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
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  • Yi Wang,

    1.  RT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute – Frederick, MD, USA
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  • Stuart F. J. Le Grice,

    1.  RT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute – Frederick, MD, USA
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  • Luis Menéndez-Arias

    1.  Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Madrid, Spain
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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.

Abbreviations
dATP

2′-deoxyadenosine 5′-triphosphate

dCTP

2′-deoxycytidine 5′-triphosphate

dGTP

2′-deoxyguanosine 5′-triphosphate

dNTP

deoxyribonucleoside-triphosphate

dTTP

2′-deoxythymidine 5′-triphosphate

fext

mismatch extension ratio

fins

misinsertion ratio

HIV-1BH10

HIV type 1 BH10 strain

Kd

equilibrium constant

kobs

observed nucleotide-incorporation rate

kpol

catalytic rate constant of the nucleotide incorporation reaction

MLV

murine leukemia virus

RT

reverse transcriptase

MoMLV

Moloney murine leukemia virus

XMRV

xenotropic murine leukemia virus-related virus

Introduction

Xenotropic murine leukemia virus-related virus (XMRV) is a recombinant gammaretrovirus that was originally detected in human prostate cancer tumors [1] and was later associated with myalgic encephalomyelitis or chronic fatigue syndrome, a disabling condition characterized by long-lasting debilitating physical and mental fatigue [2]. However, its relevance as a human pathogen has remained controversial as many reports failed to confirm the presence of XMRV in multiple cohorts of prostate cancer or chronic fatigue syndrome patients or in healthy donors (for reviews see refs [3,4]). Several studies have suggested that XMRV found in human tissues could be the result of contamination with DNA from laboratory cell lines or with mouse DNA [5–8]. The lack of diversity among XMRV sequences supposedly isolated from different individuals [7,9] was also difficult to reconcile with a genuine process of infectious transmission. The nucleotide sequences of XMRV isolated from patients [1,2] were almost identical to those obtained from virus infecting 22Rv1, a common prostate cancer cell line [10]. This cell line was developed between 1992 and 1999 by serial passage of a human prostate xenograft (CWR22) in nude mice. In an elegant study, Paprotka et al. showed that XMRV was not present in the original CWR22 tumor, but would be the likely result of a recombination event between the two endogenous murine leukemia viruses (MLVs) [11]. More recently, large collaborative studies found no evidence of murine-like gammaretroviruses in patients with chronic fatigue syndrome, after careful re-examination of blood samples previously identified as XMRV positive [9,12].

Although XMRV is unlikely to be a human pathogen, this recombinant retrovirus can infect, in vitro, human blood cells [13] as well as primary neuronal cells [14]. Moreover, intravenous inoculation of macaques with XMRV leads to persistent chronic disseminated infection [15]. A deeper knowledge of gammaretrovirus replication is important to gain a better understanding of retroviral infection, particularly in the context of potential risks associated with the xenotransplantation of tissues and organs.

The DNA polymerase domain of XMRV reverse transcriptase (RT) shares 97.5% amino acid sequence identity with the RT from Moloney MLV (MoMLV) (Fig. 1). However, amino acid differences between these enzymes are evident at the C-terminal region, which includes the RNase H domain. The DNA polymerase and the RNase H domains of XMRV RT derive from two different endogenous murine retroviruses, with the recombination site located within the nucleotide sequence encoding amino acid residues 431–455 in the RT [11]. The structural and biochemical properties of the MoMLV RT have been extensively studied [16,17]. In comparison with the MoMLV RT, the XMRV RT was less efficient in DNA synthesis and in unblocking chain-terminated primers, and showed lower processivity [18]. Estimates on the accuracy of DNA synthesis catalyzed by the XMRV RT have been limited to qualitative assessments of primer-extension products obtained in the absence of one deoxyribonucleoside-triphosphate (dNTP) and comparison of incorporation efficiencies for a single nucleotide mispair [2′-deoxyadenosine 5′-triphosphate (dATP) versus the correct 2′-deoxythymidine 5′-triphosphate (dTTP)]. These assays demonstrated a higher intrinsic fidelity for XMRV RT [18]. However, the mutation rates of XMRV and an amphotropic MLV were similar when assessed in cell culture by measuring the frequency of lacZ inactivation [18].

Figure 1.

 Amino acid sequence comparison of XMRV, MoMLV and HIV-1 RTs. Sequence alignments of gammaretroviral and HIV-1 RTs are based on sequence and structural motifs and were taken from Coté & Roth [16]. Amino acid differences between XMRV and MoMLV RTs are underlined and indicated with asterisks. In the HIV-1 RT sequence, underlined amino acids represent catalytic residues in the DNA polymerase domain (Asp110, Asp185 and Asp186) and the strictly conserved D-E-D motif in the RNase H domain (Asp443, Glu478 and Asp498). Conserved motifs A–E in the DNA polymerase domain are also indicated. The arrow shows the cleavage site rendering p51 in HIV-1 RT. Nucleotide sequences deposited in GenBank with accession numbers DQ399707 (XMRV), J02255 (MoMLV) and M15654 (HIV-1BH10) were used to obtain the RT sequences shown in the alignment.

In our study, we performed extensive analysis of the fidelity of DNA-directed DNA synthesis of XMRV RT using enzymatic assays (i.e. misincorporation and mispair extension fidelity measurements with various base pairs). In addition, we measured error rates for this enzyme in M13mp2-based lacZα forward mutation assays. The kinetic assays showed differences between XMRV and MoMLV RTs in their selectivity between correct and incorrect nucleotides, as well as in their ability to extend mispaired 3′ termini. However, our results from forward mutation assays did not reveal significant differences in the overall fidelity between XMRV and MoMLV RTs. Mutational patterns generated by XMRV and MoMLV RTs were different in relation to the nature and location of hot spots and their tendency to introduce frameshift mutations at homopolymeric sequences.

Results

Nucleotide incorporation kinetics

XMRV and MoMLV RTs were purified to homogeneity by a combination of metal-affinity and ion-exchange chromatography. In both cases, the enzymes were obtained as a single polypeptide chain containing a nonremovable His6 tag at their N terminus. Both enzymes were judged to be pure, by SDS/PAGE, and to have Mr values of around 75 000–79 000 (Fig. 2A). Their catalytic efficiency of nucleotide incorporation was determined by transient kinetics and compared with previous values obtained in the same conditions with the HIV type 1 BH10 strain (HIV-1BH10) RT [19]. Pre-steady-state burst experiments for the incorporation of a correct nucleotide (dTTP) on the DNA–DNA template-primer 31T/21P were carried out in the presence of dTTP (at concentrations ranging from 5 to 150 μm). Observed nucleotide-incorporation rates (kobs) were plotted against the corresponding dTTP concentration to obtain the catalytic rate constant of the nucleotide incorporation reaction (kpol) and the equilibrium constant (Kd). As shown in Fig. 2B, both gammaretroviral RTs had similar Kd values, but their catalytic kobs values were different. The kpol of XMRV RT was about eight times lower than the kpol obtained for the MoMLV RT. When compared with the HIV-1BH10 RT, the XMRV RT showed a reduction in catalytic efficiency of more than 100-fold (measured as kpol/Kd) (Table 1).

Figure 2.

 Analysis of purified XMRV and MoMLV RTs and kinetic parameters of DNA polymerization. (A) SDS/PAGE of purified RTs. Molecular markers, and their molecular mass values, are as follows: phosphorylase b, 97.4 kDa; BSA, 66 kDa; ovalbumin, 45 kDa; and carbonic anhydrase, 31 kDa. (B) Nucleotide concentration dependence for the incorporation of dTTP into the 31T/21P duplex DNA. The continuous line represents the best fit of the data to the Michaelis–Menten equation. The kpol values obtained were 0.19 ± 0.02 s−1 for XMRV RT and 1.55 ± 0.17 s−1 for MoMLV RT. The Kd values of XMRV RT and MoLV RT were 24.1 ± 4.3 μm and 24.9 ± 7.1 μm, respectively.

Table 1.   Pre-steady-state kinetic parameters for misincorporation. The template-primer 31T/21P was used as the substrate. Data shown are the mean values ± SD. Each of the assays was performed independently at least three times.
EnzymeNucleotidekpol (s−1)Kdm)kpol/Kdm−1·s−1)Misinsertion ratio (fins)a
  1. a  f ins = [kpol(incorrect)/Kd(incorrect)]/[kpol(correct)/Kd(correct)], where incorrect nucleotides were dCTP, dGTP or dATP, while the correct nucleotide was dTTP. Numbers in bold-face type within parentheses represent the relative increase of misinsertion fidelity relative to the HIV-1BH10 RT. b Reported values for HIV-1BH10 RT were taken from Matamoros et al. [19].

XMRV RTdTTP0.19 ± 0.0224.1 ± 4.3(7.83 ± 1.71) × 10−3 
dCTP(2.34 ± 0.24) × 10−33800 ± 1181(6.17 ± 2.02) × 10−7(7.88 ± 3.10) × 10−5 (0.56)
dGTP(8.61 ± 0.97) × 10−52433 ± 199(3.54 ± 0.49) × 10−8(4.52 ± 1.17) × 10−6 (21.5)
dATP(4.16 ± 0.17) × 10−42914 ± 832(1.42 ± 0.41) × 10−7(1.81 ± 0.66) × 10−5(0.27)
MoMLV RTdTTP1.55 ± 0.1724.9 ± 7.1(6.21 ± 1.88) × 10−2 
dCTP(2.08 ± 0.26) × 10−31466 ± 789(1.42 ± 0.78) × 10−6(2.29 ± 1.43) × 10−5(1.9)
dGTP(8.30 ± 0.83) × 10−41135 ± 325(7.33 ± 2.22) × 10−7(1.18 ± 0.51) × 10−5(8.2)
dATP(4.39 ± 0.69) × 10−44960 ± 2842(8.87 ± 5.27) × 10−8(1.42 ± 0.95) × 10−6(3.5)
HIV-1BH10 RT bdTTP11.6 ± 0.513.4 ± 1.90.863 ± 0.126 
dCTP0.291 ± 0.0527645 ± 2959(3.80 ± 1.62) × 10−5(4.40 ± 1.98) × 10−5
dGTP0.289 ± 0.0323435 ± 1099(8.40 ± 2.84) × 10−5(9.73 ± 3.58) × 10−5
dATP(2.40 ± 0.2) × 10−3559 ± 191(4.29 ± 1.50) × 10−6(4.97 ± 1.88) × 10−6

Fidelity of DNA synthesis as determined by pre-steady-state kinetics

Misinsertion and mispair extension fidelity assays were used to estimate the accuracy of DNA polymerization catalyzed by XMRV and MoMLV RTs, and the results were compared with those obtained with the previously studied HIV-1BH10 RT [19]. The kinetics of misincorporation of C, G or A opposite A into the heteroduplex 31T/21P were determined under single turnover conditions. Misinsertion kinetic parameters (kpol and Kd) are given in Table 1 and compared with those obtained in reactions carried out with the correct nucleotide, dTTP. The largest differences between RTs related to their kpol values, which were around 100 times lower for gammaretroviral RTs than for the HIV-1BH10 RT. However, these effects were less pronounced when misinsertion ratios (fins) were compared. The MoMLV RT was more accurate than the HIV-1 RT for all misinsertions tested. In the case of XMRV RT, we observed differences in fidelity that affected specific mispairs. Thus, the XMRV RT showed a very low fins for the incorporation of G opposite A (fins = 4.52 × 10−6) compared with the HIV-1 RT (fins = 9.73 × 10−5). However, differences between both RTs were relatively small for the two other misinsertions (i.e. C or A opposite A). Overall, the XMRV RT was found to be less accurate than the MoMLV RT, except for misincorporating G instead of T.

The kinetics of mispair extension were determined by measuring the incorporation of dTTP at the 3′ end of the primer, using 31-nucleotide template/21-nucleotide primer duplexes containing mismatched termini (i.e. G·T, G·G or G·A). The results are shown in Table 2. The highest mismatch extension ratios (fext) were obtained for the G·T mispair. Their values ranged from of 2.35 × 10−4 to 2.12 × 10−3. XMRV and HIV-1BH10 RTs showed similar efficiencies of G·T mispair extension. However, MoMLV RT showed a 4.8-fold increased accuracy in comparison with the HIV-1BH10 RT. Interestingly, the increased accuracy of MoMLV RT in mispair extension assays was also observed with the template-primer having a G·G mismatch. Its mispair extension ratio was 1.72 × 10−5, which is considerably lower than the values reported for XMRV and HIV-1BH10 RTs. Extension of G·A mispairs was rather inefficient with all tested enzymes, with fext values in the range of 3.58 × 10−6 to 1.78 × 10−5. The XMRV RT was 3.5 to 5 times more efficient in extending G·A mispairs in comparison with the RTs of MoMLV and HIV-1BH10.

Table 2.   Pre-steady-state kinetic parameters for mispair extension. The template-primer 31T/21P was used as the substrate. Data shown are the mean values ± SD. Each of the assays was performed independently at least three times.
EnzymeBase pair at the 3′ endakpol (s−1)Kdm)kpol/Kdm−1·s−1)Mismatch extension ratio (fext)b
  1. a  The first base corresponds to the template and the second to the primer. b fext = [kpol(mismatched)/Kd(mismatched)]/[kpol(matched)/Kd(matched)]. Numbers in bold-face type within parentheses represent the relative increase of mispair extension fidelity relative to the HIV-1BH10 RT. c Reported values for HIV-1BH10 RT were taken from Matamoros et al. [19].

XMRV RTG.C0.19 ± 0.0224.1 ± 4.3(7.83 ± 1.71) × 10−3 
G.T(4.99 ± 0.26) × 10−23012 ± 418(1.66 ± 0.25) × 10−5(2.12 ± 0.56) × 10−3 (0.53)
G.G(3.35 ± 0.30) × 10−33084 ± 714(1.09 ± 0.27) × 10−6(1.39 ± 0.46) × 10−4 (2.2)
G.A(4.27 ± 0.21) × 10−43058 ± 557(1.39 ± 0.26) × 10−7(1.78 ± 0.51) × 10−5(0.20)
MoMLV RTG.C1.55 ± 0.1724.9 ± 7.1(6.21 ± 1.88) × 10−2 
G.T(6.41 ± 0.38) × 10−24378 ± 502(1.46 ± 0.19) × 10−5(2.35 ± 0.77) × 10−4 (4.8)
G.G(4.84 ± 0.60) × 10−34535 ± 1291(1.07 ± 0.33) × 10−6(1.72 ± 0.74) × 10−5 (17.7)
G.A(8.21 ± 0.56) × 10−42816 ± 454(2.92 ± 0.51) × 10−7(4.70 ± 1.64) × 10−6 (0.76)
HIV-1BH10 RTcG.C11.6 ± 0.513.4 ± 1.90.863 ± 0.126 
G.T2.54 ± 0.132628 ± 252(9.66 ± 1.04) × 10−4(1.12 ± 0.20) × 10−3
G.G0.266 ± 0.0201014 ± 188(2.62 ± 0.52) × 10−4(3.04 ± 0.75) × 10−4
G.A0.0158 ± 0.00095110 ± 755(3.09 ± 0.49) × 10−6(3.58 ± 0.77) × 10−6

The differences between XMRV and HIV-1BH10 RTs were relatively small, except for G·A mismatch extension and misincorporation of G opposite A. However, both enzymes showed decreased misinsertion and mispair extension fidelity in comparison with the MoMLV RT. Discrimination against C or A opposite A was 3.4 times and 13.0 times more efficient in reactions catalyzed by MoMLV RT compared with XMRV RT, whereas XMRV RT showed about eight- to nine-fold decreased fidelity in extending G·T and G·G mispairs.

M13mp2 lacZα forward mutation assays

The mutation frequencies of XMRV RT and the MoMLV RT used in the kinetic assays described earlier were determined using the M13-based forward mutation assay [20], and compared with those previously reported for the HIV-1BH10 RT and a commercially available MoMLV RT [19,21]. In these assays, a gapped M13mp2 DNA was used as substrate in the polymerization reaction. The mutational target for errors made by the RT is the wild-type lacZα-complementation sequence, which is used as a template in the gap-filling reaction. Errors made by the RT may result in a decrease in α-complementation and can be detected as plaques with an altered color phenotype (pale blue or colorless) in a specific indicator strain. Silent mutations are not detected in these assays. Mutant frequencies are calculated as the ratio of mutant plaques to total plaques. MoMLV RTs obtained from different sources showed similar mutant frequencies in these assays, and were about 15-fold more accurate than the HIV-1BH10 RT (Table 3). Interestingly, the XMRV RT showed a mutant frequency of 0.157%, similar to that determined for the MoMLV RTs obtained from two different sources.

Table 3.   Accuracy of RT variants in M13mp2 lacZα forward mutation assays. The background frequencies reported in this assay (around 6 × 10−4) [19,20] are, in most cases, a consequence of rearrangements of M13mp2 DNA that resulted in the loss of the lacZ gene. Phage DNA was obtained from all mutant plaques and the sequence of the reporter gene was determined in all cases. No lacZ mutations were identified after sequencing phage DNA from more than 20 000 plaques obtained from two or three Escherichia coli electroporation reactions carried out with gapped M13mp2 DNA substrate. Four to six gap-filling reactions were performed for each enzyme
EnzymeTotal plaquesMutant plaquesMutant frequency
  1. a RT obtained from Promega Corp. (Madison, WI, USA). bRT expressed and purified using plasmid pMULVRT [22,23]. Data for MoMLV RT (Promega) were taken from Barrioluengo et al. [21]. The values reported for HIV-1BH10 RT were taken from Matamoros et al. [19]. Numbers within parentheses represent the fold-increase in accuracy, relative to HIV-1BH10 RT.

XMRV RT52 196820.00157 (13.1)
MoMLV RTa29 648400.00135 (15.3)
MoMLV RTb52 207700.00134 (15.4)
HIV-1BH10 RT67361390.0206 (1)

The mutational specificity of the XMRV RT was determined after sequencing the lacZα mutants generated in the forward mutation assays. The mutational spectrum reveals an accumulation of errors at six major hot spots, located at positions −9, +100, +104, +129, +134 and +148 (Fig. 3). Four of these are represented by a high frequency of one-nucleotide deletions (i.e. at positions −9, +100, +104 and +134) while the two others involve C→T and G→T mutations. Only one of the observed hot spots occurs in a run of identical nucleotides (a run of C nucleotides around position +134), suggesting that template-primer slippage may not be a large contributor to variability induced by the XMRV RT. The error distribution obtained with the XMRV RT differed from published mutational spectra of MoMLV and HIV-1BH10 RTs [21,24]. These two RTs generate fewer hot spots that occur at different locations in comparison with the XMRV RT. Only the sequence around positions +131/+136 emerges as a mutational hot spot for both gammaretroviral RTs. However, the types of errors found at this location are different in both enzymes. While the XMRV RT introduced one-nucleotide deletions within the run of C nucleotides, the MoMLV RT produced one-nucleotide insertions that affected the adjacent T (located at position +131) [21].

Figure 3.

 Spectrum of mutations induced by XMRV RT. Single-nucleotide substitutions are indicated by the letter corresponding to the new base (in blue) above the template sequence of the lacZα target. Inverted closed red triangles indicate deletions of one nucleotide.

As in the case of the MoMLV RT, the XMRV RT had a relatively high propensity to introduce frameshift mutations. Frameshifts represented around 60% of all errors generated by those two enzymes. However, this figure was reduced to 36.7% in the case of HIV-1BH10 RT (Table 4). Compared with the HIV-1BH10 RT, the XMRV RT error rates were reduced 17.9 times for base substitutions and 8.2 times for frameshifts. These values were similar to those obtained with MoMLV RT. Both gammaretroviral RTs showed a higher tendency to produce transversions, while HIV-1BH10 RT was prone to generate transitions. Frameshift errors produced by the HIV-1BH10 RT accumulated at homopolymeric sequences. On the other hand, insertions and deletions produced by MoMLV RT were evenly distributed between runs and nonruns of nucleotides, and frameshift errors generated by XMRV RT were less frequent in nucleotide runs. For XMRV RT, the overall −1 base frameshift error rate in homopolymeric template sequences was around 1 in 247 400. This figure was about 2.5 times lower than the corresponding value obtained with the MoMLV RT (Table 4). Further differences between XMRV and MoMLV RTs were related to the distribution and the nature of insertions and deletions. More than 30% of the errors found in the mutational spectrum of MoMLV RT were large deletions or one-nucleotide insertions [21]. However, these types of errors were not detected in our assays with the XMRV RT.

Table 4.   Summary of error rates for RTs, for various classes of mutations.
Mutation typeXMRV RTMoMLV RTaHIV-1BH10 RTb
No. of errorsError rateNo. of errorsError rateNo. of errorsError rate
  1. a MoMLV RT data were taken from Barrioluengo et al. [21]. b HIV-1BH10 RT values were taken from Álvarez et al. [24]. Numbers between parentheses represent the fold-increase in the accuracy of the RT (relative to the HIV-1BH10 RT).

All classes861/86 305 (11.7)491/86 040 (11.7)491/7369
Base substitutions381/103 018 (17.9)191/117 032 (20.3)311/5760
 Transitions10 (26.3%) 3 (15.8%) 20 (64.5%) 
 Transversions28 (73.7%) 16 (84.2%) 11 (35.5%) 
Frameshifts481/96 563 (8.2)301/87 758 (7.5)181/11 745
 Insertions0 (0%) 8 (26.7%) 3 (16.7%) 
 Deletions48 (100%) 22 (73.3%) 15 (83.3%) 
 At runs101/247 409 (39.6)141/100 380 (16.1)181/6253
 At nonruns381/56 866161/76 7140< 1/98 207

Discussion

The association between XMRV and either prostate cancer or chronic fatigue was hotly debated until Paprotka et al. provided compelling evidence on the recombinant origin of this novel gammaretrovirus [11], and multilaboratory studies revealed serious problems that affected the reproducibility of the assays, at least in the blood of patients with chronic fatigue syndrome [12]. These issues aside, other studies indicate that XMRV may infect human cells and establish a persistent infection in primates [13–15]. Gammaretroviruses could still pose a risk for human health as potential contaminants of biological products, and in particular interventions (i.e. xenotransplantation). In this context, studies on fundamental mechanisms of DNA synthesis, including fidelity of their retroviral RTs, might be necessary to gain a deeper understanding of the biology of retroviruses and provide novel tools in biotechnology.

Patient-derived XMRV is identical to a virus infecting the prostate cancer cell line CWR22Rv1, which originated from two endogenous proviruses (PreXMRV-1 and PreXMRV-2) during passage of the prostate tumor in mice [11]. The 5′ end of the XMRV genome derives from PreXMRV-2, while the 3′ end derives from PreXMRV-1. One recombination site is located in the pol gene, at the RT-coding region (between nucleotides encoding residues 431–455 of XMRV RT). Therefore, the DNA polymerase domain of XMRV RT is almost identical to that of MoMLV RT (> 97.5% sequence identity), while the differences between both enzymes are mostly concentrated at the RNase H domain (Fig. 1).

Compared with MoMLV RT, XMRV RT showed an eight-fold reduced catalytic efficiency of DNA polymerization, which was a consequence of its low kpol. Our results are broadly consistent with those recently reported by Ndongwe et al. [18], who also found that both gammaretroviral RTs had diminished DNA polymerase activity in comparison with the HIV-1BH10 RT. In their study, the authors used pre-steady-state kinetics to compare the intrinsic fidelities of XMRV, MoMLV and HIV-1 RTs. However, their analysis was limited to the misincorporation of T opposite T on a heteropolymeric 100/18mer DNA–DNA template-primer. Their reported fins values for the RTs of XMRV, MoMLV and HIV-1 were 1.76 × 10−3, 7.32 × 10−3 and 4 × 10−2, respectively [18]. These results, together with qualitative assessments of fidelity based on primer-extension reactions using three dNTPs, led the authors to conclude that the XMRV RT was more faithful than the MoMLV polymerase, while both enzymes showed increased accuracy relative to HIV-1 RT.

In our analysis, we demonstrated that MoMLV RT is more accurate than HIV-1BH10 RT in discriminating C, G or A vs. T (Table 1). In addition, we found that the HIV-1 enzyme showed a higher efficiency in extending G·T and G·G mismatches (Table 2). As pre-steady-state kinetics analyses of fidelity require relatively high concentrations of MLV RT as a result of its low catalytic efficiency of DNA polymerization, there is only one additional report comparing MoMLV and HIV-1 RTs using transient kinetics [23]. In this study, MoMLV RT was found to be 50 times less efficient than HIV-1 RT in incorporating the correct 2′-deoxycytidine 5′-triphosphate (dCTP) on an RNA-DNA template-primer.

Our results for the MoMLV RT were broadly in agreement with previous reports showing its higher accuracy, in misinsertion and mispair extension assays carried out with DNA–DNA template-primers under steady-state conditions [25–27], as well as with the results obtained by Skasko et al., who showed that the MoMLV enzyme had a 3.6-fold increased ability to discriminate between G and C, and increased fidelity of G·T mispair extension [23]. Our data were also consistent with those studies in showing that fins values were in the range of 10−4 to 10−7, lower than those reported for XMRV and MoMLV RTs by Ndongwe et al. [18]. XMRV and HIV-1BH10 RTs showed similar mispair extension efficiencies but were less faithful than MoMLV RT in these assays (Table 2). However, the XMRV RT showed remarkable selectivity against 2′-deoxyguanosine 5′-triphosphate (dGTP) when the template base was A. The fixation of a mutation involves misincorporation followed by mispair extension. Therefore, discrimination at the nucleotide-incorporation step might be critical to determine the fidelity of the XMRV RT. Nevertheless, kinetic data are strongly dependent on the sequence and the template-primer used [25], and, in this scenario, forward mutation assays may provide a better estimate of fidelity differences between different RTs because they can screen for mutations in a relatively large number of sequence contexts.

The mutant frequency obtained for XMRV RT was similar to previous estimates for other oncoretroviral RTs, such as MLV RT or avian myeloblastosis virus RT [28,29; reviewed in ref. 30]. XMRV and MoMLV RTs were 13.1–15.4 times more accurate than the HIV-1BH10 RT. HIV-1BH10 RT and other phylogenetically close HIV-1 RTs are prone to introduce frameshifts, particularly at homopolymeric nucleotide runs. More than 90% of the one-nucleotide insertions or deletions generated by the HIV-1 RT occurred in homopolymeric sequences, probably originating from the utilization of misaligned template-primers [19,31–33]. On the other hand, gammaretroviral RTs showed increased frameshift accuracy and a stronger tendency to introduce transversions. Compared with XMRV RT, the MoMLV enzyme showed a stronger tendency to introduce large deletions and one-nucleotide insertions [21]. Other significant differences between XMRV and MoMLV RTs were found in the distribution of frameshifts (i.e. one-nucleotide deletions) at run and nonrun template sequences. XMRV RT showed a 4.4-fold increase in frameshift fidelity while copying homopolymeric sequences versus heteropolymeric sequences, although similar error rates were obtained for the MoMLV RT while copying both types of sequences. Interestingly, the distribution of frameshift errors at runs vs. nonruns of nucleotides in the mutational spectrum of XMRV RT was similar to that previously reported for avian myeloblastosis virus RT [29]. These results suggest that unlike HIV-1 RT, template-primer slippage may play a secondary role in generating frameshifts during DNA synthesis catalyzed by XMRV RT.

As expected from the accuracy of their RTs in forward mutation assays, both XMRV and MoMLV showed similar mutation rates in single-cycle replication assays carried out ex vivo [18], although their mutational spectra have not been determined. In contrast to HIV-1, there is a good correlation between the intrinsic error rates of MoMLV RT and the mutation rates obtained with the virus, with estimates ranging from 2 × 10−6 to 2 × 10−5 [34,35]. Sequence analysis of XMRV obtained from prostate cancer tumors revealed extremely low variability [1,2] that was difficult to reconcile with the error-prone nature of retroviral replication. Our study demonstrates that the accuracy of the XMRV RT is not unusually high but similar to that reported for other oncoretroviral RTs.

Finally, our data also suggest a role for the connection/RNase H domain in modulating the fidelity of DNA synthesis. The differences in the catalytic efficiencies of nucleotide incorporation between XMRV and MoMLV RTs could result from altered template-primer binding. In agreement with this proposal, it has been shown that the XMRV RT has lower DNA-binding affinity and processivity in comparison with the MoMLV RT [18]. Although both RTs share identical amino acid sequences in their DNA polymerase motifs A, B, C, D and E (relevant for dNTP binding and catalysis), they differ at their connection/RNase H domains (Fig. 1). Sequence differences at those regions could affect polymerization by interfering with the positioning of the DNA at the polymerase active site, thereby influencing slippage and template switching. Further studies are warranted to elucidate the contribution of RT connection/RNase H domains to polymerization accuracy.

Materials and methods

Expression and purification of RTs

XMRV RT, derived from clone VP62 (GenBank accession number DQ399707) [1], was expressed in Escherichia coli as a single polypeptide chain of 75.9 kDa containing a nonremovable His6 tag, and then purified by a combination of immobilized metal-affinity and ion-exchange chromatography [36]. Recombinant HIV-1BH10 RT was expressed and purified as previously described [37,38]. This enzyme was co-expressed with HIV-1 protease in E. coli XL1 Blue to obtain the p66/p51 heterodimer, which was later purified by ionic exchange followed by immobilized metal-affinity chromatography on Ni2+-nitrilotriacetic acid agarose. In this enzyme, the His6 tag was introduced at the C-terminal end of p66.

MoMLV RT was overexpressed in E. coli BL21 using a plasmid (pMuLVRT) encoding the full-length enzyme with a His6 N-terminal extension [22]. Plasmid pMuLV RT was kindly provided by Dr Baek Kim (University of Rochester Medical Center, Rochester, NY, USA). The RT sequence encoded within pMuLVRT is identical to the one shown in Fig. 1, except for His8, which was replaced with Tyr. Bacterial cells obtained from 3 L of culture were harvested by centrifugation. The obtained pellet was resuspended in 20 mL of 40 mm Tris/HCl (pH 8.0) containing 10% saccharose, 1 mm phenylmethanesulfonyl fluoride, 2 mm benzamide and 1 mg·mL−1 of lysozyme, and left on ice for 15 min. The resuspended pellet was then mixed with 20 mL of a solution containing 0.8% (v/v) Nonidet P-40, 20 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride and 2 mm benzamidine, and left at 4 °C for another 15 min before adding NaCl to a final concentration of 0.5 m. Lysed cells were sonicated and centrifuged twice at 15 000 g for 15 min. The supernatant was diluted seven-fold in 40 mm Tris/HCl (pH 7.9) containing 0.5 m NaCl, and applied to a Ni2+-charged ProBondTM resin column (Invitrogen, Carlsbad, CA, USA). After extensive washing, the RT was eluted with a 0–1.0 m imidazole gradient. RT-containing fractions were combined and applied to a 3-mL Q-Sepharose column (GE Healthcare, Uppsala, Sweden), previously equilibrated in 40 mm Tris/HCl (pH 7.9) containing 0.5 m NaCl. Enzyme was recovered in the nonbinding fraction and dialyzed against 50 mm Tris/HCl (pH 7.0) containing 25 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol and 10% glycerol. Purified and concentrated RTs were stored at −20 °C in dialysis buffer containing 10% glycerol (HIV-1BH10 and MoMLV RTs) or 50% glycerol (XMRV RT). Enzyme purity was assessed by SDS/PAGE.

Active site titration

Enzymes were routinely quantified by active site titration before biochemical studies [39]. Briefly, individual RTs were incubated with 30 nm DNA–DNA template-primer [D38 (5′-GGGTCCTTTCTTACCTGCAAGAATGTATAGCCCTACCA-3′)/25PGA (5′-TGGTAGGGCTATACATTCTTGCAGG-3′)] for 10 min at 37 °C, in 10 μL of 100 mm Hepes (pH 7.0) containing 30 mm NaCl, 30 mm magnesium acetate, 130 mm potassium acetate, 1 mm dithiothreitol and 5% (w/v) polyethylene glycol. After adding 10 μL of the same buffer containing 1 mm dTTP, 4-μL aliquots were removed and quenched at 10, 20, 30 and 40 s with sample loading buffer [10 mm EDTA in 90% formamide containing xylene cyanol FF (3 mg·mL−1) and Bromophenol Blue (3 mg·mL−1)]. Products were analyzed by electrophoresis on a 20% denaturing sequencing gel and quantified by phosphorimaging. For each reaction, the percentage of elongated primer was plotted against the incubation times and the data were fit to a linear equation. As the concentration of template-primer is well above the Kd under our assay conditions [40,41], the active RT concentration was calculated from the y-intercept that represents the amount of RT bound to template-primer at time zero. Optimal RT concentrations in these experiments are usually in the range of 2.5–5 nm.

Misinsertion and mispair extension fidelity of DNA synthesis

Fidelity estimates were obtained from transient kinetics experiments carried out in 50 mm Tris/HCl buffer (pH 8.0), containing 50 mm KCl and 12 mm MgCl2 and a variable concentration of nucleotide [19,42]. Oligonucleotides 21P (5′-ATACTTTAACCATATGTATCC-3′) and 31T (5′- TTTTTTTTTAGGATACATATGGTTAAAGTAT-3′) were used as primer and template, respectively. Before annealing to the template, the primer was labeled at its 5′ terminus with [γ-32P]ATP (Perkin Elmer, Boston, MA, USA) and T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Correct nucleotide (dTTP) incorporation reactions were performed under single-turnover conditions with 100 nm (active sites) RT and 100 nm template-primer. At appropriate time-points, the reactions were quenched with EDTA (0.3 m final concentration). The products were analyzed by denaturing PAGE and quantified by phosphorimaging.

Mismatched 32P-labeled primers (i.e. 21PT, 21PG and 21PA) annealed to the 31-mer DNA template (31T) were used to determine G·T, G·G and G·A mispair extension efficiencies. In these assays, the relevant kinetic parameters were obtained for the incorporation of dTTP. Reactions involving misincorporation of dCTP, dGTP or dATP (instead of the correct dTTP) using 31T/21P, or extension of G·T, G·G or G·A mispairs, were conducted with an excess of RT (120 nm) over template-primer (100 nm). These conditions were chosen to eliminate the influence of the enzyme turnover rate (kss), which interferes in the measurements of low incorporation rates. Misincorporation reactions carried out with dGTP and dATP were carried out in the presence of 18 mm MgCl2 to ensure that enough free Mg2+ was available to bind the catalytic site A in the DNA polymerase domain of the RT [42]. Pre-steady-state kinetic data were fitted to the burst equation:

image

where A is the amplitude of the burst, kobs is the apparent kinetic constant of formation of the phosphodiester bond and kss is the kinetic constant of the steady-state linear phase. The dependence of kobs on the dNTP concentration is described by the equation:

image

where Kd is the equilibrium constant and kpol is the catalytic rate constant of the nucleotide incorporation reaction. Kd and kpol were determined from curve-fitting using Sigma Plot.

M13mp2 lacZα forward mutation assay

The assay was performed essentially as previously described [20]. Briefly, the single-stranded and replicative forms of the M13mp2 DNA were obtained from phage grown in E. coli NR9099. The replicative form was digested with PvuII to obtain a 6.8-kb double-stranded DNA that was denatured and annealed to the single-stranded M13 DNA to obtain a gapped duplex DNA substrate. This DNA has a gap of 407 nucleotides (positions −216 to +191) and lacks the lacZα-complementation target sequence in one of the strands. Gap-filling reactions (10 μL) were carried out in 25 mm Tris/HCl (pH 8.0) containing 100 mm KCl, 2 mm dithiothreitol, 4 mm MgCl2, 250 μm of each dNTP, 5 μg·mL−1 of gapped duplex DNA and 100 nm RT [24]. After 30 min of incubation at 37 °C, the reactions were stopped by adding EDTA to a final concentration of 15 mm. The obtained products were electroporated into E. coli MC1061 host cells. After a brief recovery period (10 min), transformants were plated onto M9 medium containing 0.195 mm 5-bromo-4-chloroindol-3-yl β-d-galactoside, 0.2 mm isopropyl thio-β-d-galactoside and a lawn of the α-complementation strain of E. coli, CSH50. Mutant plaques were picked and the phage double-stranded DNA replicative form was isolated. Phenotypes of mutant plaques were confirmed by nucleotide sequencing using primer 5′-GCTTGCTGCAACTCTCTCAG-3′ (Macrogen Inc., Seoul, South Korea). At least five fill-in reactions were performed for each enzyme and the nucleotide sequence of the entire gap region was determined for all mutant plaques. Mutant frequencies and specific error rates were obtained as previously described [21].

Acknowledgements

This work was supported, in part, by grants of the Ministery of Science and Innovation of Spain (BIO2010/15532), Fondo de Investigación Sanitaria (through the ‘Red Temática de Investigación Cooperativa en SIDA’ RD06/0006), and an institutional grant from the Fundación Ramón Areces. S.F.J. Le G. and Y.W. are supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health.

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