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 l eukemia virus
(BLV) [Perach, M. & Hizi, A. (1999) Virology 259, 176–189].
In the p resent study, we have studied in vitro two features of
the DNA polymerase activity of BLV RT, t he processivity of
DNA synthesis and the fidelity of DNA synthesis. These
properties were c ompared with t hose o f 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 m ispairs. 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. T hese fidelity properties are disc ussed in the context of
the v arious 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 charac-
terized by an initial persistent lymphocytosis, which is
followed by the occurrence of clonal lymphoid B-cell

the rapid emergence of drug-resistant HIV RT variants, the
development o f novel potent a nd specific inhibitors of HIV
RTs is still a principal objective in the chemotherapy of
AIDS [2,10,11]. Targeted drug d esigns 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 B LV
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 b y the pol
reading frame [12]. BLV RT was purified and studied
biochemically: it exhibits all activities typical of RTs, i.e.
both R NA- 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 s hows 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
Correspondence to Amnon Hizi, Department of Cell Biology and
Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv
69978, Israel. Fax: +972 3 6407432, Tel.: + 972 3 640 9974,
E-mail:
Abbreviations: BLV, bovine leukemia virus; HTLV, human T-cell
leukaemia virus; HIV, human immunodeficiency virus; MLV, murine

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 f act t hat a ll 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 d NTP into activated DNA (that served as t he
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 0.1–0.5 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 posi-
tions 588–602 of the /X174am3 DNA. The synthetic
template-primers used for the experiments of m isinsertion
and preformed mispair extension are shown in Figs 2 and 3.
For analysis of site-specific nucleotide misinsertion, a

MgCl
2
(for BLV and HIV-1 RTs) or 1 m
M
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
0.3–2 pmol RT per reaction (depending on activity) and
Fig. 1. DNA primer-extension and processivity of DNA synthesis
exhibited by BLV, HIV-1 and MLV RTs. All r eactions we re p erformed
with the 15-nucleotide synthetic 5¢ end-labelled oligonucleotide prime r
and a twofold excess of the template single-stranded circular
/X174am3 p hage DN A. The sequence of the primer and the experi-
mental 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 dephosp horylated double -strand ed
/X174am3 D NA frag ments (Promega) labelled with [c-
32
P]ATP at the
5¢ ends by polynucleotide k inase.
860 O. Avidan et al. (Eur. J. Biochem. 269) Ó FEBS 2002

presence of increasing concentrations of eithe r 0–1 l
M
of
the correct dNTP (dTTP) or 0–1 m
M
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 con-
centrations of dATP as th e only dNTP present (0–1 m
M
range for the mispaired AÆA, AÆCorAÆG termini or a
0–1 l
M
range for the AÆT correct terminus) [16,18].
Reactions for all kinetic analyses contained 14 m
M
Tris/
HClpH8.0,4m
M
dithiothreitol, 4 m
M
MgCl
2
and
24 lgÆmL

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 f or the mean lengths of the D NA
primers extende d by the three RTs und er the assay conditions used. Th e c orrectio n fo r th e a ctual a mount of dNTP incorporation fo r 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 16–50 nucleotide r ange, 4 2 nucleotides for the 51–100 n ucleotide r ange, 92 nucleotides for
the 101–200 nucleotide range and 342 nucleotides for the 200–700 nucleotide range). After introducing these factors, all values are expressed (as in
Table 1 ) as p ercentages of the total amounts of all primers exten ded in each length class. The values shown are the means calculated from the sam e
two independent experiments as in T able 1.
Product length
(nucleotides)
BLV RT HIV-1 RT MLV RT
Without trap With trap Without trap With trap Without trap With trap
16–50 3.3 12.4 2.4 2.1 1.4 23.8
51–100 9.3 1.3 4.7 4.0 2.5 30.2
101–200 33.2 1.8 14.5 6.3 11.7 0.7
200–700 22.5 0.8 66.2 43.4 73.0 2.2
Overall extension 68.3 16.3 87.8 55.6 88.6 56.9
Relative processivity 23.9 63.5 64.2
Ó FEBS 2002 Processivity and fidelity of BLV RT (Eur. J. Biochem. 269) 861
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

or Mn
2+
(unpublished data).
DNA synthesis under processive and nonprocessive
conditions
The processivity of a DNA or RNA polymerase is d irectly
proportional to the length of the n ascent polymeric products
formed before the enzyme molecules dissociate f rom these
product molecules and rebind the same or other template-
primer 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
Fig. 2. The pattern o f DNA mispair formation by BLV, HIV-1 and MLV RTs. The s ynthetic 50-nu cleotide t emplate w as ann ealed to the
32
P-5¢-end-
labelled primer. The primer was extended with equal DNA polymerase activities of either BL V RT, HIV-1 RT o r M LV RT in th e presence of 1 m
M
of a s ingle incorrect dNTP (i.e. C , G , o r A ) o r 1 l
M
of the co rrect dNT P (dT TP) as described in Materials an d metho ds. Th e leve l of m isinsertion i s
apparent from the elongation of the primer in the presence of the incorrect dNTP relative t o that in the presence of dTTP.

) F
ins
AÆT 0.004 25 1
AÆC 28 15.1 1/11 600
AÆG 45 4.5 1/62 500
AÆA 55 1.1 1/300 000
862 O. Avidan et al. (Eur. J. Biochem. 269) Ó FEBS 2002
as the template, which is annealed to a synthetic 5¢ end-
labelled 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 a re 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 substan-
tially lower than that w ith HIV-1 RT and MLV RT. Most
products generated by BLV RT are up to  150 nucleo-
nucleotides 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

HIV-1 R T synthesizes products that are not substantially
different in their length from those generated when multiple
rounds of synthesis were allowed. MLV RT s ynthesizes, 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
Fig. 3. The pattern of mispair extension displayed by the purified RTs of BLV, HIV-1 and MLV. The
32
P-5¢-end-labe lled 16-nucleo tide prim ers were
hybridized to the 50-nucleotide template, producing duplexes with 3¢-terminal preformed mismatches, where N at the 3¢ endofeachrepresentsthe
incorrect nucleotide (A, C or G) or the correct on (T). The primers were extended with equal DNA polymerase activities of BLV RT, M LV RT, or
HIV-1RT(asdescribedinthetextandinMaterialsandmethods)inthepresenceofeither1 m
M
dATP (when the mispaired tem plate-primers were
elongated) or 1 l
M
dATP (in the c ase where the AÆT paired substrate was extended).
Ó FEBS 2002 Processivity and fidelity of BLV RT (Eur. J. Biochem. 269) 863
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%.

of 1 l
M
dTTP with no significant further extension. The
highest extent o f 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 nucleo-
tides (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 GÆAandAÆA m ispairs, 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 s ignificant misincor-
poration 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 m
M
(to
obey steady-state kinetic conditions) and calculated the
radioactivity in g el bands relative to the total amounts of
primer present (both the unextended and the extended
ones). The rates of misincorporation (V ¼ percentage of

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/
300 000) a nd 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, 1/32 250–1/41 500 and 1/52 200–1/
75 000; and for MLV RT: 1/25 000, < 1/300 000 and
< 1 /300 000, all for the formation of AÆC, AÆG, and AÆA
mispairs, respectively [16,20].
Extension of preformed 3¢ end mispaired DNA. Misin-
sertion by itself is not su fficient 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 mis-
matched primers is an essential factor in determining the
fidelity of DNA synthesis exhibited by different polyme-
rases. 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 comple-
mentary dATP (as the only dNTP present). These standing-

m
(l
M
)
V
max
(%Æmin
)1
) F
ext
AÆT 0.037 29.9 1
AÆC 86 13.6 1/5,100
AÆG 42 10.2 1/3,400
AÆA 68 11.4 1/4,800
864 O. Avidan et al. (Eur. J. Biochem. 269) Ó FEBS 2002
mispairs to roughly the same extent. In comparison, HIV-1
RT shows a substantial preference in extending the AÆC
mispairs over the AÆAandAÆG 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 f ollowed 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 co ncentration. The relative extension
frequency (F
ext
) values are defined as apparent V

calculated previously in the same assay system were for
HIV-1 RT, 1/17 500–52 000, 1/3900–9200 and 1/35 000–
45,000, 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 proces-
sivity 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. Previ-
ous studies with various RTs have shown that the enzyme
is not highly pr ocessive while synthesizing DNA
[17,18,25,26].
The primer-extension and processivity of DNA synthe-
sis experiments shown in Fig. 1 indicate that these features
of BLV RT are significantly d ifferent th an 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 200–700 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

sized by BLV RT with the trap DNA). The variations
observed in t he experiment shown in F ig. 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 t he
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 t he 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 p rone 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

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 o f 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 ind icates that, und er 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

sivity of DNA synthesis together with a low fidelity, making
BLV RT unique among retroviral RTs. It had been
suggested a lready for mutants of HIV-1 RT that there is
an inverse correlation between the fidelity a nd processivity
of DNA synthesis (i.e. that the enhanced fidelity of
misinsertion and mispair extension is associated with a
reduced processivity [36]). The results with BLV RT in t he
present study as well as with other mutants of HIV-1 RT
[17] do not support this theory.
ACKNOWLEDGEMENT
We thank H. Be rman for typing the manuscript.
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