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Journal of Virology, October 2006, p. 9641-9650, Vol. 80, No. 19
0022-538X/06/$08.00+0     doi:10.1128/JVI.00709-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Complementation of Human Immunodeficiency Virus Type 1 Replication by Intracellular Selection of Escherichia coli Supplied in trans

Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0024

Received 7 April 2006/ Accepted 12 July 2006

Human immunodeficiency virus type 1 (HIV-1) exclusively selects tRNA₃^Lys as the primer for the initiation of reverse transcription, even though both tRNA₃^Lys and tRNA_1,2^Lys are found in HIV-1 virions. Alteration of the HIV-1 primer-binding site (PBS) to be complementary to alternate tRNAs results in the use of those tRNAs for replication, indicating that primer complementarity with the PBS is an important determinant of primer selection. In previous studies, we have exploited this fact to develop a system in which yeast (Saccharomyces cerevisiae) tRNA^Phe is provided in trans to complement the replication of HIV-1 with a PBS complementary to yeast tRNA^Phe. Recent studies have demonstrated that the presence of lysyl-tRNA synthetase in HIV-1 virions might account for the preference for the selection of tRNA₃^Lys in HIV-1 replication. To establish a complementation system more reflective of HIV-1 primer selection, we have altered the HIV-1 PBS to be complementary to the Escherichia coli tRNA₃^Lys, which shares near identity with mammalian tRNA₃^Lys except in the 3'-terminal 18-nucleotide sequence that binds to the PBS. E. coli tRNA₃^Lys expressed from a plasmid was aminoacylated in mammalian cells. Cotransfection of cells with a plasmid that encodes E. coli tRNA₃^Lys and a plasmid encoding an HIV-1 provirus with a PBS complementary to E. coli tRNA₃^Lys resulted in the production of infectious virus. A comparison of the two complementation systems revealed that higher levels of intracellular E. coli tRNA₃^Lys than of yeast tRNA^Phe were needed to achieve equal levels of infectious virus, indicating that there was no preferential selection of E. coli tRNA₃^Lys. To examine the specificity of tRNA^Lys selection, E. coli tRNA₃^Lys was modified to tRNA_1,2^Lys. This tRNA was also aminoacylated when expressed in mammalian cells and complemented the infectivity of HIV-1 at levels similar to those seen for E. coli tRNA₃^Lys. Additional mutations in the anticodon of E. coli tRNA₃^Lys were constructed; these mutations did not significantly correlate with the capacity of the tRNA primer to complement infectivity of HIV-1, even though they had a drastic effect on the aminoacylation of the tRNAs. The results of these studies demonstrate that E. coli tRNA₃^Lys provided in trans can complement HIV-1 genomes with the PBS altered to E. coli tRNA₃^Lys. However, the capacity of tRNA₃^Lys to interact with lysyl-tRNA synthetase does not entirely explain the enhanced preference for selection of tRNA₃^Lys for the replication of HIV-1.