title: Replication of avian sarcoma virus in vivo requires an interaction between the viral RNA and the TpsiC loop of the tRNA(Trp) primer.
abstract: Reverse transcription in avian sarcoma virus (ASV) initiates from the 3' end of a tRNA(Trp) primer, which anneals near the 5' end of the RNA genome. The region around the primer-binding site (PBS) forms an elaborate stem structure composed of the U5-inverted repeat (U5-IR) stem, the U5-leader stem, and the association of the tRNA primer with the PBS. There is evidence for an additional interaction between the viral U5 RNA and the T psi C loop of the tRNA(Trp) (U5-T psi C). We now demonstrate that this U5-T psi C interaction is necessary for efficient replication of ASV in culture. By randomizing specific biologically relevant regions of the viral RNA, thereby producing a library of mutant viruses, we are able to select, through multiple rounds of infection, those sequences imparting survival fitness to the virus. Randomizing the U5-T psi C interaction region of the viral RNA results in selection of largely wild-type sequences after five rounds of infection. Also recovered are mutant viruses that maintain their ability to base pair with the T psi C loop of the tRNA(Trp). To prove this interaction is specific to the tRNA primer, we constructed a second library, in which we altered the PBS to anneal to tRNA(Pro), while simultaneously randomizing the viral RNA U5-T psi C region. After five rounds of infection, the consensus sequence 5'-GPuPuCPy-3' emerged, which is complementary to the 5'-GGTTC-3' sequence found in the T psi C loop of tRNA(Pro). These observations confirm the importance of the U5-T psi C interaction in vivo.
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935, USA.
authors.forename: Shannon
authors.initials: S
authors.lastname: Morris
authors.forename: Michael
authors.initials: M
authors.lastname: Johnson
authors.forename: Ed
authors.initials: E
authors.lastname: Stavnezer
authors.forename: Jonathan
authors.initials: J
authors.lastname: Leis
mesh_terms: D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D015723:Gene Library; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012364:RNA, Transfer, Trp / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D012364:RNA, Transfer, Trp; D012367:RNA, Viral