Physiological magnesium concentrations increase fidelity of diverse reverse transcriptases from HIV-1, HIV-2, and foamy virus, but not MuLV or AMV No Access

Abstract

Reverse transcriptases (RTs) are typically assayed using optimized Mg concentrations (~5–10 mM) several-fold higher than physiological cellular free Mg (~0.5 mM). Recent analyses demonstrated that HIV-1, but not Moloney murine leukaemia (MuLV) or avain myeloblastosis (AMV) virus RTs has higher fidelity in low Mg. In the current report, -based α-complementation assays were used to measure the fidelity of several RTs including HIV-1 (subtype B and A/E), several drug-resistant HIV-1 derivatives, HIV-2, and prototype foamy virus (PFV), all which showed higher fidelity using physiological Mg, while MuLV and AMV RTs demonstrated equivalent fidelity in low and high Mg. In 0.5 mM Mg, all RTs demonstrated approximately equal fidelity, except for PFV which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to determine mutation profiles was used to examine the types of mutations made by HIV-1 RT (type B) in low (0.5 mM) and high (6 mM) Mg on a template. Unlike α-complementation assays which are dependent on LacZ activity, the NGS assay scores mutations at all positions and of every type. Consistent with α-complementation assays, a ~four-fold increase in mutations was observed in high Mg. These findings help explain why HIV-1 RT displays lower fidelity (with high Mg concentrations) than other RTs (e.g. MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing conditions that accurately represent RT’s activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV-1.

Funding
This study was supported by the:
  • National Institute of Allergy and Infectious Diseases (Award R01AI150480)
    • Principle Award Recipient: JeffreyJ DeStefano
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/content/journal/jgv/10.1099/jgv.0.001708
2021-12-14
2024-03-29
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References

  1. Telesnitsky A, Goff SP. Reverse Transcriptase Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1993
    [Google Scholar]
  2. Cowan JA, Ohyama T, Howard K, Rausch JW, Cowan SM et al. Metal-ion stoichiometry of the HIV-1 RT ribonuclease H domain: evidence for two mutually exclusive sites leads to new mechanistic insights on metal-mediated hydrolysis in nucleic acid biochemistry. J Biol Inorg Chem 2000; 5:67–74 [View Article] [PubMed]
    [Google Scholar]
  3. Johnson KA. The kinetic and chemical mechanism of high-fidelity DNA polymerases. Biochim Biophys Acta 2010; 1804:1041–1048 [View Article] [PubMed]
    [Google Scholar]
  4. Joyce CM, Steitz TA. Function and structure relationships in DNA polymerases. Annu Rev Biochem 1994; 63:777–822 [View Article] [PubMed]
    [Google Scholar]
  5. Nakamura H, Katayanagi K, Morikawa K, Ikehara M. Structural models of ribonuclease H domains in reverse transcriptases from retroviruses. Nucleic Acids Res 1991; 19:1817–1823 [View Article] [PubMed]
    [Google Scholar]
  6. Schultz SJ, Champoux JJ. RNase H activity: structure, specificity, and function in reverse transcription. Virus Res 2008; 134:86–103 [View Article] [PubMed]
    [Google Scholar]
  7. Yang W, Steitz TA. Recombining the structures of HIV integrase, RuvC and RNase H. Structure 1995; 3:131–134 [View Article] [PubMed]
    [Google Scholar]
  8. Oda Y, Nakamura H, Kanaya S, Ikehara M. Binding of metal ions to E. coli RNase HI observed by 1H-15N heteronuclear 2D NMR. J Biomol NMR 1991; 1:247–255 [View Article] [PubMed]
    [Google Scholar]
  9. Hoffman AD, Banapour B, Levy JA. Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. Virology 1985; 147:326–335 [View Article] [PubMed]
    [Google Scholar]
  10. Rey MA, Spire B, Dormont D, Barre-Sinoussi F, Montagnier L et al. Characterization of the RNA dependent DNA polymerase of a new human T-lymphotropic retrovirus (lymphadenopathy associated virus). Biochem Biophys Res Commun 1984; 121:126–133 [View Article] [PubMed]
    [Google Scholar]
  11. Starnes MC, Cheng YC. Human immunodeficiency virus reverse transcriptase-associated RNase H activity. J Biol Chem 1989; 264:7073–7077 [PubMed]
    [Google Scholar]
  12. Wondrak EM, Löwer J, Kurth R. Functional purification and enzymic characterization of the RNA-dependent DNA polymerase of human immunodeficiency virus. J Gen Virol 1986; 67 (Pt 12):2791–2797 [View Article] [PubMed]
    [Google Scholar]
  13. Delva P, Pastori C, Degan M, Montesi G, Lechi A. Intralymphocyte free magnesium and plasma triglycerides. Life Sci 1998; 62:2231–2240 [View Article] [PubMed]
    [Google Scholar]
  14. Delva P, Pastori C, Degan M, Montesi G, Lechi A. Catecholamine-induced regulation in vitro and ex vivo of intralymphocyte ionized magnesium. J Membrane Biol 2004; 199:163–171 [View Article]
    [Google Scholar]
  15. Moomaw AS, Maguire ME. The unique nature of mg2+ channels. Physiology (Bethesda) 2008; 23:275–285 [View Article] [PubMed]
    [Google Scholar]
  16. Delva P, Pastori C, Degan M, Montesi G, Brazzarola P et al. Intralymphocyte free magnesium in patients with primary aldosteronism: aldosterone and lymphocyte magnesium homeostasis. Hypertension 2000; 35:113–117 [View Article] [PubMed]
    [Google Scholar]
  17. Delva PT, Pastori C, Degan M, Montesi GD, Lechi A. Intralymphocyte free magnesium in a group of subjects with essential hypertension. Hypertension 1996; 28:433–439 [View Article] [PubMed]
    [Google Scholar]
  18. Morelle B, Salmon JM, Vigo J, Viallet P. Measurement of intracellular magnesium concentration in 3T3 fibroblasts with the fluorescent indicator Mag-indo-1. Anal Biochem 1994; 218:170–176 [View Article] [PubMed]
    [Google Scholar]
  19. Gout E, Rébeillé F, Douce R, Bligny R. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration. Proc Natl Acad Sci U S A 2014; 111:E4560–7 [View Article] [PubMed]
    [Google Scholar]
  20. Murphy E, Freudenrich CC, Levy LA, London RE, Lieberman M. Monitoring cytosolic free magnesium in cultured chicken heart cells by use of the fluorescent indicator Furaptra. Proc Natl Acad Sci U S A 1989; 86:2981–2984 [View Article] [PubMed]
    [Google Scholar]
  21. Touyz RM, Yao G. Modulation of vascular smooth muscle cell growth by magnesium-role of mitogen-activated protein kinases. J Cell Physiol 2003; 197:326–335 [View Article] [PubMed]
    [Google Scholar]
  22. Goldschmidt V, Didierjean J, Ehresmann B, Ehresmann C, Isel C et al. Mg2+ dependency of HIV-1 reverse transcription, inhibition by nucleoside analogues and resistance. Nucleic Acids Res 2006; 34:42–52 [View Article] [PubMed]
    [Google Scholar]
  23. Achuthan V, Singh K, DeStefano JJ. Physiological Mg2+ conditions significantly alter the inhibition of HIV-1 and HIV-2 reverse transcriptases by nucleoside and non-nucleoside inhibitors in vitro. Biochemistry 2017; 56:33–46 [View Article] [PubMed]
    [Google Scholar]
  24. Achuthan V, Keith BJ, Connolly BA, DeStefano JJ. Human immunodeficiency virus reverse transcriptase displays dramatically higher fidelity under physiological magnesium conditions in vitro. J Virol 2014; 88:8514–8527 [View Article] [PubMed]
    [Google Scholar]
  25. Okano H, Baba M, Hidese R, Iida K, Li T et al. Accurate fidelity analysis of the reverse transcriptase by a modified next-generation sequencing. Enzyme Microb Technol 2018; 115:81–85 [View Article] [PubMed]
    [Google Scholar]
  26. Menéndez-Arias L. Mutation rates and intrinsic fidelity of retroviral reverse transcriptases. Viruses 2009; 1:1137–1165 [View Article] [PubMed]
    [Google Scholar]
  27. Sanjuán R, Nebot MR, Chirico N, Mansky LM, Belshaw R. Viral mutation rates. J Virol 2010; 84:9733–9748 [View Article] [PubMed]
    [Google Scholar]
  28. Svarovskaia ES, Cheslock SR, Zhang W-H, Hu W-S, Pathak VK. Retroviral mutation rates and reverse transcriptase fidelity. Front Biosci 2003; 8:d117–34 [View Article] [PubMed]
    [Google Scholar]
  29. Ji JP, Loeb LA. Fidelity of HIV-1 reverse transcriptase copying RNA in vitro. Biochemistry 1992; 31:954–958 [View Article] [PubMed]
    [Google Scholar]
  30. Rezende LF, Prasad VR. Nucleoside-analog resistance mutations in HIV-1 reverse transcriptase and their influence on polymerase fidelity and viral mutation rates. Int J Biochem Cell Biol 2004; 36:1716–1734 [View Article] [PubMed]
    [Google Scholar]
  31. Sebastián-Martín A, Barrioluengo V, Menéndez-Arias L. Transcriptional inaccuracy threshold attenuates differences in RNA-dependent DNA synthesis fidelity between retroviral reverse transcriptases. Sci Rep 2018; 8:627. [View Article] [PubMed]
    [Google Scholar]
  32. Abram ME, Ferris AL, Shao W, Alvord WG, Hughes SH. Nature, position, and frequency of mutations made in a single cycle of HIV-1 replication. J Virol 2010; 84:9864–9878 [View Article] [PubMed]
    [Google Scholar]
  33. Mansky LM, Temin HM. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J Virol 1995; 69:5087–5094 [View Article] [PubMed]
    [Google Scholar]
  34. Alce TM, Popik W. APOBEC3G is incorporated into virus-like particles by a direct interaction with HIV-1 Gag nucleocapsid protein. J Biol Chem 2004; 279:34083–34086 [View Article] [PubMed]
    [Google Scholar]
  35. Malim MH, Bieniasz PD. HIV restriction factors and mechanisms of evasion. Cold Spring Harb Perspect Med 2012; 2:a006940 [View Article] [PubMed]
    [Google Scholar]
  36. Sheehy AM, Gaddis NC, Choi JD, Malim MH. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 2002; 418:646–650 [View Article] [PubMed]
    [Google Scholar]
  37. Guenzel CA, Hérate C, Le Rouzic E, Maidou-Peindara P, Sadler HA et al. Recruitment of the nuclear form of uracil DNA glycosylase into virus particles participates in the full infectivity of HIV-1. J Virol 2012; 86:2533–2544 [View Article] [PubMed]
    [Google Scholar]
  38. Mansky LM. The mutation rate of human immunodeficiency virus type 1 is influenced by the vpr gene. Virology 1996; 222:391–400 [View Article] [PubMed]
    [Google Scholar]
  39. Mansky LM, Le Rouzic E, Benichou S, Gajary LC. Influence of reverse transcriptase variants, drugs, and Vpr on human immunodeficiency virus type 1 mutant frequencies. J Virol 2003; 77:2071–2080 [View Article] [PubMed]
    [Google Scholar]
  40. Mansky LM, Preveral S, Selig L, Benarous R, Benichou S. The interaction of vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 In vivo mutation rate. J Virol 2000; 74:7039–7047 [View Article] [PubMed]
    [Google Scholar]
  41. O’Neil PK, Sun G, Yu H, Ron Y, Dougherty JP et al. Mutational analysis of HIV-1 long terminal repeats to explore the relative contribution of reverse transcriptase and RNA polymerase II to viral mutagenesis. J Biol Chem 2002; 277:38053–38061 [View Article] [PubMed]
    [Google Scholar]
  42. Hou EW, Prasad R, Beard WA, Wilson SH. High-level expression and purification of untagged and histidine-tagged HIV-1 reverse transcriptase. Protein Expr Purif 2004; 34:75–86 [View Article] [PubMed]
    [Google Scholar]
  43. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001
    [Google Scholar]
  44. Abram ME, Ferris AL, Das K, Quinoñes O, Shao W et al. Mutations in HIV-1 reverse transcriptase affect the errors made in a single cycle of viral replication. J Virol 2014; 88:7589–7601 [View Article] [PubMed]
    [Google Scholar]
  45. Potapov V, Ong JL. Correction: Examining Sources of Error in PCR by Single-Molecule Sequencing. PLoS One 2017; 12:e0181128 [View Article] [PubMed]
    [Google Scholar]
  46. Shah FS, Curr KA, Hamburgh ME, Parniak M, Mitsuya H et al. Differential influence of nucleoside analog-resistance mutations K65R and L74V on the overall mutation rate and error specificity of human immunodeficiency virus type 1 reverse transcriptase. J Biol Chem 2000; 275:27037–27044 [View Article] [PubMed]
    [Google Scholar]
  47. Boyer PL, Stenbak CR, Hoberman D, Linial ML, Hughes SH. In vitro fidelity of the prototype primate foamy virus (PFV) RT compared to HIV-1 RT. Virology 2007; 367:253–264 [View Article] [PubMed]
    [Google Scholar]
  48. Ahn EH, Lee SH. Detection of Low-Frequency Mutations and Identification of Heat-Induced Artifactual Mutations Using Duplex Sequencing. IJMS 2019; 20:199 [View Article]
    [Google Scholar]
  49. Arbeithuber B, Makova KD, Tiemann-Boege I. Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications. DNA Res 2016; 23:547–559 [View Article] [PubMed]
    [Google Scholar]
  50. Schmitt MW, Kennedy SR, Salk JJ, Fox EJ, Hiatt JB et al. Detection of ultra-rare mutations by next-generation sequencing. Proc Natl Acad Sci U S A 2012; 109:14508–14513 [View Article] [PubMed]
    [Google Scholar]
  51. Chen L, Liu P, Ettwiller LM. DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification. Science 2017; 355:752–756 [View Article] [PubMed]
    [Google Scholar]
  52. Costello M, Pugh TJ, Fennell TJ, Stewart C, Lichtenstein L et al. Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation. Nucleic Acids Res 2013; 41:e67 [View Article] [PubMed]
    [Google Scholar]
  53. Stoler N, Arbeithuber B, Povysil G, Heinzl M, Salazar R et al. Family reunion via error correction: an efficient analysis of duplex sequencing data. BMC Bioinformatics 2020; 21:96 [View Article] [PubMed]
    [Google Scholar]
  54. Wang TT, Abelson S, Zou J, Li T, Zhao Z et al. High efficiency error suppression for accurate detection of low-frequency variants. Nucleic Acids Res 2019; 47:e87 [View Article]
    [Google Scholar]
  55. Bebenek K, Abbotts J, Wilson SH, Kunkel TA. Error-prone polymerization by HIV-1 reverse transcriptase. Contribution of template-primer misalignment, miscoding, and termination probability to mutational hot spots. J Biol Chem 1993; 268:10324–10334 [View Article] [PubMed]
    [Google Scholar]
  56. Yasukawa K, Iida K, Okano H, Hidese R, Baba M et al. Next-generation sequencing-based analysis of reverse transcriptase fidelity. Biochem Biophys Res Commun 2017; 492:147–153 [View Article] [PubMed]
    [Google Scholar]
  57. Rawson JMO, Gohl DM, Landman SR, Roth ME, Meissner ME et al. Single-strand consensus sequencing reveals that HIV type but not subtype significantly impacts viral mutation frequencies and spectra. J Mol Biol 2017; 429:2290–2307 [View Article] [PubMed]
    [Google Scholar]
  58. Boyer JC, Bebenek K, Kunkel TA. Unequal human immunodeficiency virus type 1 reverse transcriptase error rates with RNA and DNA templates. Proc Natl Acad Sci U S A 1992; 89:6919–6923 [View Article] [PubMed]
    [Google Scholar]
  59. Bebenek K, Beard WA, Casas-Finet JR, Kim HR, Darden TA et al. Reduced frameshift fidelity and processivity of HIV-1 reverse transcriptase mutants containing alanine substitutions in helix H of the thumb subdomain. J Biol Chem 1995; 270:19516–19523 [View Article] [PubMed]
    [Google Scholar]
  60. Gong S, Kirmizialtin S, Chang A, Mayfield JE, Zhang YJ et al. Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis. J Biol Chem 2021; 296:100184 [View Article] [PubMed]
    [Google Scholar]
  61. Bebenek K, Kunkel TA. Streisinger revisited: DNA synthesis errors mediated by substrate misalignments. Cold Spring Harb Symp Quant Biol 2000; 65:81–91 [View Article] [PubMed]
    [Google Scholar]
  62. Kunkel TA. DNA replication fidelity. J Biol Chem 2004; 279:16895–16898 [View Article] [PubMed]
    [Google Scholar]
  63. Kerr SG, Anderson KS. RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase: evidence of discrimination between DNA and RNA substrates. Biochemistry 1997; 36:14056–14063 [View Article] [PubMed]
    [Google Scholar]
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