Mechanistic Role of Residue Gln151 in Error Prone DNA Synthesis by Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase (RT)

Abstract

It has previously been reported that mutations in the Gln 151 residue of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) greatly enhance RT fidelity. In this study, we employed pre-steady state kinetic assays to elucidate the mechanistic role of residue Gln 151 in highly error prone DNA synthesis by HIV-1 RT. Using our Q151N high fidelity mutant, which is structurally altered in its ability to interact with the 3-OH on the sugar moiety of the incoming deoxynucleotide triphosphate (dNTP), we examined how this change in RT-dNTP interaction affects HIV-1 RT fidelity. First, we found the binding affinity (K D ) of wild type and Q151N RT proteins to different template/primers to be similar. These results indicate that the Gln 151 residue is not involved in the formation of the binary complex (RT⅐template/primer) during DNA polymerization. We also found that by changing residue 151 from a Gln3 Asn, the maximum rate of dNTP incorporation (k pol ) for both correct and incorrect dNTPs was not affected. In contrast, the ability of the Q151N mutant to bind both correct and incorrect dNTPs (K d ) was diminished. The Q151N mutant was 120-fold less efficient at binding correct dNTP than wild type RT, and its decrease in binding was such that we were unable to measure the actual binding affinity of Q151N for incorrect dNTPs. Presumably, the fidelity increase observed during the steady state is explained by this defect in Q151N binding to incorrect dNTP. In wild type RT, residue Gln 151 is important for tight binding of incorrect dNTPs and may contribute to the low fidelity nature of HIV-1 RT. Since the Q151N mutation also alters RT binding to correct dNTPs, the wild type Gln 151 residue may play an important role in efficient binding of RT to correct dNTPs. Our findings suggest that residue Gln 151 is an important element for the execution of both highly error prone and efficient DNA synthesis by HIV-1 RT.It is becoming more apparent that many organisms employ multiple DNA polymerases to replicate their genomes. Some of these DNA polymerases are specifically involved in error prone DNA synthesis required for either spontaneous mutagenesis or bypassing DNA damage (1-3). Recent biochemical studies of DNA polymerases and and their bacterial UmuC/DinB homologs (4 -6) show that these are actually very low fidelity polymerases. It is possible that these organisms have evolved functionally diverse DNA polymerases for the different activities of genomic replication and mutagenesis. As demonstrated in a series of kinetic experiments with various DNA polymerases, it is clear that the ability to incorporate incorrect dNTPs 1 affects the efficiency of DNA synthesis (7). In other words, low fidelity and poor ability to discriminate between correct and incorrect dNTPs are detrimental to efficient DNA polymerization, which is likely essential for chromosomal DNA replication. The fact that efficient DNA synthesis and error prone DNA synthesis are kinetically at odds with one another may explain why possession of separat...

Macrophage Tropism of HIV-1 Depends on Efficient Cellular dNTP Utilization by Reverse Transcriptase

“…The dNTP binding to the active site of the mutant RTs likely becomes a rate-limiting step during the steady-state primer extension reaction at low dNTP concentrations. Overall DNA polymerization capability at the dNTP concentrations found in macrophages is greatest for wild-type RT, followed by V148I RT and then Q151N RT, which displayed the most significant decrease in dNTP binding affinity (14,17). Our kinetic analysis demonstrated that, like these HIV-1 dNTP binding mutants, MuLV RT also has a 40 -75-fold higher k d than wild-type HIV-1 (data not shown).…”

“…Reaction products were immediately denatured by incubating at 95°C for 5 min, and 4 l of each 30 l of final reaction mixture was quantitated by phosphorimaging analysis (PerkinElmer Life Sciences) of 14% acrylamide-urea denaturing gels (SequaGel, National Diagnostics; model S2 sequencing gel electrophoresis apparatus, Labrepco). Note that in our purification protocol, ϳ30 -60% of the purified RT protein was active as determined by our pre-steady-state kinetic assay using a similar 18-mer/40-mer primer/ RNA template (17,18). The reaction with less RT (10 nM), which is still a higher concentration than the highest dNTP concentration used (6.4 nM), gave a similar incorporation profile under the assay condition.…”

“…Target Cells with Different dNTP Concentrations-To further examine the relationship between cellular dNTP content and retroviral replication, we took advantage of two mutated derivatives of the HIV-1 RT, V148I and Q151N, that had recently been shown by pre-steadystate kinetic analysis to have severely reduced dNTP binding affinity (k d , 25.6-and 234.2-fold respectively) compared with wild-type HIV-1 RT, even though these mutants possess wildtype levels of catalysis (k pol ) (14,17). As seen in Fig.…”

Frequent Incorporation of Ribonucleotides during HIV-1 Reverse Transcription and Their Attenuated Repair in Macrophages

“…HIV-1 RT was subsequently expressed in Escherichia coli BL21 (DE3) and purified with nickel-nitrilotriacetic acid chromatography followed by DEAE and SP anion exchange as described previously (23). These RT proteins were quantified and stored in 10% glycerol dialysis buffer as described previously (24,25).…”

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

“…We recently isolated two HIV-1 RT mutants, V148I and Q151N, that specifically exhibit reduced dNTP binding affinity (1/K d ) without a change in catalysis (k pol ) (16,17). Kinetic and structural analyses suggested that these mutations interfered with H-bond formation between the side chain of the Gln-151 residue and the 3Ј-OH group of the incoming dNTP substrate (18).…”