Replication of Avian Sarcoma Virus In Vivo Requires an Interaction between the Viral RNA and the TψC Loop of the tRNA Trp Primer

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Abstract
Reverse transcription in avian sarcoma virus (ASV) initiates from the 3′ end of a tRNATrp 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 TψC loop of the tRNATrp (U5-TψC). We now demonstrate that this U5-TψC 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-TψC 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 TψC loop of the tRNATrp. To prove this interaction is specific to the tRNA primer, we constructed a second library, in which we altered the PBS to anneal to tRNAPro, while simultaneously randomizing the viral RNA U5-TψC 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 TψC loop of tRNAPro. These observations confirm the importance of the U5-TψC interaction in vivo.