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Figure 1 | Virology Journal

Figure 1

From: Mutations in the TΨC Loop of E. coli tRNALys,3 Have Varied Effects on In Trans Complementation of HIV-1 Replication

Figure 1

Complementation of HIV-1 infectivity with E. coli tRNALys,3 A58U mutant. Panel A. Diagram of E. coli tRNALys,3 A58U mutant. The base change A58U is indicated. Boldface nucleotides indicate the 3' 18-nuleotides complementary to the PBS of HIV-1 (E. coli tRNALys,3).

Panel B. Expression and aminoacylation of E. coli tRNALys,3 and E. coli tRNALys,3 A58U in 293T cells following transfection. Total RNA was isolated under acidic conditions to stabilize the amino acid tRNA bond. Approximately one-half was treated with high pH as to break the amino acid-tRNA bond (deAA). Samples were run on an acid polyacrylamide gel and blotted into nitrocellulose. All samples were analyzed with a probe specific for tRNALys,3 [15]. The migration of aminoacylated tRNA (Lane 2) and deacylated controls (Lane 1) are denoted as AA and deAA, respectively. NT is RNA from non-transfected 293T cells.

Panel C. Infectivity of NL4-EcoLys3 complemented by E. coli tRNALys,3 or E. coli tRNALys,3 A58U. 0.5 μg NL4-EcoLys3 was co-transfected with 0.1, 0.25, 0.5, 1.0 μg plasmids encoding E coli tRNALys,3 or mutant into 293T cells. Virus was collected 48 hours post transfection. The amounts of infectious virus produced from transfection were determined using the JC53-BL bioassay which measures luciferase activity [19]. The infectivity is determined by the amount of luciferase is divided by the amount of virus as determined by p24 ELISA, to give RLu per nanogram. Values are the average (+SD) from three assays.

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