Mutation of the Methylated tRNA3Lys Residue A58 Disrupts Reverse Transcription and Inhibits Replication of Human Immunodeficiency Virus Type 1

Abstract

Cellular tRNA 3Lys serves as the primer for reverse transcription of human immunodeficiency virus type 1 (HIV-1). tRNA 3 Lys interacts directly with HIV-1 reverse transcriptase (RT), is packaged into viral particles, and anneals to the primer-binding site (PBS) of the HIV-1 genome in order to initiate reverse transcription. Residue A58 of tRNA 3 Lys , which lies outside the PBS-complementary region, is posttranscriptionally methylated to form 1-methyladenosine 58 (M 1 A58). This methylation is thought to serve as a pause signal for plus-strand strong-stop DNA synthesis during reverse transcription. However, formal proof that the methylation is necessary for the pausing of RT has not been obtained in vivo. In the present study, we investigated the role of tRNA 3Lys residue A58 in the replication cycle of HIV-1 in living cells. We have developed a mutant tRNA 3 Lys derivative, tRNA 3 Lys A58U, in which A58 was replaced by U. This mutant tRNA was expressed in CEM cells. We demonstrate that the presence of M 1 A58 is necessary for the appropriate termination of plus-strand strongstop DNA synthesis and that the absence of M 1 A58 allows RT to read the tRNA sequences beyond residue 58. In addition, we show that replacement of M 1 A58 with U inhibits the replication of HIV-1 in vivo. These results highlight the importance of tRNA primer residue A58 in the reverse transcription process. Inhibition of reverse transcription with mutant tRNA primers constitutes a novel approach for therapeutic intervention against HIV-1.Retroviruses contain two copies of an RNA genome but replicate via a DNA intermediate (18). Reverse transcription of the RNA genome into DNA is performed by the viral enzyme reverse transcriptase (RT). The primer for reverse transcription is a cellular tRNA. Retroviruses, long terminal repeat (LTR) retrotransposons, and long interspersed nucleotide element retrotransposons use cellular tRNAs to initiate cDNA synthesis. Different tRNAs are used by different retroviruses and retrotransposons (12,14).Lentiviruses, such as feline and simian immunodeficiency viruses and human immunodeficiency virus (HIV), use tRNA 3 Lys as their primers. tRNA 3 Lys interacts directly with the HIV-1 RT, is packaged into viral particles, and anneals to the PBS of the HIV-1 genome in order to initiate reverse transcription.Early in the viral life cycle, tRNA 3 Lys primes minus-strand strong-stop DNA synthesis. Plus-strand strong-stop DNA synthesis is primed by the polypurine tract. During plus-strand strong-stop DNA synthesis, elongation terminates at a 1-methyladenosine at position 58 (M 1 A58) of tRNA 3 Lys . The precise mechanism of termination at this stage of reverse transcription is not fully characterized. After termination of plus-strand strong-stop DNA synthesis, the tRNA primer is removed by RNase H, allowing the second-strand transfer and subsequent completion of reverse transcription.The role of M 1 A58 of tRNA 3 Lys in the retroviral life cycle was first proposed by Gilboa et al. (8), who suggested that termination of ...

The bipartite structure of the tRNA m1A58 methyltransferase from S. cerevisiae is conserved in humans

“…Past work has shown that the presence of m 1 A58 leads to proper termination of + strand strong-stop DNA synthesis during HIV replication, presumably because m 1 A can not base pair correctly (Burnett and McHenry 1997;Renda et al 2001). The significance of m 1 A58 is further demonstrated by in vivo studies in which HIV replication is inhibited in T cells expressing tRNA 3 Lys with A58 changed to U (Renda et al 2001(Renda et al , 2004. Since tRNA 3…”

“…Along with the function of m 1 A58 in tRNA i Met stability, it has been reported that m 1 A58 in human tRNA 3 Lys is important for efficient replication of human immunodeficiency virus type 1 (HIV-1) (Gilboa et al 1979;Burnett and McHenry 1997;Renda et al 2001). All retroviruses contain a primer binding site (PBS) of 18 nucleotides that is complementary to the 3 0 -end of a host tRNA, allowing the tRNA to be used as a primer for À strand DNA synthesis and as a template for replication of the PBS on the + strand.…”

Modification of Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Target Cells with Elevated Cellular dNTP Concentrations

“…The HIV-1 packaging-GFP plasmid, pHIV-GFP⌬Env (a gift from Dr. Vicente Planelles, University of Utah), expresses all strain NL4-3 proteins except for Env and Nef, which have been replaced by EGFP (25). A plasmid expressing vesicular stomatitis virus envelope protein, pVSV-G, was also obtained from Dr. Planelles.…”

“…Pseudotyped HIV-1 Vector Production-pHCMV-VSVG envelope vector (32) and pHIV-EGFP⌬Env transfer vector (25), which encodes the HIV-1 NL4-3 genome with the EGFP gene in place of HIV-1 Nef and a deletion of the env gene, were used for preparing pseudotyped HIV-1. The Q151N mutations were cloned into the pD3-EGFP (pNL4-3-based) vector using overlapping PCR with proper primers that were designed based on the pNL4-3 RT sequence as described previously (2).…”

Publisher's Note

“…During plusstrand strong-stop synthesis, however, N 1 -methylation at adenosine 58 (A58) in tRNA Lys3 is required to properly terminate DNA synthesis. 47,48 Replacement of A58 with U, which is not methylated to produce the block, allows HIV-1 RT to read past this point and inhibits HIV-1 replication.…”