The processivity and fidelity of DNA synthesis exhibited by the reverse transcriptase of bovine leukemia virus

Transcription

1 Eur. J. Biochem. 269, (2002) Ó FEBS 2002 The processivity and fidelity of DNA synthesis exhibited by the reverse transcriptase of bovine leukemia virus Orna Avidan, Michal Entin Meer, Iris Oz and Amnon Hizi Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel We have recently expressed in bacteria the enzymatically active reverse transcriptase (RT) of bovine leukemia virus (BLV) [Perach, M. & Hizi, A. (1999) Virology 259, ]. In the present study, we have studied in vitro two features of the DNA polymerase activity of BLV RT, the processivity of DNA synthesis and the fidelity of DNA synthesis. These properties were compared with those of the well-studied RTs of human immunodeficiency virus type 1 (HIV-1) and murine leukaemia virus (MLV). Both the elongation of the DNA template and the processivity of DNA synthesis exhibited by BLV RT are impaired relative to the other two RTs studied. Two parameters of fidelity were studied, the capacity to incorporate incorrect nucleotides at the 3 end of the nascent DNA strand and the ability to extend these 3 end mispairs. BLV RT shows a fidelity of misinsertion higher than that of HIV-1 RT and lower than that of MLV RT. The pattern of mispair elongation by BLV RT suggests that the in vitro error proneness of BLV RT is closer to that of HIV-1 RT. These fidelity properties are discussed in the context of the various retroviral RTs studied so far. Keywords: bovine leukaemia virus; fidelity; processivity; reverse transcriptase. Bovine leukaemia virus (BLV) is a naturally occurring exogenous B-cell lymphotropic retrovirus, which is the aetiological agent of cattle leukosis. This disease is characterized by an initial persistent lymphocytosis, which is followed by the occurrence of clonal lymphoid B-cell tumours after a long latency period [1]. This virus is related to human T-cell leukemia viruses type I and type II (HTLV-I and HTLV-II, respectively), forming a subfamily of transactivating retroviruses [2]. The genomes of these complex retroviruses have close to their 3 ends the regulatory genes tax and rex and the presence of both Tax and Rex proteins, encoded by these genes, is required for viral replication. These viruses also show nucleotide sequence similarities, although BLV and HTLVs do not infect the same cell types, because they probably bind different cell receptors [2 4]. The process of reverse transcription is the major early intracellular event critical to the life cycle of all retroviruses. The synthesis of the proviral DNA is catalysed entirely by the reverse transcriptase (RT). The plus-strand viral RNA is Correspondence to Amnon Hizi, Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Fax: , Tel.: , ahizy@post.tau.ac.il Abbreviations: BLV, bovine leukemia virus; HTLV, human T-cell leukaemia virus; HIV, human immunodeficiency virus; MLV, murine leukaemia virus, MMTV, mouse mammary tumour virus; AMV, avian myeloblastosis virus; EIAV, equine infectious anaemia virus; AIDS, acquired immunodeficiency syndrome; F ins, frequency of insertion; F ext, frequency of extension; DDDP, DNA-dependent DNA polymerase; RNase H, ribonuclease H. Note: M. E. Meer and O. Avidan contributed equally to the research described in this manuscript. The results presented are in partial fulfilment of a PhD thesis (M.E.M.) at Tel Aviv University. (Received 6 August 2001, revised 14 November 2001, accepted 3 December 2001) copied by the RNA-dependent DNA polymerase activity of RT, producing RNA/DNA hybrids. The intrinsic ribonuclease H (RNase H) activity of RT specifically hydrolyses the RNA in these heteroduplexes. Finally, the plus-sense DNA strand is synthesized by copying of the minus-sense DNA strand by the DNA-dependent DNA polymerase (DDDP) activity of RT [2,5]. As RT is a preferred target for the development of viral inhibitors as antiretroviral drugs, the structural and catalytic properties of RTs have been the focus of many recent studies, including three-dimensional crystal studies [6 9]. A major effort was devoted to the research of the RTs of the human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2, respectively), which are responsible for acquired immunodeficiency syndrome (AIDS); most of the anti-aids drugs approved so far for the treatment of AIDS are inhibitors of the viral RT. Due to the rapid emergence of drug-resistant HIV RT variants, the development of novel potent and specific inhibitors of HIV RTs is still a principal objective in the chemotherapy of AIDS [2,10,11]. Targeted drug designs rely on a better understanding of the structure and function of retroviral RT. Therefore, the investigation of RT of other retroviruses should expand our understanding of the catalytic properties of these closely related proteins. We have recently expressed the recombinant RT of BLV in bacteria. The gene encoding the RT was designed to start at its 5 end next to the last codon of the mature viral protease; namely, the amino terminus of the RT matches the last 26 codons of the pro gene and is encoded by the pol reading frame [12]. BLV RT was purified and studied biochemically: it exhibits all activities typical of RTs, i.e. both RNA- and DNA-dependent DNA polymerases and RNase H activity. Unlike most RTs, the BLV RT is enzymatically active as a monomer even after binding a DNA substrate. The enzyme shows a preference for Mg 2+ over Mn 2+ in both its DNA polymerase and RNase H activities. BLV RT was shown to have a relatively low

2 860 O. Avidan et al. (Eur. J. Biochem. 269) Ó FEBS 2002 sensitivity to nucleoside triphosphate analogues, known to be potent inhibitors of other RTs such as that of HIV [12]. In the present study we have extended the investigation of BLV RT by an in vitro analysis of the processivity and the fidelity of DNA synthesis (namely, the ability of BLV RT to misincorporate nucleotides at the 3 end of the growing DNA strand and the further extending of the preformed mismatches). MATERIALS AND METHODS Recombinant RTs and DNA polymerase activities Monomeric BLV RT was expressed in bacteria and purified to homogeneity after modifying the method published recently [12] These modifications were as follows: (a) the puc12n6h BLV RT-transformed Escherichia coli were grown in Terrific Broth (without glycerol) instead of NZYM medium; (b) the carboxymethyl Sepharose column buffer was at ph 6.5 (instead of ph 7.0); (c) after the purification had been carried out the BLV RT was further concentrated in an Amicon Centriprep 30 concentrator. Recombinant heterodimeric HIV-1 RT was expressed in bacteria as described [13]. Recombinant murine leukaemia virus (MLV) RT was also expressed in E. coli [14]. The recombinant proteins containing six histidines at their amino termini were purified as described above for BLV RT, except for the fact that all buffers used to purify MLV RT included 0.2% (v/v) Triton X-100 (instead of 0.1%). The DNA polymerase activities were assayed as described previously [15]. One unit of activity was defined as the amount of enzyme that catalyses the incorporation of 1 pmol dntp into activated DNA (that served as the template-primer) in 30 min at 37 C, under the assay conditions. Similar BLV, HIV-1 or MLV RT DNA polymerase activities were used in all experiments described, using lg RT protein (according to the specific activities of the different enzymes). Template primers For the experiments of DNA primer extension and processivity, we used single-stranded circular /X174am3 DNA (from New England Biolabs) as the DNA template, which was primed with a 15-residue synthetic primer (5 -AAAGCGAGGGTATCC-3 ) that hybridizes at positions of the /X174am3 DNA. The synthetic template-primers used for the experiments of misinsertion and preformed mispair extension are shown in Figs 2 and 3. For analysis of site-specific nucleotide misinsertion, a synthetic 50-residue template (with a sequence derived from nucleotides of /X174am3 DNA) was primed with the same 15-residue oligonucleotide used for extension and processivity. This primer hybridizes to the sequence at positions (in the 5 fi3 direction) of the 50-residue template DNA (Fig. 2). For the extension of DNA from a mispaired terminus, the set of template-primers used is composed of the same 50-residue oligonucleotide template used for the misinsertion studies (Fig. 3), primed with a set of 16-residue oligonucleotides (that hybridize to the nucleotides at positions of the template). Four versions of 16-residue primers were used, each differing from the other only at its 3 -terminal nucleotide (Fig. 3). All primers used in Fig. 1. DNA primer-extension and processivity of DNA synthesis exhibited by BLV, HIV-1 and MLV RTs. All reactions were performed with the 15-nucleotide synthetic 5 end-labelled oligonucleotide primer and a twofold excess of the template single-stranded circular /X174am3 phage DNA. The sequence of the primer and the experimental details are described in Materials and methods. The symbols for the DNA synthesis experiments are as follows: ( ) DNA extension performed with no DNA trap; (+) DNA extension experiments conducted in the presence of unlabeled DNA trap. Molecular mass markers are HinfI-cleaved dephosphorylated double-stranded /X174am3 DNA fragments (Promega) labelled with [c- 32 P]ATP at the 5 ends by polynucleotide kinase. this study were labelled at their 5 ends with c[ 32 P]ATP (using T4 polynucleotide kinase) and were annealed to the templates with a twofold molar excess of each template over its primer as described previously [16]. DNA polymerization and processivity experiments The reactions were conducted in a final volume of 12.5 ll 6.6 mm Tris/HCl, 4 mm dithiothreitol, 24 lgæml )1 BSA, 6mM MgCl 2 (for BLV and HIV-1 RTs) or 1 mm MnCl 2 (for MLV RT), final ph 8.0, supplemented by the /X174am3 template-primer at a final concentration of 30 lgæml )1. For processivity studies, the BLV, HIV-1 and MLV RTs, at equal DNA polymerase activities, were incubated with the annealed template primer for 5 min at 30 C. In all polymerization experiments shown we used pmol RT per reaction (depending on activity) and

3 Ó FEBS 2002 Processivity and fidelity of BLV RT (Eur. J. Biochem. 269) 861 Table 1. Quantitative analysis of DNA primer-extension and relative processivity of DNA synthesis. The radioactivity in the DNA bands in all polynucleotide length ranges were summed and then the values obtained were divided by the sums of all extended and unextended primers (detected in the phosphoimaging scanning of the gels as shown in Fig. 1). The values given are the extended primers in each product length range expressed as percentages of the total amounts (all extended and unextended primers) of the DNA products. The calculations were conducted separately for gel lanes of reactions carried out in the absence or presence of an excess of the unlabeled DNA trap (see Materials and methods). The overall extensions in the presence of the DNA trap, divided by the comparable figures obtained with no trap present, yielded the relative processivity values expressed as percentages (see text). The values are the menas calculated from two independent experiments (one of which is shown in Fig. 1) and the variations were usually < 15%. Product length (nucleotides) BLV RT HIV-1 RT MLV RT Without trap With trap Without trap With trap Without trap With trap Overall extension Relative processivity Table 2. Quantitative analysis of DNA synthesis and processivity after correcting for the relative length of the DNA products. The data shown were derived from the same two independent experiments as in Table 1. Here, the data were evaluated after correcting for the mean lengths of the DNA primers extended by the three RTs under the assay conditions used. The correction for the actual amount of dntp incorporation for a given DNA product was achieved by multiplying the radioactivity in each 5 end-labelled polynucleotide product length class by the median of the number of nucleotides added in each range (i.e. 17 nucleotides for the nucleotide range, 42 nucleotides for the nucleotide range, 92 nucleotides for the nucleotide range and 342 nucleotides for the nucleotide range). After introducing these factors, all values are expressed (as in Table 1) as percentages of the total amounts of all primers extended in each length class. The values shown are the means calculated from the same two independent experiments as in Table 1. Product length (nucleotides) BLV RT HIV-1 RT MLV RT Without trap With trap Without trap With trap Without trap With trap Overall extension Relative processivity pmol of the template primer. The reaction mixtures were divided into two, without or with a DNA trap of a large excess of unlabelled activated herring sperm DNA, at a final concentration of 0.6 mgæml )1 (prepared as described previously [15]). All reactions were initiated immediately afterwards by adding the four dntps, each at a final concentration of 20 lm, followed by incubation at 37 C for 30 min. The reactions were stopped by adding an equal volume of formamide dye mix, denatured at 100 C for 3 min, cooled on ice and analysed by electrophoresis through 8% polyacrylamide/urea sequencing gels in 90 mm Tris/borate, 2 mm EDTA ph 8.0, as described previously [17]. The extension products were quantified by densitometric scanning of gel autoradiograms and the amounts of primer extended were calculated. Fidelity of DNA synthesis For site-specific nucleotide misinsertion, we assayed dntp incorporation opposite to the A residue at position 23 of the template as described [16,18] (see also Fig. 2). The 32 P-5 -end-labelled 15-residue primer was extended in the presence of increasing concentrations of either 0 1 lm of the correct dntp (dttp) or 0 1 mm each of the incorrect dntps (datp, dctp or dgtp). All dntps used were of the highest purity available (Pharmacia) with no detectable traces of contamination by other dntps. For mispair extension (Fig. 3), elongation of 32 P-5 -end-labelled 16-nucleotide primers was measured with increasing concentrations of datp as the only dntp present (0 1 mm range for the mispaired AÆA, AÆC oraæg termini or a 0 1 lm range for the AÆT correct terminus) [16,18]. Reactions for all kinetic analyses contained 14 mm Tris/ HCl ph 8.0, 4 mm dithiothreitol, 4 mm MgCl 2 and 24 lgæml )1 BSA. The reactions were incubated at 37 C for either 2 min (for the correct incorporation or correctly matched DNA elongation), or 5 min (for misincorporation or extension of formed mismatched DNA). Kinetic reactions were performed with an 10-fold molar excess of template-primer over BLV RT to ensure steady-state kinetics in the linear range. All reaction products were analysed by electrophoresis through 14% polyacrylamide/ urea in Tris/borate and EDTA sequencing gels, and band intensities were quantified as described above. This allowed the calculation of reaction velocities, i.e. the amount of

4 862 O. Avidan et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Fig. 2. The pattern of DNA mispair formation by BLV, HIV-1 and MLV RTs. The synthetic 50-nucleotide template was annealed to the 32 P-5 -endlabelled primer. The primer was extended with equal DNA polymerase activities of either BLV RT, HIV-1 RT or MLV RT in the presence of 1 mm of a single incorrect dntp (i.e. C, G, or A) or 1 lm of the correct dntp (dttp) as described in Materials and methods. The level of misinsertion is apparent from the elongation of the primer in the presence of the incorrect dntp relative to that in the presence of dttp. total 32 P-labelled primer extended per minute in the conditions used. The V max and K m values were calculated from the double-reciprocal linear plots of velocity vs. dntp concentrations [16,18]. RESULTS We have analysed in vitro two basic properties of DNA synthesis by BLV RT, i.e. processivity and fidelity, both in comparison with the well-studied RTs of HIV-1 (representing a low fidelity RT from the lentivirus subfamily of retroviruses) and of MLV (representing a relatively high fidelity RT from the mammalian type C retroviruses) Table 3. Kinetic parameters for site-specific misincorporation by BLV RT. The 15-residue c) 32 P-5 -end-labelled primer was hybridized to a fourfold molar excess of the 50-residue template derived from the sequence of nucleotides of /X174am3 DNA (Fig. 2). In each set of the kinetic experiments, the template-primer was incubated with BLV RT in the presence of increasing concentrations of a single dntp. The oligonucleotide products were analysed and described in Materials and methods. The apparent K m and V max values were determined from at least two independent experiments performed as described in Materials and methods and in the text and the variations were usually < 20%. The values of relative frequency of insertion (F ins )werecalculated as described in the text. Pair or mispairs formed K m (lm) V max (%Æmin )1 ) F ins AÆT AÆC / AÆG / AÆA / [16,19 21]. The assays were performed with templateprimers already used in our laboratory with other RTs, allowing comparison of information. Similar to all RTs studied so far, BLV RT was found to lack a 3 fi5 exonuclease (proofreading) activity (data not shown), thereby permitting direct kinetic analysis of primer-extension. Previous data show that BLV RT, like HIV-1 RT, prefers Mg 2+ over Mn 2+ [12]. Therefore, all assays carried out with these RTs were performed in the presence of Mg 2+ ions. For MLV RT, we have evidence that overall extension of primers by this RT in the presence of Mn 2+ is far better than with Mg 2+, whereas the fidelity of DNA synthesis by MLV RT (both misinsertion and mispair extension; see below) is similar with Mg 2+ or Mn 2+ (unpublished data). DNA synthesis under processive and nonprocessive conditions The processivity of a DNA or RNA polymerase is directly proportional to the length of the nascent polymeric products formed before the enzyme molecules dissociate from these product molecules and rebind the same or other templateprimer molecules [17,22]. The extent of product elongation in one cycle of synthesis (before the polymerase disassociates from the growing strand) may depend on kinetic parameters that affect binding, single nucleotide addition, translocation, pausing, etc. It is apparent that retroviral RTs are far from performing totally processive events (where the entire template molecule is copied as a consequence of a single binding event of the enzyme) [17]. Therefore, we have tested the processivity of the BLV RT in comparison with the two well-studied RTs of HIV-1 and MLV. In the primer-extension assay, described in Fig. 1, we used the heteropolymeric single-stranded /X174am3 DNA

5 Ó FEBS 2002 Processivity and fidelity of BLV RT (Eur. J. Biochem. 269) 863 Fig. 3. The pattern of mispair extension displayed by the purified RTs of BLV, HIV-1 and MLV. The 32 P-5 -end-labelled 16-nucleotide primers were hybridized to the 50-nucleotide template, producing duplexes with 3 -terminal preformed mismatches, where N at the 3 end of each represents the incorrect nucleotide (A, C or G) or the correct on (T). The primers were extended with equal DNA polymerase activities of BLV RT, MLV RT, or HIV-1 RT(as described in the text and in Materials and methods)in the presence of either 1 mm datp (when the mispaired template-primers were elongated) or 1 lm datp (in the case where the AÆT paired substrate was extended). as the template, which is annealed to a synthetic 5 endlabelled primer. The extension of the primer by the RTs was carried out in the absence or presence of a DNA trap, added to the reaction mixture after the RT is given the opportunity to bind the template-primer and before polymerization starts (see Materials and methods). As the trap is added in a vast excess, only prebound RT molecules are allowed to extend the labelled primer. This restricts the extension reaction to only one round of synthesis, hence once RT falls off, it binds the trap and is not capable of performing further rounds of extending the labelled primer. As expected, all three RTs produce longer DNA products when multiple rounds of synthesis are allowed. All RTs used have been calibrated to have the same DDDP activity using activated DNA as the substrate (see Materials and methods). Yet, the extent of elongation obtained with BLV RT with no trap present is substantially lower than that with HIV-1 RT and MLV RT. Most products generated by BLV RT are up to 150 nucleonucleotides in length, whereas for the other two RTs the majority of the products are substantially longer than 200 nucleotides. The primer-extension labelled products were quantified and the extent of elongation was calculated by two methods. In the first, we calculated the amount of product as a percentage of the total radioactivity detected (Table1).BothHIV-1RTandMLVRTshow,withno trap present, overall extensions of 73 76% which is significantly higher than that of BLV RT (61%). The majority of the products of the former two RTs (28 35%) are longer than 200 nt, whereas only 6% of the products synthesized by BLV RT are longer than 200 nt. These differences are more remarkable after quantifying the products generated by introducing a correction of the lengths of the polynucleotides synthesized (Table 2). In this method the average lengths of products was taken into account in the calculation, as by being 5 end-labelled all oligonucleotides have the same level of label per molecule, irrespective of their lengths. This method corrects for the actual amount of dntps incorporated per given product. The figures calculated by this second method show also that the overall extension of BLV RT is significantly lower than the extension calculated for the other RT studied (68% for the BLV RT and 88% for HIV-1 and MLV RTs). As expected, when a DNA trap is present and only one round of DNA synthesis is permitted, all RTs synthesize less, as well as shorter, product when compared with multiple-round synthesis (Fig. 1). The analysis of the processivity of DNA synthesis in the presence of a DNA trap suggests that BLV RT has a processivity that is substantially different from that of HIV-1 and MLV RTs. It is apparent that BLV RT produces very short products, most of them < 30 nucleotides in length. In comparison, HIV-1 RT synthesizes products that are not substantially different in their length from those generated when multiple rounds of synthesis were allowed. MLV RT synthesizes, in the presence of the trap DNA, products that are shorter than those produced without a trap (but longer than those generated by BLV RT). The quantitative analysis of the relative processivity depends on the method of calculation. When the overall extensions were calculated by the first method outlined above (Table 1) MLV RT shows a superb processivity of almost 100%, whereas BLV RT has substantially lower processivity (54%) which is somewhat

6 864 O. Avidan et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Table 4. The kinetics of the extension of 3 end matched or preformed mismatched primer termini by BLV RT. The 32 P-5 -end-labelled 16-nucleotide primers were hybridized to a 50-nucleotide template derived from the sequence of nucleotides of /X174am3 DNA, producing duplexes with a 3 -terminal paired (AÆT) or mismatched (AÆC, AÆG or AÆA) primers (Fig. 3). Each template-primer was incubated with BLV RT in the presence of increasing concentrations of datp. The products were analysed as described in the text. The apparent K m and V max values were the means calculated from at least two independent experiments and the variations were usually < 15%. The relative frequency F ext valuesaretheratiooftherateconstants (V max /K m ) for the mispair divided by the ratio of the corresponding constants for the paired AÆT terminus. Pair or mispairs terminus K m (lm) V max (%Æmin )1 ) F ext AÆT AÆC /5,100 AÆG /3,400 AÆA /4,800 higher than that of HIV-1 RT (46%). However, by calculating the level of extension after correcting for the lengths of the products synthesized, the data obtained is substantially different (Table 2). HIV-1 and MLV RTs exhibit relative processivity values, which are practically identical ( 64%) whereas BLV RT shows a much lower processivity of 24%. The fidelity of DNA synthesis All our previous studies with a variety of retroviral RTs have shown that the parameters for fidelity of DNA synthesis in vitro (i.e. 3 end misinsertion and the extension of the performed 3 end mispaired primers) depend primarily on the sequences of nucleic acids copied, rather than whether DNA or RNA templates were copied [16,18,20,23]. Subsequently, in the present study we have analysed DNA templates as representing both DNA and RNA substrates. The formation of 3 mispaired DNA. To study the fidelity of misinsertion, we used an assay system that measures the standing-start reaction of 3 end misinsertion. This is achieved by following the misincorporation of incorrect dntps opposite the template A nucleotide, which corresponds to position 23 in the 50-nucleotide template used, in comparison with the incorporation of dttp (see Fig. 2 and Materials and methods). The elongation of the 32 P-5 -endlabelled 15-nucleotide primer was performed with either 1 lm of the correct dntp (dttp) or 1 mm of each of the incorrect dntps. Fig. 2 shows the gel analysis of the elongation products with the correct or incorrect dntps. It is apparent that the general pattern of primer extension obtained with BLV RT is quite similar to this with HIV-1 RT. There is an elongation of one nucleotide in the presence of 1 lm dttp with no significant further extension. The highest extent of misincorporation is observed with dctp, forming CÆA mispairs, which are elongated further creating, in the case of BLV RT, CÆT mispairs followed by the correct pairs CÆG (18 nt). In comparison, HIV-1 RT is capable of elongating further the 18-nucleotide primers to 19 nucleotides (with a CÆT mispair at the 3 end). With both BLV and HIV-1 RTs, the extent of mispair formation with dgtp and datp (forming GÆA and AÆA mispairs, respectively) is lower than with dctp. MLV RT shows, on the other hand, a substantially lower level of misincorporation relative to the other two RTs studied. The only significant misincorporation by MLV RT is apparent with dctp, forming CÆA mispairs, with no significant further elongation of the 16-mer products with this mispair at its 3 end. To quantify the capacity of BLV RT to form 3 end mispairs, four separate sets of primer-extension reactions were carried out and analysed. In each case, we used increasing concentrations of a single dntp, thereby determining the standing-start rate of synthesis of the correct pair vs. the three possible mispairs. We used a range of dntp concentrations always below 1 mm (to obey steady-state kinetic conditions) and calculated the radioactivity in gel bands relative to the total amounts of primer present (both the unextended and the extended ones). The rates of misincorporation (V ¼ percentage of primers elongated per minute) were calculated as a function of dntp concentrations, as described in Materials and methods. The apparent K m and V max values for each dntp were all derived from the double-reciprocal curves of the initial velocities of primer extension vs. the substrate concentrations (data not shown) and are given in Table 3. To calculate the frequencies of misinsertions (F ins values) we used the method used previously [16,18,19]: F ins ¼ ðv max=k m Þ w ðv max =K m Þ R where (w) denotes the incorrect nucleotide (datp, dctp or dgtp) and (R) is dttp. As expected from the pattern of primer extensions (Fig. 2), the highest F ins values calculated for BLV RT is for dctp (1/11 600, see Table 3), whereas, the formation of AÆA mispairs is very rare (F ins 1/ ) and the value calculated for dgtp incorporation is slightly higher (1/62 500). The parallel F ins values calculated by us previously in the same assay system for HIV-1 RT were: 1/3460 1/9000, 1/ / and 1/ / ; and for MLV RT: 1/25 000, < 1/ and < 1/ , all for the formation of AÆC, AÆG, and AÆA mispairs, respectively [16,20]. Extension of preformed 3 end mispaired DNA. Misinsertion by itself is not sufficient to create stable site-specific mutations, unless the terminally mispaired DNA is further extended, leading to the fixation of the mistaken sequence. Therefore, the efficiency of extending 3 preformed mismatched primers is an essential factor in determining the fidelity of DNA synthesis exhibited by different polymerases. We have evaluated the ability of BLV RT to extend preformed 3 end mispaired 16-residue primers (AÆA, AÆC, AÆG) by analysing the extension of these primers during DNA polymerization in the presence of the next complementary datp (as the only dntp present). These standingstart reactions were performed in comparison to HIV-1 RT and MLV RT analysed with the same mispair extension reactions. The gel analysis of the extension products shown in Fig. 3 shows that BLV RT is capable of elongating all

7 Ó FEBS 2002 Processivity and fidelity of BLV RT (Eur. J. Biochem. 269) 865 mispairs to roughly the same extent. In comparison, HIV-1 RT shows a substantial preference in extending the AÆC mispairs over the AÆA andaæg mispairs. MLV RT shows the same preference in extending the mispairs (AÆC > AÆA >AÆG) although the extent of elongating these mispairs is significantly lower than the extensions observed with HIV-1 RT. To study the kinetics we followed primer elongation as a function of increasing concentrations of datp as the only dntp present (Table 4). The ratios of all extended products were calculated relative to the total amount of the primers as a function of datp concentration. The relative extension frequency (F ext ) values are defined as apparent V max /K m values, calculated for the formed mismatches, divided by the corresponding V max /K m values obtained for the correctly paired terminus (AÆT). The results show that the apparent K m values for the extension of all three studied mispairs by BLV RT are similar. As expected, the V max value calculated for the corrected paired terminus is higher than those values determined for the mispaired termini. Also expected is the finding that this RT exhibits K m values for the extension of the AÆA, AÆC andaæg mismatches that are much higher than the comparable value calculated for the correct AÆT pair. As the extension of all three mispairs is about the same (Fig. 3) it is not surprising that the relative extension frequencies calculated for all mispairs are quite similar, ranging from 1/3400 (for the AÆG terminus) to 1/5100 (for the AÆC mispair). On the other hand the F ext values calculated previously in the same assay system were for HIV-1 RT, 1/ , 1/ and 1/ ,000, for the formation of AÆA, AÆC, and AÆG mispairs, respectively [16,20]. DISCUSSION Polymerases are processive, i.e. they can attach to the polymeric substrates and perform polymerization cycles without intervening dissociations [21,24]. A total processivity of synthesis of either DNA or RNA is accomplished when the entire DNA or RNA template is copied as a consequence of only one polymerase-binding event. Previous studies with various RTs have shown that the enzyme is not highly processive while synthesizing DNA [17,18,25,26]. The primer-extension and processivity of DNA synthesis experiments shown in Fig. 1 indicate that these features of BLV RT are significantly different than those of both HIV-1 RT and MLV RT. The data were quantified by two methods (Tables 1 and 2). It is apparent that BLV RT has a processivity substantially lower than that of the two other RTs studied. Even without an excess of unlabelled trap DNA, BLV RT is not capable of synthesizing significant amounts of product DNA longer than 120 nucleotides, with strong pausings between 90 and 120 nucleotides. In comparison, HIV-1 and MLV RTs synthesize a relatively large amount of longer product DNA molecules of nucleotides in length, and the majority of the products are in this length range. This difference between BLV RT and the two other RTs suggests that BLV RT has weaker binding to the DNA substrate than the other RTs studied. It might also be that the premature pausings observed with BLV RT are sequence-dependent. It will be of interest to study other sequences than those used here to identify any unique sequences that cannot be copied easily by BLV RT. The processivity experiment was conducted with a large excess of trap DNA to prevent rebinding of RT molecules to the nascent DNA. The extent of DNA synthesis with BLV RT is low and most products are very short (Fig. 1 and Tables 1 and 2). This shows that these products, generated under single-cycle conditions were synthesized by those BLV RT molecules that were bound to the DNA before the addition of the trapping agent (and were dissociated from the growing chain after incorporating only few nucleotides) suggesting a poor processivity of this RT. The pattern of the processivity seen with HIV-1 RT is entirely different. Despite exhibiting a moderate processivity, the distribution of the elongation products in the presence of the trap DNA is very similar to that seen in its absence (although, as expected, the total amount of product generated with the trap is lower, only 46 64% of those synthesized without the trap). This phenomenon might suggest that those HIV-1 RT molecules that can withstand dissociation are capable of completing the synthesis (or show a high ÔpersistenceÕ of elongation without further dissociation). MLV RT shows a behaviour that is intermediate between the apparent features of BLV RT and HIV-1 RT. The products formed in the presence of the trap DNA are substantially shorter than those synthesized with no trap present (though they are much longer than those synthesized by BLV RT with the trap DNA). The variations observed in the experiment shown in Fig. 1 necessitated the use of the two quantification methods, summarized in Tables 1 and 2. BLV RT shows an overall processivity of DNA synthesis, which is significantly lower than the values calculated for both HIV-1 and MLV RTs (see Table 2). Yet, based on the amount of primers extended in the processivity experiments, BLV RT is capable of extending about the same amount of primers as HIV-1 RT ( 50%), despite the very significant differences in the ÔpersistenceÕ of elongation (see Fig. 1 and Table 1). MLV RT is capable of extending many more primer molecules (showing a value of almost 100% of relative processivity). It is possible that these results may vary slightly depending of the sequence of the DNA copied and the conditions used in the experiments. None of the RTs studied so far have any 3 fi5 proofreading exonuclease activities, thus, making RTs more error prone than other DNA polymerases with this activity [5,16,18,27,28]. Yet, a comparison of the overall fidelity of DNA synthesis exhibited by RTs from different retroviruses reveals significant differences among them. It was reported that the RTs of HIV-1 and HIV-2 are relatively more error prone than other RTs, such as those of avian myeloblastosis virus (AMV) or MLV [19,20,23,29,30], explaining the extensive genetic heterogeneity of both HIV-1 and HIV-2, which affects viral pathogenesis, the rapid emergence of drug-resistant variants and, hence, the progression of AIDS [2,10,11,31]. We have also found that the relatively low fidelity of DNA synthesis exhibited by HIV RTs is shared by the RT of equine infectious anaemia virus (EIAV), which belongs to the lentivirus subfamily of retroviruses [16]. In general, the fidelity of DNA polymerases results from the combination of nucleotide insertion and extension (in addition to the presence or absence of proofreading activities). Base substitution mutations during reverse transcription can arise from the incorporation of a

8 866 O. Avidan et al. (Eur. J. Biochem. 269) Ó FEBS 2002 noncomplementary nucleotide at the 3 end of the nascent DNA strand, followed by an extension of the preformed mispair [32,33]. Therefore, using parameters of both the capacity to misincorporate an incorrect nucleotide and the ability to extend the preformed 3 mispairs, it was suggested that the overall rates of the in vitro error proneness in the RTs studied is as follows: lentiviral RTs > AMV RT > MLV RT [16,19,29,30,34]. A more recent study carried out with the RT of mouse mammary tumour virus (MMTV) has shown some deviation between the efficiency of misincorporating an incorrect nucleotide and the ability to elongate such a mispaired DNA [18]. We have studied the error proneness of BLV RT using the defined template-primers and steady-state kinetics methods used previously in our laboratory to study various RTs [16,18 20,23,29,34]. The misinsertion frequencies observed with BLV RT show that the specificity of mismatch formation is AÆT > AÆC > AÆG >AÆA, compatible with the pattern observed with the other RTs [16,18 20,23,29]. This misinsertion is affected by a major increase in the K m values and a less significant reduction in the V max values. The F ins values are somewhat different than those observed previously with HIV-1, HIV-2 and EIAV RTs. The fidelity of misincorporation of MLV RT is substantially higher than both BLV and lentiviral RTs (Fig. 2) and [19,23]. Therefore, the overall order of error proneness of the retroviral RTs studied, based on the site-specific misincorporation experiment, is lentiviral RTs > BLV RT MMTVRT>AMVRT>MLVRT. As to the capacity of BLV RT to extend preformed mispairs, it is apparent from Fig. 3 and Table 4 that BLV RT extends all mismatches studied (i.e. AÆA, AÆC, and AÆG) to approximately the same extent. The enzyme can extend the mispairs by only one correct nucleotide (A) with no further extension by misincorporating A opposite to G. This is in contrast with the pattern of elongation observed here with HIV-1 and MLV RTs (Fig. 3) and previously by these RTs and the RTs of HIV-2, EIAV, MMTV and AMV [16,18,19,23,29]. With all other RTs the efficiency of preformed mispair extension with the same mispairs was found always to be in the order AÆC > AÆA P AÆG. Moreover, all RTs except for BLV RT were capable of extending the AÆC mispair beyond the addition of only one A. This indicates that, under the assay conditions used, all other RTs can incorporate A opposite to G at position 18. This is true even for MLV RT which has the highest fidelity of all RTs studied. The steady-state kinetics analysis of the mispair extension by BLV RT shows that the V max and the K m values are relatively close for all mismatched substrates (Table 4). Moreover, major discrimination can be attributed to the relatively large K m differences governing the extension of matched vs. mismatched base pairs, with much smaller differences in the V max values. The high frequency of extending the studied mispairs by BLV RT, relative to our previous results, puts this RT on the top of the list with lentiviral RTs in the in vitro error proneness of RTs in the following order: BLV RT lentiviral RTs > AMV RT > MMTV RT > MLV RT. However, the fact that the mispaired DNA can be extended by BLV RT by only one nucleotide beyond the mismatched 3 end may explain, at least in part, why virions of BLV grown in culture show a relatively low mutation rate per replication cycle [35]. If extension of mispairs stops after the addition of one nucleotide also in vivo, there will not be synthesis of fulllength mutated DNA and the overall fidelity will be relatively high. This may also explain the observed in vitro reduced processivity of BLV RT. 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