Identification of critical elements in the tRNA acceptor stem and T(Psi)C loop necessary for human immunodeficiency virus type 1 infectivity - PubMed (original) (raw)

Identification of critical elements in the tRNA acceptor stem and T(Psi)C loop necessary for human immunodeficiency virus type 1 infectivity

Q Yu et al. J Virol. 2001 May.

Abstract

A mutant human immunodeficiency virus type 1 (HIV-1) with a primer binding site (PBS) complementary to yeast tRNA(Phe) (psHIV-Phe), which relies on exogenous yeast tRNA(Phe) as reverse transcription primer, was used to investigate elements in the tRNA acceptor stem and T(Psi)C stem-loop required for the tRNA primer selection and use in HIV-1 replication. tRNA(Phe) mutants with two- or four-nucleotide deletions in the 3' end retained the capacity to complement replication of psHIV-Phe. tRNA(Phe) mutants with an extended 5' end had reduced capacity for complementation, which could be restored by extension of the 3' end of these tRNA(Phe) mutants with sequences complementary to the HIV-1 U5 region. Further analysis of mutations in the acceptor stem of tRNA(Phe) suggested that an intact acceptor stem RNA structure is important for complementation. Analysis of single-nucleotide changes in the T(Psi)C stem-loop of tRNA(Phe) revealed an unexpected, essential role of this region for rescue of psHIV-Phe.

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Figures

FIG. 1

Effect of tRNAPhe acceptor stem mutations on complementation of psHIV-Phe. (A) psHIV-Phe proviral genome. The defective psHIV-Phe proviral genome contains a PBS complementary to yeast tRNAPhe (PBSPhe). The env gene of HIV was substituted by the gpt gene under the control of simian virus 40 early promoter (SV-gpt). To generate pseudoviruses, this plasmid was cotransfected with the plasmid encoding vesicular stomatitis virus G protein either with or without tRNA. Successful infection of cells with the pseudoviruses confers resistance to mycophenolic acid. (B) Illustration of tRNAPhe mutants with nucleotide changes at the acceptor stem. The added or substituted nucleotides are depicted in bold. Only the terminal portion of each tRNAPhe mutant (circled) is illustrated. (C and D) Numbers of drug-resistant colonies derived from infection of the psHIV-Phe pseudoviruses complemented by indicated mutants. Values are means of data obtained from three independent experiments, with standard deviations depicted (error bars).

FIG. 2

Mutations in the tRNAPhe TΨC stem-loop affect complementation of psHIV-Phe. (A) Illustrations of the positions of the nucleotides mutated in designated tRNAPhe mutants. (B) The psHIV-Phe rescue capacity of the tRNAPhe mutants as measured by drug-resistant colony numbers. Values are means of data obtained from three independent experiments, with standard deviations depicted (error bars).

FIG. 3

RNA secondary structures of the PBS and surrounding regions in psHIV-Phe (A) and in wild-type HIV-1 (HXB2 strain) (B) predicted by the M-fold structure modeling program. The PBS regions are in bold. The boxed nucleotides in a loop region represent the PBS-downstream sequence complementary to the TψC loop sequence of tRNAs. Potential loop-loop interactions between the tRNA TΨC loop and the PBS-downstream sequence of the viral RNA genome are depicted. The drawings are for illustration only and are not to scale.

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Identification of critical elements in the tRNA acceptor stem and T(Psi)C loop necessary for human immunodeficiency virus type 1 infectivity - PubMed (original) (raw)