Intrinsic DNA synthesis fidelity of xenotropic murine leukemia virus-related virus reverse transcriptase

Authors

  • Verónica Barrioluengo,

    1.  Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Madrid, Spain
    Search for more papers by this author
  • Yi Wang,

    1.  RT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute – Frederick, MD, USA
    Search for more papers by this author
  • Stuart F. J. Le Grice,

    1.  RT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute – Frederick, MD, USA
    Search for more papers by this author
  • Luis Menéndez-Arias

    1.  Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Madrid, Spain
    Search for more papers by this author

L. Menéndez-Arias, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), c/Nicolás Cabrera 1, Campus de Cantoblanco, 28049 Madrid, Spain
Fax: +34 91 196 4420
Tel: +34 91 196 4494
E-mail: lmenendez@cbm.uam.es

Abstract

Although recent reports have provided strong evidence to suggest that xenotropic murine leukemia virus-related virus (XMRV) is unlikely to be the causative agent of prostate cancer and chronic fatigue syndrome, this recombinant retrovirus can nonetheless infect human cells in vitro and induce a chronic infection in macaques. In the present study, we determined the accuracy of DNA synthesis of the reverse transcriptases (RTs) of XMRV and Moloney murine leukemia virus (MoMLV) using a combination of pre-steady-state kinetics of nucleotide incorporation and an M13mp2-based forward mutation assay. The results obtained were compared with those previously reported for the HIV type 1 BH10 strain (HIV-1BH10) RT. MoMLV and XMRV RTs were 13.9 and 110 times less efficient [as determined by the catalytic rate constant of the nucleotide incorporation reaction (kpol)/equilibrium constant (Kd)] than the HIV-1BH10 RT in incorporating correct nucleotides. Misinsertion and mispair extension kinetic studies demonstrated that MoMLV RT was more accurate than the HIV-1BH10 RT. In comparison with the MoMLV RT, the XMRV RT showed decreased mispair extension fidelity and was less faithful when misincorporating C or A opposite A. However, the XMRV RT showed stronger selectivity against G in misinsertion fidelity assays. Forward mutation assays revealed that XMRV and MoMLV RTs had similar accuracy of DNA-dependent DNA synthesis, but were > 13 times more faithful than the HIV-1BH10 enzyme. The mutational spectra of XMRV and MoMLV RTs were similar in having a relatively higher proportion of frameshifts and transversions compared with the HIV-1BH10 RT. However, the XMRV polymerase was less prone to introduce large deletions and one-nucleotide insertions.

Ancillary