA transcription catalyzed by a host RNA polymerase.
Gudima
Severin O
SO
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, USA.
Chang
Jinhong
J
Taylor
John M
JM
D002460:Cell Line; D016679:Genome, Viral; D003698:Hepatitis Delta Virus / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011995:Recombination, Genetic; D014158:Transcription, Genetic; D014162:Transfection; D014779:Virus Replication / Q000235:genetics*
D012367:RNA, Viral