(Received for publication, May 22, 1995; and in revised form, July 26, 1995)
From the
A set of five mutations (A62V, V75I, F77L, F116Y, and Q151M) in
the polymerase domain of reverse transcriptase (RT) of human
immunodeficiency virus type 1 (HIV-1), which confers on the virus a
reduced sensitivity to multiple therapeutic dideoxynucleosides (ddNs),
has been identified. In this study, we defined the biochemical
properties of RT with such mutations by using site-directed
mutagenesis, overproduction of recombinant RTs, and steady-state
kinetic analyses. A single mutation, Q151M, which developed first among
the five mutations in patients receiving therapy, most profoundly
reduced the sensitivity of RT to multiple ddN 5`-triphosphates
(ddNTPs). Addition of other mutations to Q151M further reduced the
sensitivity of RT to ddNTPs. RT with the five mutations proved to be
resistant by 65-fold to 3`-azido-2`,3`-dideoxythymidine 5`-triphosphate
(AZTTP), 12-fold to ddCTP, 8.8-fold to ddATP, and 3.3-fold to
2`,3`-dideoxyguanosine 5`-triphosphate (ddGTP), compared with wild-type
RT (RT). Steady-state kinetic studies revealed comparable
catalytic efficiency (k
/K
) of RTs
carrying combined mutations as compared with that of RT
(<3-fold), although a marked difference was noted in inhibition
constants (K
) (e.g.K
of a mutant RT carrying the five
mutations was 62-fold higher for AZTTP than that of RT
).
Thus, we conclude that the alteration of RT's substrate
recognition, caused by these mutations, accounts for the observed
multi-ddN resistance of HIV-1. The features of multi-ddNTP-resistant
RTs should provide insights into the molecular mechanism of RT
discriminating ddNTPs from natural substrates.
The accumulating data suggest that the development of HIV-1 ()variants with reduced susceptibility to reverse
transcriptase (RT) inhibitors is related to clinical deterioration in
patients receiving RT
inhibitors(1, 2, 3, 4, 5, 6) .
It has been shown that the high error rate of HIV-1 RT, approximately
1-10 misincorporations/HIV-1 genome/round of
replication(7, 8, 9) , suggests that
misincorporation by HIV-1 RT is responsible for the hypermutability of
HIV-1, enabling HIV-1 to rapidly acquire drug resistance. This notable
diversity of HIV-1 genome resulting from error-prone RT is likely to be
associated with ``natural'' drug resistance seen in HIV-1
from patients receiving no prior antiretroviral therapy(10) .
In this regard, the combined use of multiple antiretroviral agents has
been postulated to block or retard the emergence of HIV-1 less
susceptible to therapeutic agents. However, several recent reports have
demonstrated that HIV-1 can acquire resistance to multiple drugs in
vitro and in vivo(11, 12, 13, 14, 15, 16) .
These data may suggest that even combination therapy may ultimately
fail to suppress the replication of this hypermutable virus, although
it is possible that combination chemotherapy continues to be
efficacious if HIV-1 variants have a substantial replication
disadvantage due to the altered enzymatic conformation/function as
compared with wild type HIV-1; thus, the progression of the disease may
be significantly delayed(17) .
We and others have recently demonstrated that HIV-1 can develop a novel set of five mutations (A62V, V75I, F77L, F116Y, and Q151M) during combination chemotherapy with 3`-azido-2`,3`-dideoxythymidine (AZT; zidovudine) plus 2`,3`-dideoxyinosine (ddI; didanosine) or 2`,3`-dideoxycytidine (ddC; zalcitabine) and that these mutations confer resistance to various antiretroviral 2`,3`-dideoxynucleoside analogs (ddNs) on HIV-1(11, 13, 18) . In this study, we attempted to define the biochemical properties of RT carrying all or a subset of the five mutations. We constructed a cartridge mutagenesis system, which enabled us to introduce the desired mutations into relatively short fragments to generate both infectious HIV-1 clones (18) and recombinant RTs. We then examined the susceptibility of mutant RTs to multiple ddNTPs and determined steady-state kinetic constants. We also discuss the relationships of the observed HIV-1 resistance to multiple ddNs and altered RT functions caused by all or a subset of the five mutations.
Figure 1:
A
linear representation of HIV-1 RT (amino acids 1-560) with an
expanded region (amino acids 10-280) containing the polymerase
catalytic site. Two cloning sites, XmaI and NheI,
were introduced by mutating the nucleotide sequences, CCAGGA and
GCAAGT, which encode Pro-Gly
and
Ala
-Ser
(BH10; (47) ) to CCCGGG and
GCTAGC, respectively, without changing the deduced amino acids. The XmaI-NheI fragment (759 base pairs) encoding
Gly
-Ala
(thickline)
was used as a cartridge to construct mutant RT expression vectors (see
``Experimental Procedures''). Amino acid substitutions
introduced are all located within the cartridge and are designated by
amino acid numbers of HIV-1 RT.
Figure 2:
SDS-polyacrylamide gel analysis of
RT and mutant RT preparations. An equal amount (2 pmol of
active RT molecules) of each RT preparation was loaded onto 4-15%
linear gradient polyacrylamide gel (Bio-Rad), electrophoresed, and
stained with Coomassie Brilliant Blue. Lane1,
RT
; lane2, RT
; lane3, RT
; lane4, RT
. Note a major 66-kDa polypeptide
(>90% of the total protein) and two truncated polypeptides at 62 and
55 kDa (<5%). All 11 preparations listed in Table 1were
analyzed, and virtually identical patterns were observed (not
shown).
In order to confirm that p66 homodimer RT preparations
were suitable for the study of substrate recognitions between RT and mutant RT preparations, we subjected a p51/p66 heterodimer
RT
(21) to our assay system. With regard to dTMP
incorporation into poly(rA)
(dT)
, the K
and k
values of the
p51/p66 heterodimer RT
were 3.3 ± 0.8 µM and 2.0 ± 0.1 s
, respectively (data not
shown). These values were comparable with the K
and k
values of our p66 homodimer
RT
, being 6.5 ± 0.5 µM and 1.8
± 0.3 s
, respectively (Table 2).
Indeed, Beard et al.(24) have recently demonstrated
that the two forms of RT
, homodimer (p66/p66) and
heterodimer (p51/p66), have comparable kinetic parameters of
enzyme-template/primer interactions, concluding that the homodimeric
form of RT can serve as a model for the interactions of heterodimeric
form of RT with template-primers.
In order to examine the effects of various combinations of mutations
on the sensitivity of RT against ddNTPs, we introduced four mutations
(A62V, V75I, F77L, F116Y) in addition to the Q151M mutation. In
contrast to inconsiderable changes observed with single substitution of
each of these four amino acids, combined mutations brought about marked
increases in IC values (Table 1, Fig. 3). RT
with four mutations (RT
) and that with all
five mutations (RT
) had the most profound
effect on the sensitivity of RT to ddNTPs tested (Table 1).
Figure 3:
Sensitivities of various RT preparations
against selected dideoxynucleotides. Each plot represents the mean
percentage of activity of quadruplicate determinations of RT (
), RT
(
), RT
(
), RT
(
), and
RT
(
) in the presence of ddATP (A), ddCTP (B), ddGTP (C), or AZTTP (D). Substrate concentrations used are described under
``Experimental Procedures.'' The IC
values
determined are summarized in Table 1.
We
also examined the sensitivity of selected RTs to a non-nucleoside RT
inhibitor, nevirapine(22) , using [H]dGTP
and poly(rC)
(dG)
as a nucleotide substrate
and a template-primer, respectively. The IC
values of
nevirapine with RT
and RT
were
0.50 and 0.59 µM, respectively. These results are
consistent with our previous observations that the infectious mutant
HIV-1 carrying all five mutations was as sensitive to nevirapine as
wild-type HIV-1(18) .
Figure 4:
Time course of dAMP incorporation into
MS2/22A mediated by various RT preparations. Reaction mixture (100
µl) contained 1.8 (), 2.4 (
), 3.0 (
), and 3.6
(
) µl of RT
(A) or RT
(B), template-primer MS2/22A (0.5 µM), 5
µM [
H]dATP, 50 mM Tris-HCl,
pH 7.8, 6 mM MgCl
, 0.01% Triton X-100, and 150
mM KCl. Reaction was initiated by the addition of enzyme at 37
°C. A portion (15 µl) of the reaction mixture was subjected to
analysis at the indicated times. The product formed was measured by the
DE81 filter binding assay. Insets show the replot of the
extrapolated y intercept (burst amplitude) versus RT
added in the reaction. Concentrations of RT
and
RT
, determined from the slope of the replot,
were 0.22 ± 0.001 and 0.22 ± 0.002 µM,
respectively.
Figure 5:
Lineweaver-Burk plots of ddATP inhibition.
The reaction was of the distributive synthesis mode in which only
[H]dATP was present as a nucleotide substrate in
the reaction mixture as described under ``Experimental
Procedures.'' Enzymes examined were RT
(7
nM; panelA), RT
(7
nM; panelB), RT
(10
nM; panelC), and RT
(6.5 nM; panelD). Concentrations of
ddATP used for examining RT
were 0, 0.1, 0.2, 0.4
µM; for RT
, 0, 2.0, 4.0, and 8.0
µM; for RT
and
RT
, 0, 4.0, 8.0, and 16 µM.
Replot of the slope versus ddATP concentrations is shown in
each inset. K
and k
values, determined from the plot in the
absence of ddATP, are summarized in Table 3. K
values determined from the x intercept of the replot are summarized in Table 4.
We also
determined steady-state kinetic constants using
[H]ddATP as a nucleotide substrate. The K
value of RT
and
mutant RTs (Table 4) was virtually identical to its K
value with ddATP (Table 3), consistent
with a previous report demonstrating that the K
value of ddNTP against dNMP incorporation into a heteropolymeric
template-primer was almost equal to K
(27) , indicating that
ddNTP and dNTP behave as classical competitive substrates to each other
under single nucleotide incorporation assay conditions(27) .
RT Carrying Q151M and T215Y Mutations-Most of the
HIV-1 variants carrying all or several of the five mutations, isolated
from patients receiving combination chemotherapy with AZT plus ddC or
AZT plus ddI, had the previously reported AZT, ddC, or ddI-associated pol gene mutations except for K219Q and K219E, and none had
the most potent AZT-associated mutation,
T215Y(11, 13, 18) . In this regard, it is
possible that one or more of the five mutations are incompatible with
AZT, ddC, or ddI-associated mutations. In order to test this
possibility, we produced a recombinant RT (RT)
carrying the Q151M mutation and T215Y(32) . We found, however,
that RT
had an enzymatic activity comparable with
that of RT
and was less susceptible to all four ddNTPs
tested, ddATP, ddCTP, ddGTP, and AZTTP (Table 5).
The mutations responsible for resistance of HIV-1 against
nucleoside RT inhibitors have been mapped to the RT-encoding region of
the pol gene, and accumulated data suggest that altered
substrate recognition by RT is associated with drug
resistance(29, 30, 33) . However, the changes
in the sensitivity of RT to the triphosphate of a ddN appear to account
partly for in vitro resistance of HIV-1 to the
ddN(29, 34) . Indeed, several enzymatic studies have
demonstrated that the sensitivities of RT to ddNTPs differ from the in vitro viral sensitivities to
ddNs(31, 34, 35) . For instance, a
recombinant infectious HIV-1 carrying four AZT-associated amino acid
substitutions (D67N, K70R, T215Y, and K219Q) is about 100-fold less
sensitive to AZT than the wild-type HIV-1 strain in
vitro(34) ; however, an RT with the same four amino acid
substitutions is as sensitive to the inhibition of AZTTP as is the
wild-type RT (RT)(31) . Prasad et al.(35) have also demonstrated that HIV-1 carrying a mutant
RT with an E89G substitution, which showed cross-resistance to various
ddNTPs including ddGTP, was sensitive to ddG in a cell culture system.
These observations do not appear to be easily explained under one
unifying theory at present. In the present study, we demonstrate that a
unique set of five mutations (A62V, V75I, F77L, F116Y, and Q151M)
alters RT's substrate recognition and confers resistance to
various ddNTPs on RT, which may fully account for the observed in
vitro resistance of HIV-1 to multiple ddNs(18) .
It is
evident that Gln plays a critical role in the enzymatic
function of RT and is linked to the development of multi-ddN resistance
of HIV-1(18) . The amino acid Gln
consists of the
highly conserved sequence, Leu-Pro-Gln-Gly, in the corresponding region
within motif B of RT from various animal and human retroviruses (36) ; however, the substituted amino acid, methionine, is
found in the corresponding region of hepatitis B viruses (HBVs),
suggesting that Q151M is not overly detrimental to the function of RT.
We found in this study that the Q151M substitution decreases the
polymerase activity by 30% (as assessed with k
/K
; Table 2) with
several dNTPs and significantly reduces the sensitivity of RT to
ddNTPs. In this regard, various groups have examined the effect of
amino acid substitution of
Gln
(37, 38, 39) . A Q151N
substitution has shown no significant alteration in the polymerase or
RNase H activities of RT(38) , A Q151H substitution, however,
has been shown to reduce the polymerase activity by 30% and the
sensitivity of RT to AZTTP by 4-50-fold(39) , and a Q151E
substitution has been reported to reduce the polymerase activity by 30% (37) as compared with RT
.
The amino acid
Phe, which is located close to the proposed dNTP-binding
site of HIV-1 RT(40) , is also part of a highly conserved
region (motif A) in RTs from various retroviruses, but the
corresponding amino acid in RT in hepatitis B viruses has been found to
be tyrosine(36) , which may explain why the RT with such a
substitution at a highly conserved amino acid remains functional. In
fact, Boyer et al.(38) have recently reported that
F116Y alone does not affect the polymerase and RNase H activity of RT .
In our study, RT
carrying F116Y was also functional and
behaved similarly to RT
against ddNTPs (Table 1). It
is not clear how F116Y affects the function of RT when other mutations
are added. The addition of the V75I mutation to
RT
, generating RT
, further
reduced the sensitivity to ddNTPs, although V75I alone had little
effect on the sensitivity (Table 1). Codon 75 mutation with a
different amino acid, V75T, has been identified in HIV-1 variants less
susceptible to 2`,3`-didehydro-2`,3`-dideoxythymidine, ddC, and ddI in vitro(41) . These data suggest that the amino acid
at codon 75 of HIV-1 RT plays a crucial role in substrate recognition.
Detailed steady-state kinetic analyses revealed that the altered
nucleotide substrate recognition of RT is evidently caused by all or a
subset of the five mutations, suggesting that these amino acids
interact with an incoming nucleotide substrate, although presently it
is not possible to distinguish direct effects caused by amino acid
substitutions from those mediated via template-primer
interactions(40, 42) . It has been reported that
Gln is located close to the first single-stranded base of
the template and that three amino acids (Val
,
Phe
, and Gln
) form part of the
``template grip''(43) . This may lead to
repositioning or conformational change of the template-primer, causing
a distortion of the geometry of the polymerase active site, enabling RT
to discriminate natural substrates from multiple
ddNTPs(40, 43, 44) . In this regard, we
attempted to define the difference between RT
and mutant
RTs with respect to the RT's interaction with the
template-primers. We then found that all RT
and mutant RTs
examined had a virtually identical profile in the time course of dAMP
incorporation into MS2/22A (Fig. 4). This steady-state rate,
dominated by the dissociation of enzyme
template-primer complex (26, 27, 28) , was found comparable in all
mutant RTs as compared with that of RT
(within 2-fold),
suggesting that none of the five mutations significantly affects the
dissociation of the enzyme from the enzyme
template-primer
complex. We also found that there was no significant difference in k
of ddAMP-forced terminated template-primer for
all RTs tested (Table 5; k
= k
), suggesting that the resistant phenotypes of
RTs do not involve the dissociation of the ddAMP-forced terminated
template-primer. Furthermore, among RTs examined we have found no
significant difference (3-fold) in their K
values
to poly(rA)
(dT)
estimated by apparent K
values for template-primer determined with low
concentrations of a nucleotide substrate(45) ; their K
values were 4.2 ± 0.3, 5.0 ± 1.2,
5.1 ± 0.6, 2.8 ± 0.7, and 2.2 ± 0.4 nM for RT
, RT
, RT
,
RT
, and RT
, respectively. (
)Moreover, we have found no significant changes in the
processivity of RTs as measured by dTMP incorporation into
poly(rA)
d(T)
(data not shown). It is worth noting
that in the present work, bacterially expressed p66 homodimer RTs were
employed; thus, the observed changes caused by amino acid substitutions
might differ from changes that may occur in the p51/p66 heterodimer
RTs. In this regard, Becerra et al. have reported a difference
in association constants upon dimerization of p66/p66 and p51/p66
subunits of RT(46) , which may produce changes in
enzyme-substrate interactions differently between heterodimer and
homodimer RTs when amino acid substitutions occur. Further experiments
using proper heterodimer RTs and pre-steady kinetic analysis are needed
to define the structures and functions of HIV-1 RT when these mutations
develop.
It is noteworthy that HIV-1 strains carrying Q151M often do not bear amino acid mutations that have been associated with viral resistance to AZT, although HIV-1 variants were isolated from patients receiving long-term therapy including AZT(11, 13, 18) . It is possible that certain nucleotide or amino acid sequence(s) inherent to HIV-1 strains in certain patients predispose these viruses not to acquire any AZT-related mutations but to develop the Q151M substitution and ultimately the rest of the five mutations. Comparative nucleotide sequence analysis of pretherapy HIV-1 that later acquired Q151M and pretherapy strains that later developed the AZT-related mutations may define such predisposing nucleotide or amino acid sequence(s). It remains to be determined whether HIV-1 carrying Q151M can subsequently acquire the AZT-associated amino acid substitutions. It is also of interest to study the mechanisms of substrate recognition or discrimination of ddNTPs by three-dimensional structure analysis using mutant RTs with all or a subset of the five mutations. Such studies may provide us with insight to the understanding of molecular mechanisms of substrate recognition and catalytic function of HIV-1 RT.