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Journal of Virology, August 2002, p. 7571-7577, Vol. 76, No. 15
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.15.7571-7577.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Replication of Avian Sarcoma Virus In Vivo Requires an Interaction between the Viral RNA and the T{psi}C Loop of the tRNATrp Primer

Shannon Morris,1 Michael Johnson,2 Ed Stavnezer,1 and Jonathan Leis2*

Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935,1 Department of Microbiology and Immunology, Northwestern University School of Medicine, Chicago, Illinois 606112

Received 1 February 2002/ Accepted 22 April 2002

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 T{psi}C loop of the tRNATrp (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 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-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 tRNAPro. These observations confirm the importance of the U5-T{psi}C interaction in vivo.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Northwestern University School of Medicine, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-0338. Fax: (312) 503-2790. E-mail: j-leis{at}northwestern.edu.

Journal of Virology, August 2002, p. 7571-7577, Vol. 76, No. 15
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.15.7571-7577.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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