(Received for publication, July 28, 1995; and in revised form, November 15, 1995)
From the
We have previously described the in vitro and in
vivo characterization of a panel of mutations affecting the RNase
H domain of Moloney murine leukemia virus reverse transcriptase (Blain,
S. W., and Goff, S. P.(1993) J. Biol. Chem. 268,
23585-23592; Blain, S. W., and Goff, S. P. (1995) J. Virol. 69, 4440-4452). We were intrigued by a discrepancy between in vitro and in vivo RNase H results for two of the
mutants. While C and
5E appeared to have nearly wild-type
RNase H activity in vitro, they were unable to degrade their
genomic RNA in vivo and thus were effectively RNase H null
mutants in this context. In this present report, we describe the
differential effects of these mutations on RNase H activity in
vitro in the presence of Mg
versus Mn
: mutants
C and
5E were active in
the presence of the less biologically relevant Mn
and
not in the presence of Mg
. We also describe three
mutants with only partial activity in Mg
. The
presence of the different cations can also affect DNA polymerization
and processivity of an RNase H-deficient mutant.
Reverse transcriptase (RT) ()is responsible for
converting the single-stranded RNA genome of a retrovirus into
double-stranded DNA(1, 2) . RT accomplishes this
process using two activities: a DNA polymerase activity that is able to
synthesize DNA from both RNA and DNA templates and a ribonuclease H
activity (RNase H) that is able to degrade RNA present in RNA-DNA
hybrid form (for reviews, see (3) and (4) ). RNases H
release short oligonucleotide products with 5`-PO
and 3`-OH
groups and show a divalent cation requirement for catalysis (for
reviews, see (5, 6, 7, 8) ). The two
activities of MMLV RT reside in separable domains: the N-terminal
two-thirds of the enzyme contains the DNA polymerase domain, while the
RNase H domain is in the C-terminal one-third(9) . The RNase H
domain of MMLV RT is highly homologous to other RNases H, including Escherichia coli(10, 11) and HIV-1 (12, 13, 14) RTs. Thus, although the
structure of MMLV RNase H has not been determined, it is likely that
the RNase H domain of this enzyme will be similar to those of other
RNases H(15) .
RNase H activity has been implicated in several steps in reverse transcription: the enzyme is essential for the viral life cycle, and mutant viruses that lack RNase H activity are noninfectious(16) . RT initiates DNA synthesis from a tRNA primer bound to a region near the 5`-end of the genomic RNA termed the primer-binding site (PBS). Elongation of this tRNA to the 5`-end of the genome results in formation of(-)-strand strong stop DNA, the first DNA intermediate to appear during reverse transcription(17) . The newly synthesized (-)-strand strong stop DNA forms an RNA-DNA hybrid with the (+)-strand genomic RNA, which is then degraded to permit translocation to the 3`-end of the RNA. Analysis of abortive replication products produced by virions that lack RNase H has shown that the(-)-strand strong stop DNA remains in hybrid form with the genomic RNA, accounting for the observed reduction in translocation and elongation for these mutants (16) . In addition to degradation of the genomic RNA, RNase H performs several specialized functions at later times, including the creation and removal of the polypurine tract primer and the removal of the (-)-strand tRNA primer(18) .
We
previously described the in vitro characterization of a panel
of mutations made in the RNase H domain of MMLV RT(19) . The
design of these mutant enzymes was based on sequence alignments and the
crystal structures of E. coli and HIV-1 RNases H and the
predicted structure of the MMLV RNase H
domain(10, 11, 12, 13, 14, 15) .
Most of the RNase H mutants analyzed retained full or at least partial
RNase H activity in vitro as assayed by in situ gel
techniques. We additionally characterized these mutants in vivo in the context of the full-length retroviral
provirus(20) . Two mutants, 5E and
C RTs, which
appeared to retain significant RNase H activity in vitro (50
and 100% activity, respectively), were completely noninfectious as
virus in vivo. These mutant viruses were further analyzed in
the endogenous assay, in which reverse transcription is carried out in vitro in purified virions in the presence of radiolabeled
dNTPs, and various radiolabeled DNA products can be detected. This
analysis demonstrated that
5E and
C left
their(-)-strand strong stop DNA in hybrid form with the genomic
RNA and thus were effectively RNase H null mutants in the context of
the endogenous reaction.
We were intrigued by the discrepancy
between the presence of RNase H activity in the in situ gel
assay and the absence of RNase H activity in the endogenous assay. We
hypothesized that the differential activity detected in these two
assays might result from the following: 1) a difference between the
recombinant and virion-associated RTs analyzed, 2) a difference between
the substrates degraded in these two assays (random heteropolymeric
radiolabeled RNA-DNA hybrid in the in situ gel assay versus genomic RNA hybridized to the newly
synthesized(-)-strand DNA during the endogenous reaction), 3) the
presence of other viral proteins that might affect RT activity during
reverse transcription, or 4) a difference between the different cations
used in these two assays (Mnversus Mg
).
Tests revealed that the basis for the
difference in the activity of these mutant enzymes in vitro and in vivo resulted from differential RNase H activity
when assayed in the presence of Mg or
Mn
. In particular, we describe two mutants that
appear to be active only in the presence of the less biologically
relevant Mn
. The presence of the different cations
also appears to affect DNA polymerization and processivity.
pRT30-2 (the wild-type enzyme), D524N,
5E, and
C were analyzed as E. coli-expressed
proteins, purified as described previously (19, 21) .
Mutants R657S, Y598V, S526A, Y586F,
5E, and H7 were analyzed as
virion-associated RTs. To prepare the mutant RTs, these mutations were
moved into the context of the full-length provirus pNCA (16, 20, 22) . Stable producer lines were
generated for these mutant proviruses by the calcium phosphate-mediated
cotransformation method as described
previously(16, 20) . Maintenance of cells in
Dulbecco's modified Eagle's medium supplemented with 10%
calf serum was as described previously(16, 20) . To
prepare virions, producer cells were fed Dulbecco's modified
Eagle's medium supplemented with 10% NuSerum (Collaborative
Biomedical Products, Bedford, MA) for 12 h prior to harvest. The
virions were pelleted for 3 h at 25,000 rpm, resuspended, layered over
a 25/45% sucrose step gradient, and sedimented to the interface. The
viral band was collected and repelleted for 2 h at 25,000 rpm following
dilution in TNE buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA). Viral RTs were assayed without further
purification; virions were lysed in Nonidet P-40 present in the
reaction mixture as described below. The enzyme diluent was TNE buffer
for all of the dilutions.
Both bacterially expressed and
virion-associated RTs were quantitated to determine the
units/milliliter of DNA polymerase activity by oligo(dT)poly(rA)
primer template assays in Mn
(23) , and
enzymes to be compared were normalized by this assay. Since all of the
mutant RTs had previously been shown to have wild-type DNA polymerase
activity in this assay(19) , normalization by the measure of RT
activity was appropriate. Protein levels were also compared by Western
blot analysis with anti-30-2, a polyclonal rabbit anti-RT
antibody(19, 20) . In all cases, the protein levels
and thus the polymerase-specific activity of the mutants were the same
as those of wild-type RT.
Concentrated virions were
incubated for 30 min on ice in the presence of 0.3% Nonidet P-40, 50
mM NaCl, and 1 mM dithiothreitol in a 15-µl
volume. Reaction mixtures were then diluted to a total of 85 µl
with buffer lacking Nonidet P-40 to give final concentrations of 0.05%
Nonidet P-40, 50 mM NaCl, 1 mM dithiothreitol, and
either 6 mM MgCl with 50 mM Tris-HCl, pH
8.3, or 2 mM MnCl
with 50 mM Tris-HCl, pH
7.5. The substrate was added to a final concentration of
1
pM. The reaction was allowed to proceed at 37 °C for 1 h,
followed by SDS and proteinase K treatment for 30 min at 37 °C; and
reaction products were extracted with phenol and ethanol-precipitated
in the presence of 1 µg of tRNA. The products were analyzed by
electrophoresis on 8% nondenaturing polyacrylamide gels. Purified
recombinant enzymes were incubated in a 50-µl reaction containing
0.05% Nonidet P-40, 50 mM NaCl, 1 mM dithiothreitol,
3 pM substrate, and either 6 mM MgCl
with
50 mM Tris-HCl, pH 8.3, or 2 mM MnCl
with
50 mM Tris-HCl, pH 7.5, for 1 h at 37 °C. Following
incubation, the reactions were treated as described above for the
virion reactions.
Figure 1:
RNase H activity of recombinant
proteins in the defined substrate assay. Purified recombinant enzymes
were incubated as described under ``Experimental Procedures''
in either 6 mM MgCl or 2 mM MnCl
. Lane 1, TNE buffer control; lane
2, 1 unit of E. coli RNase H (RH) in
Mg
; lanes 3-5, wild-type (WT)
RT in Mg
; lanes 6-8, wild-type RT in
Mn
; lanes 9-11,
5E in
Mn
; lanes 12-14,
C in
Mn
; lane 15, D524N in Mg
; lanes 16-18, D524N in Mn
; lane
19, untreated substrate; lane 20, denatured substrate.
The RT preparations were normalized by DNA polymerase activity in
oligo(dT)
poly(rA) primer template assays in Mn
as well as by Western blot analysis to compare protein levels.
Thus, the 1:32 dilution in lane 5 corresponds to the same
amount of protein in lanes 7, 10, 13, and 18. Enzymes were diluted in TNE buffer. ds,
double-stranded; ss, single-stranded. Sizes of marker DNAs are
indicated to the right.
Purified recombinant RTs were added to this substrate
and incubated at 37 °C for 1 h (Fig. 1). Purified E.
coli RNase H was able to degrade this substrate efficiently,
producing the single-stranded slower species, which corresponded to
full RNA degradation and/or release (Fig. 1, lane 2).
Purified wild-type RT (pRT30-2) was able to degrade the substrate
efficiently in both Mn and Mg
(Fig. 1, lanes 3-8; and Fig. 2, lanes 3-6). However, wild-type RT was
8-16-fold more active in the presence of Mn
compared with Mg
(Fig. 1, compare lanes 6 and 7 in Mn
to lanes 3 and 4 in Mg
).
Figure 2:
RNase H activity of recombinant proteins
in the defined substrate assay in Mg. Purified
recombinant enzymes were treated as described under ``Experimental
Procedures.'' All of the enzymes in this panel were analyzed in
the presence of Mg
. Lane 1, TNE buffer
negative control; lane 2, 1 unit of E. coli RNase H (RH); lanes 3-6, wild-type (WT) RT; lanes 7-10,
5E; lanes 11-14,
C; lane 15, D524N; lane 16, untreated substrate; lane 17, denatured substrate; lane 18, marker DNAs.
The wild-type and mutant preparations can be compared with the
dilutions used in Fig. 1. ds, double-stranded; ss, single-stranded.
Virion-associated
wild-type RT was compared with the recombinant enzyme. Wild-type RT
from virions was able to efficiently degrade the substrate in both
Mn and Mg
, and a 16-fold increase
in activity in the presence of Mn
was observed (Fig. 3, lanes 2-4; and Fig. 4, lanes
3-6), as with the recombinant enzyme. This result suggests
that MMLV RNase H was indeed significantly more active in the presence
of Mn
compared with Mg
and that
similar results could be obtained with the recombinant and
virion-associated enzymes analyzed in this assay.
Figure 3:
RNase H activity of
detergent-permeabilized virions in the defined substrate assay in
Mn. Purified virions were permeabilized and assayed
as described under ``Experimental Procedures.'' All of the
mutants in this panel were analyzed in the presence of
Mn
. Lane 1, H7; lanes 2-4,
wild-type (WT) RT; lanes 5-7, S526A; lanes
8-10, R657S; lanes 11-13, Y598V; lanes
14-16,
5E; lane 17, untreated substrate; lane 18, denatured substrate. The dilutions listed for the
wild type and mutants were normalized with respect to DNA polymerase
activity in oligo(dT)
poly(rA) primer template assays and by
Western blot analysis. It should be noted that the wild-type and mutant
preparations are not the same as the recombinant preparations in Fig. 1and Fig. 2.
Figure 4:
RNase H activity of
detergent-permeabilized virions in the defined substrate assay in
Mg. Purified virions were permeabilized and assayed
as described under ``Experimental Procedures.'' All of the
mutants in this panel were analyzed in the presence of
Mg
. Lane 1, TNE buffer control; lane
2, 1 unit of E. coli RNase H (RH); lanes
3-6, wild-type (WT) RT; lanes 7-10,
S526A; lanes 11-14, R657S; lanes 15-18,
Y598V. The wild-type and mutant preparations can be compared with those
used in Fig. 3. ds, double-stranded; ss,
single-stranded.
Since these enzymes were purified from E. coli,
we could not rule out the possibility that the residual activity of
5E and
C at higher enzyme concentrations was due to
contaminating E. coli RNase H activities, although by in
situ gel techniques, these enzyme preparations did not contain any
other detectable RNase H activities(19, 21) . To
address this concern,
5E was analyzed as a virion-associated
enzyme.
5E exhibited wild-type activity in Mn
,
but little or no activity in Mg
(Fig. 3, lanes 14-16; and Fig. 5, lanes
1-4). As a virion-associated enzyme, the slower migrating
smear was not detected, even at high enzyme concentrations (Fig. 5, lanes 1-4). Thus,
5E behaved
effectively as an RNase H null mutant in Mg
. This
result might account for the discrepancy in the activities seen in the
endogenous reaction and the in situ gel assay for mutants
5E and
C (Table 1). While
5E and
C do retain
nearly wild-type RNA-DNA nuclease activity in Mn
,
they are essentially inactive in the presence of Mg
.
Figure 5:
RNase H activity of
detergent-permeabilized virions in the defined substrate assay.
Purified virions were permeabilized and assayed as described under
``Experimental Procedures.'' Lanes 1-4,
5E in Mg
; lanes 5-7, Y586F in
Mg
; lanes 8 and 9, wild-type (WT) RT in Mn
; lanes 10 and 11, Y586F in Mn
; lane 12, H7 in
Mn
; lane 14, untreated substrate; lane
15, denatured substrate. The dilutions listed for the wild type
and mutants can be compared with the dilutions used in Fig. 3and Fig. 4. ds, double-stranded; ss, single-stranded.
Several other mutants were analyzed to determine whether the
differential ability to degrade RNA depending on the divalent cation
used was a common feature of many RNase H mutants. Mutant D524N,
containing a change in a residue implicated in catalytic activity, was
unable to degrade the defined substrate efficiently in either
Mg or Mn
(Fig. 1, lanes
15-18; and Fig. 2, lane 15). Even when
almost 16-fold more D524N enzyme was analyzed, no activity on the
defined substrate was observed in the presence of Mn
(Fig. 1, compare lane 16 for D524N with lane
7 for the wild type). Likewise, mutant Y586F, assayed as a
virion-associated enzyme, was almost completely inactive in
Mg
and Mn
(Fig. 5, lanes
5-7, 10, and 11). At a 1:128 dilution,
where the wild type was able to degrade the substrate efficiently,
Y586F was completely inactive (Fig. 5, lane 11); at a
1:32 dilution, the mutant was slightly active in Mn
(lane 10). As mutant Y586F has a tendency to revert to a
more active RNase H form during cell culture(24) , this slight
activity may be the result of trace amounts of this reverted enzyme
contaminating this viral preparation. It is clear that D524N and Y586F
were essentially inactive RNases H in the presence of both cations.
These results suggest that the differential activity of
5E and
C in the presence of Mg
is a defect specific to
these two, and not a phenotype intrinsic to all RNase H mutants.
Mutants S526A, R657S, and Y598V were previously shown to be active
in the in situ assay and are able to degrade their genomic RNA
as well as the wild type in the endogenous reaction (Table 1)(19, 20) . When these mutants were
analyzed as virion-associated RTs on the defined substrate, all had
wild-type activity in Mn (Fig. 3, lanes
5-13). In the presence of Mg
, however,
only partial activity was observed, and a novel intermediate
degradation species was detected (Fig. 4, lanes
7-18). The double-stranded substrate migrating at 240 base
pairs disappeared when the mutants were assayed at the 1:8 enzyme
concentration, but instead of releasing the single-stranded product
corresponding to complete RNA digestion, these mutants appeared to
chase much of the hybrid to a form that migrated only slightly more
slowly (Fig. 4, compare lane 4 for the wild type to lanes 9, 13, and 17 for the mutants). Mutant
5E left the substrate completely double-stranded at this 1:8
concentration and at higher concentrations, ruling out the possibility
that this was due to a contaminating background activity (Fig. 5, lanes 1-4). We suspect that this form
corresponds to a discrete DNA-RNA species, but cannot estimate how much
RNA remains associated with the DNA. S526A did degrade the substrate to
the fully single-stranded form when increasing amounts of enzyme were
assayed (Fig. 4, lane 7). The intermediate form can
also be seen for the wild type at very low enzyme concentrations (Fig. 4, lane 6). These results suggest that while
these mutants are active RNases H, they may be less effective than
wild-type RT at removing all the RNA from these substrates in
Mg
.
As described previously, mutant 5E synthesizes full-length
(-)-strand strong stop DNA, but this species remains in hybrid
form with the genomic RNA (Table 1)(20) . Treatment of
this species with RNase A under high salt concentrations produced a
perfect duplex, termed the FF (fast form) DNA, which migrated at a
characteristic position on a nondenaturing polyacrylamide gel (Fig. 6, lane 7). When this FF species was denatured, a
163-nucleotide form was detected, corresponding to the 145-nucleotide
(-)-strand strong stop DNA plus 18 nucleotides of the tRNA primer
that were resistant to RNase A treatment in high salt due to base
pairing with the genomic PBS sequences (Fig. 6, lane
9). A small amount of a smaller species was also visible.
Treatment with RNase A in low salt degraded all of the genomic RNA,
allowing detection of the 145-nucleotide(-)-strand strong stop
DNA species free of any RNA (Fig. 6, lane 8) as well as
a small amount of a shorter product. These experiments show that the
assays are functioning as expected and provide marker DNAs for the
various products.
Figure 6:
RNase A treatment in high and low salt of
endogenous reaction products synthesized in Mn. The
endogenous reaction was performed as described under
``Experimental Procedures.'' Lanes 1-6, H7; lanes 7-9,
5E. Lanes 1-3 and 7-9 were performed in the presence of 6 mM MgCl
. Lanes 4-6 were performed in the
presence of 2 mM MnCl
. The endogenous reaction
products were treated with RNase A in high (H; lanes
1, 3, 4, 6, 7, and 9)
or low (L; lanes 2, 5, and 8) salt
buffer prior to electrophoresis on 8% nondenaturing polyacrylamide
gels. Lanes 2, 3, 5, 6, 8,
and 9 were denatured prior to loading by suspension in
dye-containing formamide. Lanes 1, 4, and 7 were analyzed without denaturation. The VFF and FF forms are
labeled. nt, nucleotide.
RNase H null mutants do not make full-length
strong stop DNA because of defects in the processivity of
RT(20, 21) . Mutant H7 has a frameshift between the
DNA polymerase and RNase H domains and is thus effectively a single
domain DNA polymerase, i.e. an RNase H null mutant (9, 16) . This mutant produced predominantly a
truncated form of (-)-strand strong stop DNA, termed VFF for
``very fast form,'' in the endogenous reaction in
Mg (Fig. 6)(20, 21, 25) . Little or
no completed full-length DNA was detected after RNase A treatment in
high salt (Fig. 6, lane 1). When the VFF species was
denatured, very little of the 163-nucleotide product was detected,
confirming the tendency of this mutant to pause prematurely (Fig. 6, lane 3). However, assays using the other
cation showed that mutant H7 was indeed able to synthesize full-length
(-)-strand strong stop DNA in Mn
. Treatment of
the Mn
reaction products with RNase A in high salt
produced the FF species (Fig. 6, lane 4); after
denaturation, the 163-nucleotide form was detected (lane 6).
Treatment with RNase A in low salt produced the 145-nucleotide form (Fig. 6, lane 5). Thus, the DNA polymerase activity of
the mutant was enhanced in Mn
such that efficient
formation of the (-)-strand strong stop DNA was induced. The
signature of this DNA, the FF species, was in double-stranded form.
Thus, although Mn
improved the ability of the DNA
polymerase to synthesize the full-length strong stop DNA, it could not
restore RNase H activity to this null mutant.
Previous analyses indicated that the mutant RTs 5E and
C had considerable RNase H activity in vitro as assayed
by in situ gel techniques, but left the(-)-strand strong
stop DNA in hybrid form in the endogenous reaction. The experiments
described here show that
C and
5E could efficiently degrade
an RNA-DNA substrate with Mn
but not with
Mg
as divalent cation (Table 1). As
Mg
is probably the biologically relevant divalent
cation, these two mutants are effectively RNase H null mutants in
vivo, consistent with the observed loss of infectivity and
reduction in strand translocation in the mutant viruses(20) .
The effects seen in these two mutants are more extreme versions of
effects seen with other enzymes and mutants. In the defined substrate
assay, wild-type MMLV RT RNase H was 16-fold more active in the
presence of Mn
compared with Mg
,
assayed both as a purified recombinant protein and as a
detergent-permeabilized virion-associated enzyme. Differential
activities with the two cations have been observed for other RNases H
in other assays as well. The HIV-1 RNase H single domain, expressed as
a hexahistidine-tagged fusion protein independently of the DNA
polymerase domain, similarly exhibits Mn
- but not
Mg
-dependent RNase H activity(26) .
Furthermore, the addition of an appropriate C-helix into an inactive
single domain version of the HIV-1 RNase H, which normally lacks this
helix, restores Mn
- but not
Mg
-dependent activity(27, 28) .
However, it should be noted that E. coli RNase H and HIV-1
RNase H as assayed in the intact RT prefer Mg
for
optimal catalysis.
Analogous divalent-dependent behavior has been
reported for many nucleic acid-binding enzymes (e.g.(29) and (30) ; very recently, (31) ). The
basis for the differential activity of these enzymes with the two
divalent cations is unclear, but could be due to aspects of the cation
binding with the enzyme or with the substrate. Determination of cation
requirements is complicated by the fact that they bind not only to
enzymes, but also to nucleic acids and dNTPs, leading to different
template and substrate complexes depending on the cation
used(32) . There is some evidence that these mutant enzymes,
assayed in Mg, may be affected in substrate binding.
It is interesting to note that the
C mutation removes a basic
handle region that has been implicated in preferential substrate
binding(21, 33) . Mutation and substitution of the
lysines in this region in the E. coli RNase H enzyme raise the K
without changing the V
(33) . However, the
5E mutation removes a loop
between the fifth
-sheet and the last
-helix, near a
conserved histidine residue that has been implicated in catalysis
rather than substrate binding (34, 35) .
Mn and Mg
may bind to RNase H in
two different positions. While the crystal structures of several RNases
H are known, it remains unclear whether one or two divalent cations are
involved in catalysis. A two-cation mechanism, similar to that for the
3`
5` exonuclease of DNA polymerase I, has been suggested for E. coli RNase H(10, 36) . Additionally,
crystallographic analysis of the HIV-1 RNase H single domain revealed
two divalent cations (Mn
) bound at the enzyme's
active site(12) . But in other studies, a single Mg
was observed bound at the active site of E. coli RNase
H(11, 37) . As all of these structures were determined
in the absence of substrate, it is difficult to ascertain whether the
observed cation binding is productive or rather inappropriate binding
that would be altered when the substrate was present.
Co-crystallization of RNase H with an RNA-DNA substrate would help
address this issue.
We also detected significant differences between
RNase H activities measured in the in situ gel assay and the
defined substrate assay even when both assays were carried out with
Mn. D524N had
10% activity in the in situ gel assay, but was inactive in the defined substrate assay, and
conversely, S526A was fully active in the defined substrate assay, but
had only 25% activity in the in situ assay. During the in
situ assay, the gel is typically allowed to renature for several
days, during which time the enzyme is constantly surrounded by
substrate. Thus, this assay may not be sensitive to subtle defects in
affinity. An additional difference that may account for the
discrepancies between the two assays is the fact that proteins must
renature in the in situ gel assay, and mutations may
specifically affect this step.
With amounts of enzyme sufficient for
wild-type RT to fully degrade the RNA from the radiolabeled DNA
substrate in the RNase H defined substrate reaction, mutants S526A,
R657S, and Y598V showed some activity in Mg, but were
unable to degrade all of the RNA from the substrate. The full-length
double-stranded species completely disappeared, and a new discrete
species was detected. While we cannot tell how much RNA was removed by
these mutants, the major product was the same for all three, and at low
concentrations, this product was detected with wild-type RT. This
result suggests that these mutants may be less processive RNases H than
the wild type or that they only cleaved efficiently up to a specific
site in the RNA. The structural features of the substrate that might
determine the accumulation of this intermediate are unknown. It should
be noted that this cleavage is not due to background activity seen for
all the mutants: similar products were not seen with mutant
5E,
H7, or Y586F. The defects seen in this assay may be among those
responsible for the delayed replication of viruses carrying these
mutations(20) .
In the endogenous reaction, mutants S526A, R657S, and Y598V were able to degrade their genomic RNA completely, like wild-type RT. What then is the difference between the endogenous reaction and the RNase H defined substrate reaction? One main difference is the presence of a more intact capsid in the endogenous reaction and the resulting increased effective concentration of other viral proteins. Virions were permeabilized with 0.01% Nonidet P-40 in the endogenous reaction and were more fully lysed with 0.3% Nonidet P-40 in the in vitro defined substrate reactions. Thus, processive RNase H activity may be effected by the presence of other retroviral proteins (most notably NC, the basic single-strand nucleic acid-binding protein), which are diluted out during the defined substrate reaction. Although we do not favor the idea, we cannot rule out the possibility that the mutant enzymes are simply more sensitive to high Nonidet P-40 concentrations.
A second significant difference between the two assays is the fact that DNA polymerization and RNA degradation may occur simultaneously during the endogenous reaction. It is possible that these mutants are more active in degradation when it is coupled to polymerization, a feature that we are not testing during the defined substrate reaction. There is evidence that RNase H may behave differently when coupled with polymerization(38, 39) . Further analysis of these mutations may help us understand their differential effects in the endogenous and defined substrate assays, in an attempt to understand polymerization-dependent and -independent RNase H activity.
Previous
work has shown that the DNA polymerase activity of most RTs, including
HIV-1, Rous sarcoma virus, and MMLV RTs, prefers Mg for full activity. The DNA polymerase activity of MMLV RT is
unusual in preferring Mn
over Mg
as
a divalent cation(40, 41, 42, 43) .
MMLV RT shows a reduced rate of synthesis in Mg
, and
the DNA products are also generally shorter, suggesting that the enzyme
may be less processive ( (40) and data not shown). Mutations in
the RNase H domain can also affect the DNA polymerase activity and can
particularly reduce its ability to form long products(25) . In
these studies, we found that Mn
is able to
qualitatively influence DNA polymerase processivity during the
endogenous reaction. In the presence of MnCl
, the normally
nonprocessive mutant H7 was converted to a more processive form,
efficiently synthesizing full-length(-)-strand strong stop DNA.
Thus, assays of MMLV RT in Mn
show alterations in
both the DNA polymerase and RNase H activities, generally showing
enhanced activity and masking significant defects. These results
suggest that RT should be assayed in Mg
to detect
biologically significant effects with the greatest sensitivity.