Essential regions of the tRNA primer required for HIV-1 infectivity

Abstract

Human immunodeficiency virus (HIV), like all retroviruses, requires a cellular tRNA as a primer for initiation of reverse transcription. In a previous study, we demonstrated that an HIV-1 with a primer binding site complementary to yeast tRNA(Phe) (psHIV-Phe) was not infectious unless yeast tRNA(Phe) was supplied in trans. This unique in vivo complementation system has now been used to define the elements of the tRNA required for HIV-1 replication. Mutant tRNA(Phe) with deletions in TPsiC stem-loop, anticodon stem-loop or D stem-loop of the tRNA were generated and assessed for the capacity to rescue psHIV-Phe. Mutant tRNA(Phe) with disrupted TPsiC stem-loop did not rescue psHIV-Phe. In contrast, a mutant tRNA(Phe) without the D stem-loop was fully functional for the rescue. The tRNA anticodon stem-loop region was found to be important for efficient complementation. The results of our studies demonstrate for the first time the importance of specific structural and sequence elements of the tRNA primer for HIV-1 reverse transcription and define new targets for interruption of HIV-1 replication.

Figure 1

psHIV-Phe proviral genome. The defective psHIV-Phe proviral genome with a PBS complementary to yeast tRNA^Phe is depicted. The gene encoding xanthine-guanosine phosphoribosyl transferase (gpt) under the control of SV40 early promoter was substituted for the env gene of HIV. The 18-nt sequence of the PBS complementary to yeast tRNA^Phe is referred to as PBS^Phe. To generate pseudoviruses, this plasmid was co-transfected with the plasmid encoding VSV-G protein either with or without tRNA. Successful infection of cells with the pseudoviruses confers resistance to mycophenolic acid.

Figure 2

The capacity of the yeast tRNA^Phe deletion mutants to restore psHIV-Phe infectivity. (A) The RNA nucleotide sequences of yeast tRNA^Phe, mutant 35mer and 62mer. The 3′-terminal 18-nt complementary to the PBS of psHIV-Phe are in bold. (B) The numbers of drug-resistant colonies derived from the infection of psHIV-Phe pseudoviruses generated by cotransfecting psHIV-Phe provirus and VSV-G expression plasmid (pLGRNL) along with wild-type yeast tRNA^Phe, 35mer or 62mer. psHIV-Phe virus without tRNA complementation served as a negative control. (C) The stability of biotin-labeled tRNA^Phe mutants following cotransfection. Biotin-labeled yeast tRNA^Phe or mutants were cotransfected with psHIV-Phe provirus and pLGRNL into 293T cells by incubating the transfection mixtures with the cells for 8–10 h. At 0 or 36 h after cotransfection, total RNAs were isolated from the transfected cells, spotted on a Nylon membrane and visualized using a biotin luminescent detection kit. Biotin-labeled tRNA^Phe was spotted directly on the membrane as a positive control (control 1). The cotransfection of unlabeled tRNA^Phe with psHIV-Phe and pLGRNL served as a negative control (control 2); cells incubated with biotin-labeled tRNA^Phe, psHIV-Phe and pLGRNL in the absence of transfection reagent were also included as a control (control 3). The RNA stocks used for cotransfection were diluted and then spotted on the membrane as controls (labeled as input RNA). (D) Stability of ³⁵S-labeled tRNA^Phe mutants following cotransfection. Similar experiments as described in (C) were carried out with ³⁵S-labeled tRNAs in place of the biotin-labeled tRNAs. Nucleic acids were precipitated from the cells at 0, 18 or 36 h after cotransfection and collected to determine radioactivity by counting. The counts obtained at 0 h (time 0 corresponds to 8 h after the incubation of transfection mixtures with cells), 18 and 36 h were divided by the input amounts for each sample. The values presented represent percentages of the input amount.

Figure 3

Complementation of psHIV-Phe virus by cotransfected yeast tRNA^Phe mutants. (A) The sequence of four yeast tRNA^Phe mutants generated by further deletion within the 62mer. The 3′-terminal 18-nt that are complementary to the PBS of psHIV-Phe are in bold. (B) The numbers of drug-resistant colonies derived from infection of the complemented pseudoviruses; psHIV-Phe virus without complementation was used as a control. (C) The stability of the tRNA^Phe mutants following cotransfection. The biotin-labeled yeast tRNA^Phe or mutants cotransfected with psHIV-Phe and pLGRNL were recovered from the transfected cells and analyzed as described in Figure 2C.

Figure 4

Comparison of the tRNA^Phe mutants with different structures in the anticodon stem–loop region. (A) Two mutants were generated by altering nucleotides in the anticodon stem of mutant 62mer. The C₁₄-G₁₆ to G₁₄-C₁₆ (outlined) change in the 62-loop was predicted to disrupt the anticodon stem. The 62-stem was generated by introducing mutations (C₂₆-G₂₈ to G₂₆-C₂₈) (outlined) into mutant 62-loop to restore the complementarity between the two strands. (B) Drug-resistant colonies derived from the infection of psHIV-Phe virus complemented by 62-loop or 62-stem; psHIV-Phe virus without tRNA complementation was used as a control. (C) Stability of the tRNA^Phe mutants following cotransfection. See Figure 2C for description of the procedure.

Figure 5

Complementation of psHIV-Phe by a tRNA^Phe mutant with the anticodon of tRNA^Lys3. (A) The anticodon of tRNA^Phe (GAA) was substituted with the anticodon of tRNA^Lys3 (UUU, in bold) in the 62mer (Fig. 2A) to generate mutant 62-UUU. (B) The numbers of drug-resistant colonies derived from the infection of psHIV-Phe virus rescued by 62-UUU, 62mer or tRNA^Phe.

Figure 6

Summary of the effect of tRNA^Phe mutations on primer selection and use in HIV-1 reverse transcription. The tertiary or ‘L-shape’ representation of yeast tRNA^Phe is depicted. Regions of the tRNA important for complementing the psHIV-Phe virus are indicated by triangles. The region not required for virus complementation is labeled with an x. Circles indicate the positions of the point mutations affecting the secondary structure of the anticodon stem while not interfering with the virus complementation.