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. 2012 Jan;40(1):345-59.
doi: 10.1093/nar/gkr694. Epub 2011 Sep 8.

Biochemical, Inhibition and Inhibitor Resistance Studies of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

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Free PMC article

Biochemical, Inhibition and Inhibitor Resistance Studies of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

Tanyaradzwa P Ndongwe et al. Nucleic Acids Res. .
Free PMC article

Abstract

We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (k(off)) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1.

Figures

Figure 1.
Figure 1.
Assessment of KD.DNA, kon and koff using surface plasmon resonance. SPR was used to measure the binding affinity of RTs to a nucleic acid substrate. Increasing concentrations of each RT (0.2, 0.5, 1, 2, 5, 10, 20, 50, 100 and 200 nM) were injected over a streptavidin chip with biotinylated double-stranded DNA immobilized on its surface as described in ‘Materials and Methods’ section. The experimental trace (red) shown is the result of a subtraction of the data obtained from the channel containing the immobilized nucleic acid minus the signal obtained from an empty channel. The black curve represents the fitted data according to the ‘heterogeneous ligand’ model that assumes two different binding modes for RT on the nucleic acid.
Figure 2.
Figure 2.
Pre-steady state kinetics of nucleotide incorporation by XMRV RT. 150 nM XMRV RT was pre-incubated with 40 nM Td31/5′-Cy3-Pd18a rapidly mixed with a solution containing MgCl2 (5 mM) and varying concentrations of dATP: 25 µM (filled square), 35 µM (filled triangle), 50 µM (filled inverted triangle), 75 µM (filled rhombus), 100 μM (filled circle), 125 μM (open square) and 150 µM (open triangle); and incubated for 0.1 to 6 s before being quenched with EDTA. The DNA product for each dATP concentration was fit to the burst equation (A). The burst amplitudes generated for each dATP concentration were then fit to a hyperbola equation (B) yielding the optimal rates of dNTP incorporation; kpol (8.9 s−1) and dNTP binding to the RT-DNA complex; Kd.dATP (26.6 µM).
Figure 3.
Figure 3.
Comparison of in vitro fidelity of HIV-1, MoMLV and XMRV RTs. Extension of 10 nM Td100/5′-Cy3-Pd18a by HIV-1 RT, MoMLV RT or XMRV RT (20, 50 and 50 nM, respectively) in the presence of 150 µM each of three out of four nucleotides (the missing nucleotide is marked at the bottom of each lane). Reactions were run for 30 min for HIV-1 RT and 45 min for XMRV RT and MoMLV RT. For each enzyme the first lane in each set shows the position of unextended primer, the second lane shows full extension in the presence of all four dNTPs, and each consecutive lane shows extension in the presence of three dNTPs. The arrows on the right mark the expected pauses based on the indicated composition of the template strand.
Figure 4.
Figure 4.
Comparison of in vivo fidelity of XMRV with amphotropic MLV. The ANGIE P cells used for this assay contain a retroviral vector (GA-1), which encodes a bacterial β-galactosidase gene (lacZ) and a neomycin phosphotransferase gene. Replication fidelity is measured by the frequency of lacZ inactivation resulting in an increase in white colonies. The fidelity differences between the two viruses are not statistically significant (error bars represent standard error from three independent experiments).
Figure 5.
Figure 5.
Processivity (trap assay) of HIV-RT, MoMLV RT and XMRV RT. DNA synthesis was monitored in the presence of calf thymus DNA as an enzyme trap. Each enzyme (30 nM HIV RT, 100 nM MoMLV RT or 100 nM XMRV RT) was pre-incubated with 40 nM Td100/Cy3-Pd18a. Lanes 1 and 2 of each set show unlimited DNA synthesis in the absence of trap for 5 and 10 min for HIV-1 RT and 10 and 40 min for XMRV RT and MoMLV RT. In Lanes 3 and 4 the reaction is initiated by the addition of dNTPs (100 µM each) together with the calf thymus DNA trap (0.5 µg/µl) such that the products generated represent a single processive synthesis event for the respective time points for each enzyme. Lane 5 shows the effectiveness of the trap determined by incubating the calf thymus DNA with the enzyme before addition of labeled template-primer. Processive primer extension by HIV-1 RT and MoMLV RT in Lanes 4–6 of the left and middle panel is higher than by XMRV RT in Lanes 4–6 of the right panel.
Figure 6.
Figure 6.
Single-turnover processivity assays. 30 nM Td31/Cy3-Pd18a was combined with 100 nM XMRV RT or 50 nM MoMLV RT in RT buffer before rapidly mixing with all four dNTPs (100 µM each) and 5 mM MgCl2 for varying incubation times (0.05–45 s) and quenching with EDTA. Extension of the 18-mer primer (open rhombus) ((open triangle) for XMRV RT) into 19-mer (filled square) and 22-mer (filled square), by MoMLV RT (A) and XMRV RT (B) was fit to a double exponential equation to determine rates of product appearance, and subsequent processive extension of those products (rates shown in Table 5).
Figure 7.
Figure 7.
Inhibition of XMRV RT by RNA aptamers. 10 nM XMRV RT was incubated with increasing amounts of RNA aptamer in Reaction Buffer for 5 min at 37°C followed by addition of 20 nM Td31/Cy3-Pd18a and 50 µM of each dNTP. (A) The reactions were stopped after 30 min and resolved on a polyacrylamide gel. The predicted secondary structures of each aptamer were generated by mfold. (B) The percent full extension was quantified for m.1.1FL (filled inverted triangle), m.1.3 (filled circle) and m.1.4 (filled square) and data points fit to one-site competition non-linear regression using GraphPad Prism 4 to calculate IC50. HIV-1 RT was not susceptible to m.1.1FL (open triangle). (Errors represent data deviation from the fit).
Figure 8.
Figure 8.
PPi-mediated unblocking of AZT-(A) and EFdA-(B) terminated DNA. About 20 nM of (A) AZT- or (B) EFdA-terminated Td31/Cy3-Pd18c (T/PAZT-MP or T/PEFdA-MP) was incubated with HIV-1 RT (60 nM) or XMRV RT (200 nM) in the presence of 150 µM PPi and 6 mM MgCl2. Aliquots of the reactions were stopped at different time points (0–90 min) and resolved on a 15% polyacrylamide–7M urea gel as described in the ‘Materials and Methods’ section.
Figure 9.
Figure 9.
Molecular model of XMRV RT. Ribbons diagram of XMRV RT with the conserved polymerase Motifs color-coded: Motif A (green), B (brown), C (purple), D (red), E (orange) and F (blue). The residues that differ from MoMLV’s polymerase domain are shown in ball and stick representation.

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