Role of Post-transcriptional Modifications of Primer tRNALys,3 in the Fidelity and Efficacy of Plus Strand DNA Transfer during HIV-1 Reverse Transcription

Abstract

During HIV reverse transcription, (؉) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3 end of tRNA Lys,3 hybridized to the viral primer binding site (PBS). Before (؉) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3 end of primer tRNA Lys,3 . Since the detailed mechanism of (؉) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV Lys,3 . Thus, modified nucleoside m 1 A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (؉) strand DNA transfer. We show that other factors such as the nature of the (؊) PBS of the acceptor template and the RNase H activity of RT also influence the efficacy of (؉) strand DNA transfer.The distinctive characteristic of retroviruses is the conversion of their RNA genome into double-stranded DNA, which is subsequently integrated into the cellular genome. Reverse transcription is a complex process composed of multiple steps catalyzed by the viral enzyme reverse transcriptase (RT) 1 (reviewed in Ref. 1). The general model of reverse transcription includes two obligatory DNA transfer reactions (Fig. 1A). RT initiates minus strand DNA synthesis by elongation of a specific cellular tRNA primer annealed to the primer binding site (PBS) and continues as far as the 5Ј end of the RNA genome, generating the so-called strong stop cDNA (ss-cDNA). The concomitant degradation of the RNA template by the RNase H activity of RT makes the ss-cDNA single stranded. ss-cDNA is transferred by NC protein (2, 3) to the 3Ј end of either the template RNA or the other RNA molecule present in the virus particle to complete (Ϫ) strand DNA synthesis. (ϩ) strand synthesis is primed by an RNase H-resistant sequence, the polypurine tract (PPT), and continues as far as a doublestranded RNA region corresponding to the 18-nt tRNA-viral PBS RNA complex. RT needs to unwind this tRNA-PBS RNA to reverse-transcribe the 3Ј end of primer tRNA. The stop signal for this (ϩ) strand synthesis has been proposed to be the methylated adenosine at position 58 (m 1 A-58) of primer tRNA (4, 5) but it is not the absolute termination site of RT in the case of HIV (6, 7). Termination at m 1 A-58 results in the (ϩ) strong stop DNA species carrying a complete (ϩ) PBS sequence at its 3Ј terminus. Before (ϩ) strand DNA transfer, the tRNA primer is thought to be removed by the RT-associated RNase H activity (7-11); thus, the (ϩ) PBS at the 3Ј end of (ϩ) ssDNA becomes available for hybridization to the (Ϫ) PBS synthesized at the 3Ј end of the (Ϫ) strand DNA, and synthesis of both strands resumes with strands copying each other (Fig. 1A).In this report, we analyzed the following factors that influence the efficiency of HIV-1 (ϩ) strand DNA transfer: the modified nucleosides of primer tRNA Lys,3 , the RNase H activity...

Identification of Eukaryotic and Prokaryotic Methylthiotransferase for Biosynthesis of 2-Methylthio-N6-threonylcarbamoyladenosine in tRNA

“…Combining these phylogenomic results with the previously established occurrence of hypermodified adenosines ms 2 i 6 A and ms 2 t 6 A in both B. subtilis and human tRNAs (26,27), we hypothesized that both MtaB and e-MtaB enzyme families are likely to catalyze the methylthiolation of t 6 A to form ms 2 t 6 A (Scheme 2). Because previously characterized MiaB family members all catalyze biosynthesis of ms 2 i 6 A (22, 28), the MiaB family members encoded in the B. subtilis and human genomes are likely to be responsible for the observed biosynthesis of ms 2 i 6 A.…”

NMR and biochemical characterization of recombinant human tRNA3 Lys expressed in Escherichia coli: Identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription

“…Lys are essential for its function as a primer of reverse transcription (Barat et al+, 1991;Isel et al+, 1993Isel et al+, , 1996Lanchy et al+, 1996) (Björk, 1996) and none of these have been described as crucial for priming reverse transcription+ Only m 1 A 58 is important for efficacy and fidelity of (ϩ) strand DNA transfer (Burnett & McHenry, 1997;Auxilien et al+, 1999) (Roy et al+, 1984)+ The methyl of m 7 G 46 (3+86 ppm) resonates at a lower field because of the nearness of the positively charged N7 atom+ It gives a strong NOESY cross-peak to its aromatic H8 proton, which is also shifted downfield at 9+25 ppm (Fig+ 2) and is slowly exchangeable with the solvent, as previously described for m 7 G (Leroy et al+, 1985)+ The methyl at 1+35 ppm corresponds to that of a threonine side chain+ Connectivities within the threonine spin system were confirmed in a TOCSY experiment (not shown)+ The NOE cross-peak between the methyl group of the threonine side chain and its amide proton can be seen in Figure 2+ Finally, the methyl at 2+95 ppm must correspond to the mnm 5 s 2 U 34 nucleotide because the NH at 10+05 ppm has two NOEs with a methyl at 2+95 ppm and a CH 2 group at 3+60 ppm+ The presence of the two dihydrouridines (D) could be detected in the NOESY and the TOCSY experiments, through the observation of correlations between the ring CH 2 methylene groups (not shown)+ These were straightforward to identify, as they resonate between 2+0 and 3+0 ppm, in a region of the spectrum that is otherwise devoid of signal in unmodified RNA+ However, individual assignment of these protons could not be achieved+ Pseudouridines (⌿) contain two imino groups (HN1 and HN3) separated by a CO group+ In an HNCO type experiment performed on a doubly labeled 13 C/ 15 N tRNA 3 Lys , these two imino protons must thus correlate to the same intervening carbonyl+ We were able to detect such a double correlation for ⌿ 55 (data not shown)+ For ⌿ 39 , only the HN1 imino proton was observed, presumably because the other imino group is in a fast exchange rate with solvent+ Confirmation of the ⌿ assignments came from 1 H{ 15 N} HMQC experiment (Fig+ 2B) in which both N1 imino groups of ⌿ 39 and ⌿ 55 resonate, respectively, at 133 ppm and 136 ppm in the nitrogen dimension, quite far from both the N3 iminos of Us (159-163 ppm) and the N1 iminos of Gs (146-151 ppm)+ Furthermore, the HNCO experiment also revealed the correlation originating from the threonine moiety of the t 6 A 37 nucleotide, which is traditionally observed across an amide bond+ As sulphur nuclei are not readily amenable to NMR studies, the presence of thiolated nucleotides was investigated by looking for their sensitivity to chemical Lys oxidation+ Iodine treatment of tRNA selectively promotes the formation of disulphide bridges between 2-thio-uridine residues (Carbon et al+, 1965)+ This, in turn, inactivates the tRNA, which forms covalent dimers+ When applied to our recombinant tRNA, this treatment induces a dimerization of a fraction of the molecules (15-30%), as observed by denaturing gel electrophoresis (not shown)+ This indicates that a significant fraction of the recombinant tRNA contains thiolated nucleotides+ This must correspond to s 2 U 34 , because we checked that s 4 U 8 is not reactive to iodine under the same co...…”

The Arginine Clusters of the Carboxy-Terminal Domain of the Core Protein of Hepatitis B Virus Make Pleiotropic Contributions to Genome Replication

“…Similarly, formation of long-range base paired structures may be facilitated by a NAC akin to the way retroviral NC or NC-like proteins (in LTR retrotransposons) facilitate the rearrangements that occur during genomic RNA dimerization and primer tRNA binding (14,26,27,29,54,55). Finally, the actual DNA strand transfers may be catalyzed, akin to how retroviral NC proteins catalyze the minus-and plus-strand transfers for those viruses (4,46,67). The results of the present study do not directly test the hypothesis that the CTD is a NAC.…”