Morris Shannon, Johnson Michael, Stavnezer Ed, Leis Jonathan
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935, USA.
J Virol. 2002 Aug;76(15):7571-7. doi: 10.1128/jvi.76.15.7571-7577.2002.
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.
禽肉瘤病毒（ASV）中的逆转录从tRNA(Trp)引物的3'端起始，该引物在RNA基因组的5'端附近退火。引物结合位点（PBS）周围的区域形成了一个复杂的茎结构，由U5反向重复序列（U5-IR）茎、U5前导茎以及tRNA引物与PBS的结合组成。有证据表明病毒U5 RNA与tRNA(Trp)的TψC环之间存在额外的相互作用（U5-TψC）。我们现在证明这种U5-TψC相互作用对于ASV在培养物中的高效复制是必需的。通过随机化病毒RNA的特定生物学相关区域，从而产生一个突变病毒文库，我们能够通过多轮感染选择那些赋予病毒生存适应性的序列。随机化病毒RNA的U5-TψC相互作用区域导致在五轮感染后选择出大部分野生型序列。还回收了能够与tRNA(Trp)的TψC环进行碱基配对的突变病毒。为了证明这种相互作用对tRNA引物具有特异性，我们构建了第二个文库，在其中我们改变PBS以使其与tRNA(Pro)退火，同时随机化病毒RNA的U5-TψC区域。经过五轮感染后，出现了一致序列5'-GPuPuCPy-3'，它与tRNA(Pro)的TψC环中发现的5'-GGTTC-3'序列互补。这些观察结果证实了U5-TψC相互作用在体内的重要性。
