The Accuracy of Reverse Transcriptase from HIV-1

Abstract

A study was conducted to determine the fidelity of DNA synthesis catalyzed in vitro by the reverse transcriptase from a human immunodeficiency virus type 1 (HIV-1). Like other retroviral reverse transcriptases, the HIV-1 enzyme does not correct errors by exonucleolytic proofreading. Measurements with M13mp2-based fidelity assays indicated that the HIV-1 enzyme, isolated either from virus particles or from Escherichia coli cells infected with a plasmid expressing the cloned gene, was exceptionally inaccurate, having an average error rate per detectable nucleotide incorporated of 1/1700. It was, in fact, the least accurate reverse transcriptase described to date, one-tenth as accurate as the polymerases isolated from avian myeloblastosis or murine leukemia viruses, which have average error rates of approximately 1/17,000 and approximately 1/30,000, respectively. DNA sequence analyses of mutations generated by HIV-1 polymerase showed that base substitution, addition, and deletion errors were all produced. Certain template positions were mutational hotspots where the error rate could be as high as 1 per 70 polymerized nucleotides. The data are consistent with the notion that the exceptional diversity of the HIV-1 genome results from error-prone reverse transcription.

Hybrid origin of a B chromosome (PSR) in the parasitic wasp Nasonia vitripennis

“…A large number of nucleotide substitutions have occurred in the RT region among the elements on PSR, with preferential accumulation at synonymous sites (McAllister 1995). Although errorprone replication during retrotransposition may increase the substitution rate (Preston et al 1988;Roberts et al 1988;Li and Loeb 1992), a large number of replications would be required to generate the amount of sequence diversity observed among the elements on PSR, but there are only about nine copies of NATE on PSR (McAllister et al, in preparation). In addition to sequence divergence among elements in this internal region, sequence differences between the two LTR sequences from a single element (PSR c16) indicate that this element inserted into its present location approximately 120,000 years ago (McAllister 1995).…”

A possible role for cysteine residues in the fidelity of DNA synthesis exhibited by the reverse transcriptases of human immunodeficiency viruses type 1 and type 2

“…All RTs wild type and mutant used in this study were recombinant enzymes expressed by us in E. coll[l [3][4][5][6][7][8][9][10][11][12][13][14][15] and purified to homogeneity according to Clark et al [16]. The specific activities of the different RTs used were 4,000-5,000 units per/.t$.…”

“…In contrast, MLV RT (with 671 amino acids) has 8 cysteines while avian sarcoma virus and avian ~ayelo-blastosis/sarcoma virus RTs have 12 cysteines in the fl subunit (895 residues long) and 8 cysteines in the ~t subunit (572 residues long) [9]. Interestingly, 'c3,s~eine rich' RTs, that are sensitive to suifhydryl reagent,~, were shown to have a relatively higher fidelity of DNA synthesis than HIV-1 RT [10]. Cysteine residues were found to be involved in the biological functions of various enzymes [11,12].…”

Mutational studies of human immunodeficiency virus type 1 reverse transcriptase : the involvement of residues 183 and 184 in the fidelity of DNA synthesis

“…Unlike many cellular DNA polymerases, retroviral RTs are devoid of 3'~ 5' exonuclease proofreading activity [7]. However, the fidelity of both the RNA-dependent and DNA-dependent DNA polymerase activities of HIV-1 and HIV-2 RTs is up to 10-fold lower than that detected for other proofreading-deficient RTs, i.e., RTs of murine leukemia virus (MLV) and avian myeloblastosis virus (AMV) [9][10][11][12][13][14]. It has been proposed that efficient misinsertion and extension of mismatched 3'-termini of the nascent DNA are major determinants for the low fidelity of HIV-1 and HIV-2 RTs [11,15].…”