Selection of functional mutations in the U5-IR stem and loop regions of the Rous sarcoma virus genome.
BACKGROUND
The 5' end of the Rous sarcoma virus (RSV) RNA around the primer-binding site forms a series of RNA secondary stem/loop structures (U5-IR stem, TpsiC interaction region, U5-leader stem) that are required for efficient initiation of reverse transcription. The U5-IR stem and loop also encode the U5 integrase (IN) recognition sequence at the level of DNA such that this region has overlapping biological functions in reverse transcription and integration.


RESULTS
We have investigated the ability of RSV to tolerate mutations in and around the U5 IR stem and loop. Through the use of viral libraries with blocks of random sequence, we have screened for functional mutants in vivo, growing the virus libraries in turkey embryo fibroblasts. The library representing the U5-IR stem rapidly selects for clones that maintain the structure of the stem, and is subsequently overtaken by wild type sequence. In contrast, in the library representing the U5-IR loop, wild type sequence is found after five rounds of infection but it does not dominate the virus pool, indicating that the mutant sequences identified are able to replicate at or near wild type levels.


CONCLUSIONS
These results indicate that the region of the RNA genome in U5 adjacent to the PBS tolerates much sequence variation even though it is required for multiple biological functions in replication. The in vivo selection method utilized in this study was capable of detecting complex patterns of selection as well as identifying biologically relevant viral mutants.
Johnson
Michael
M
Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. michaeljohnson@northwestern.edu
Morris
Shannon
S
Chen
Aiping
A
Stavnezer
Ed
E
Leis
Jonathan
J
D001358:Avian Sarcoma Viruses / Q000737:chemistry / Q000235:genetics*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000821:virology; D015723:Gene Library; D016678:Genome; D009154:Mutation; D009690:Nucleic Acid Conformation; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D048348:Reverse Transcription; D014162:Transfection; D014421:Turkey
D012342:RNA, Small Nuclear; D012367:RNA, Viral; C405485:U5 small nuclear RNA