(Received for publication, November 10, 1995; and in revised form, December 20, 1995)
From the
We describe catalytically active mutants of HIV RT (human
immunodeficiency virus reverse transcriptase) generated by random
sequence mutagenesis and selected in Escherichia coli for
ability to complement the temperature-sensitive phenotype of a DNA
polymerase I (Pol I) mutant. We targeted amino acids
Asp-67 through Arg-78 in HIV RT, which form part of the
3-
4
flexible loop and harbor many of the currently known mutations that
confer resistance to nucleoside analogs. DNA sequencing of 109 selected
mutants that complement the Pol I
phenotype revealed
substitutions at all 12 residues targeted, indicating that none of the
wild-type amino acids is essential. However, single mutations were not
observed at Trp-71, Arg-72, and Arg-78, consistent with evolutionary
conservation of these residues among viral RTs and lack of variation at
these positions among isolates from patients. The mutations we
recovered included most of those associated with drug resistance as
well as previously unidentified mutations. Purification and assay of 14
mutant proteins revealed correlation between their DNA-dependent DNA
polymerize activity in vitro and ability to complement the Pol
I
phenotype. Activity of several mutants was resistant to
3`-azidothymidine triphosphate. We conclude that random sequence
mutagenesis coupled with positive genetic selection in E. coli yields large numbers of functional HIV RT mutants. Among these are
less active variants which are unlikely to be isolated from
HIV-infected individuals and which will be informative of the roles of
individual amino acids in the catalytic functions of the enzyme.
Infection by human immunodeficiency virus 1 (HIV-1) ()is the pathogenic precursor to clinical development of
acquired immunodeficiency syndrome (AIDS). HIV-1 contains a reverse
transcriptase (HIV RT) that catalyzes synthesis of both a single- and
double-stranded DNA copy of the viral genome, and is required for viral
replication. As such, HIV RT provides a central target for chemotherapy
of AIDS. Current chemotherapeutic agents that target HIV RT, such as
AZT, ddC, and ddI, are incorporated into viral DNA but cannot be
further elongated, thus terminating DNA synthesis (Mitsuya and Broder,
1986). Unfortunately, the exceptionally high mutation rate of the virus
drives rapid emergence of resistant strains, rendering these nucleoside
analogs ineffective (Boucher et al., 1992; Larder and Kemp,
1989; Mitsuya and Broder, 1986). Single and multiple base substitutions
within the HIV RT gene are responsible for emergence of these
drug-resistant mutants that escape chemotherapy. In order to develop
drugs that more effectively target HIV RT, we require a clearer
understanding of the potential of different sites within the enzyme to
produce drug-resistant mutations.
Many of the mutations that render
HIV resistant to nucleoside analogs are located in the 3-
4
loop,
9-
10 turn, and
11a/b regions of HIV RT (Tantillo et al., 1994). The
3-
4 region in the fingers domain
is believed to function as a template grip that interacts with
single-stranded (ss) DNA and RNA templates (Boyer et al.,
1994; Tantillo et al., 1994). Mutations within the
3-
4 loop that confer drug resistance include K65R (resistant
to ddC, ddI, and 3TC (Gu et al., 1994)), D67N (resistant to
AZT (Larder and Kemp, 1989), Larder and Kemp, 1989)), T69D (resistant
to ddC (Fitzgibbon et al., 1992)), K70R (resistant to AZT
(Larder and Kemp, 1989)), and L74V (resistant to ddI (St. Clair et
al., 1991)). Recently, two additional mutations, V75I and F77L,
have been isolated from patients treated with dideoxynucleosides
(Shirasaka et al., 1995). The diverse locations of mutations
in HIV RT that render the virus resistant to the same drug has prompted
the suggestion that many of these mutations act indirectly, at a
distance. It is postulated that such mutations affect conformation near
the catalytic site, modifying interactions between the fingers domain
and the template DNA or RNA (Boyer et al., 1994; Tantillo et al., 1994) to enhance discrimination between natural
substrates and inhibitors. Other drug-resistant mutations, not in
3-
4 loop region, such as M184V (Gu et al., 1992),
that are located close to the catalytic site are proposed to directly
affect dNTP binding (Tantillo et al., 1994).
We recently
demonstrated that HIV RT can substitute for DNA polymerase I in Escherichia coli (Kim and Loeb, 1995a). In our genetic
complementation system, expression of HIV RT allows mutant E. coli cells harboring a temperature-sensitive DNA polymerase I (Pol
I) to grow at non-permissive temperature. Pol I
cells expressing an inactive mutant (D186N) fail to grow at
nonpermissive temperature, demonstrating the requirement for
catalytically functional HIV RT. Based on the observation that
complementation by HIV RT is specifically inhibited by nucleoside
analogs such as AZT, we proposed that the system can be used to screen
potential anti-HIV RT drugs in bacteria (Kim and Loeb, 1995b).
In
the present study, we used the bacterial complementation system to
select active HIV RT mutants from libraries that encode random sequence
substitutions in the wild type gene. We replaced the 36 contiguous
nucleotides near the 3-
4 loop that encode amino acids 67
through 78 with random sequences and selected from this library mutants
that complement the Pol I
mutation. This positive genetic
selection allowed us to recover most of the active HIV RT mutations
thus far observed in naturally occurring variants and drug-resistant
mutants. Our large library of active mutants includes previously
unidentified mutations and mutations in the
3-
4 loop that
confer resistance to AZTTP.
Figure 1:
Scheme for random sequence mutagenesis
of HIV RT coupled with genetic selection for functional mutants.
Construction of a plasmid-borne library of HIV RT variants containing
random nucleotide substitutions at codons 67 through 78, and selection
of active mutants by complementation of the temperature-sensitive
phenotype of an E. coli DNA polymerase I mutant, is described
under ``Experimental Procedures.'' Synthesis of random
nucleotide-containing oligonucleotides (insets) is illustrated
in steps 1-4; [N] in oligomer 2
denotes 36 contiguous residues containing 12% random nucleotides at
each residue. Ligation of the inserts into the stuffer plasmid pBK18,
to replace the wild-type HIV RT sequence at codons 67-78 and
bring HIV RT under control of the lac promoter, is shown in step 5. Preparation of the plasmid library and selection for
functional mutants in Pol I
cells is shown in steps
6-8.
To confirm the
temperature-independent phenotype of transformed cells that formed
colonies at 37 °C, plasmids were prepared from 205 such colonies
and retransformed individually into Pol I cells. To assess
the ability of each selected HIV RT mutant to substitute for E.
coli DNA polymerase I, the plating efficiency and colony size were
assayed following an 18-h incubation at 37 °C. Of the
re-transformed plasmids, 176 (86%) showed greater than 75% plating
efficiency (number of colonies at 37 °C relative to the number at
30 °C). Approximately 3% of transformants formed no colonies at 37
°C; this false positive value presumably reflects the background
growth of Pol I
cells observed on high cell density plates
(Kim and Loeb, 1995a). In separate experiments, Pol I
cells transformed with pBK18 (the stuffer plasmid without HIV RT)
showed plating efficiency of 1.5% at 37 °C. Of the 176
retransformed cells that showed plating efficiency greater than 75%, 57
(32%) formed large colonies at 37 °C equivalent in size to that of
Pol I
cells expressing wild-type HIV RT
(+++), 81 (46%) formed colonies of medium size
(++), and 38 (22%) produced small colonies.
The levels of substitution among active mutants, together with the associated complementation efficiencies, are listed in Table 1. Overall, there were fewer amino acid changes among mutants that exhibited high efficiency of complementation (large colony size equivalent to that of wild-type HIV RT) than among mutants with lower complementation efficiency. Thus, the average number of amino acid substitutions in the 37 mutants that formed large colonies at 37 °C (+++) was 1.0. Among the 49 mutants that formed medium sized colonies (++), the average number of amino acid substitutions was 3.0. Among the 23 mutants forming small colonies (+) the average number of substitutions was 4.1. All of the single mutants formed colonies that were the same size as that of wild type, whereas most of the active mutants having more than 4 amino acid changes produced smaller colonies at 37 °C.
Figure 2:
Amino acid substitutions in functional HIV
RT mutants. The amino acid sequences of 109 mutants that complemented
the temperature-sensitive phenotype of Pol I cells were
determined by DNA sequencing of the random nucleotide containing
inserts. Italicized type indicates substitution of an amino
acid similar to the wild-type. Underlining indicates a
naturally occurring variation. Bold type indicates a
substitution found, either singly or together with others, in drug
resistant mutants. In B, the number to the right of individual mutant amino acids indicates the number of
occurrences at the designated position. The number given at (1) below each position is the total number of substitutions
at that position; the number at (2) is the number of different
amino acids identified at that position. In C, complementation
efficiency denotes the average colony size relative to wild type of all
mutants containing substitutions at the designated residue; wild-type
efficiency is denoted by +++ on the vertical
scale.
Figure 3: Inhibition of DNA polymerase activity of HIV RT mutants by AZTTP. The DNA-dependent DNA polymerase activity of HIV RT mutants was assayed on a gapped DNA template in the presence of AZTTP as described under ``Experimental Procedures.''
In this work, we tested whether complementation of a
replication defective E. coli DNA polymerase I mutant by HIV
RT (Kim and Loeb, 1995a) can be used to select active HIV RT mutants
from a population of variants containing random nucleotide
substitutions (Horwitz and Loeb, 1986). In our functional
complementation system, expression of HIV RT enables an E. coli mutant harboring temperature-sensitive DNA polymerase I to grow at
a nonpermissive temperature (Kim and Loeb, 1995a). In these initial
experiments with random sequence substitutions in HIV RT, we targeted a
region that has little secondary structure and would likely permit a
large number of substitutions that yield functional mutants. The
crystal structure of HIV RT indicates that the 12 amino acid target
forms a flexible loop between the 3 and
4 domains (Tantillo et al., 1994). In addition, many viral isolates that are
resistant to nucleoside analogs contain mutations within this region.
The inherent flexibility of the target and the occurrence of variants
in the natural host is consistent with our finding that all 12 residues
are tolerant of substitutions, either singly or in combination with
others.
We observed in the experiments described here that
approximately 12% of Pol I cells transformed with a
library encoding random substitutions at amino acids 67-78 formed
colonies at 37 °C. The fraction of a library that supports colony
formation at nonpermissive temperature, i.e. the fraction of
encoded sequences that can complement the Pol I
phenotype,
depends on several factors. These include the upper and lower limits of
intracellular HIV RT activity compatible with growth of the bacterial
host (the window for selection), the proportion of random nucleotides
relative to wild-type nucleotides within the substituted segment, and
the function of the targeted amino acids in supporting DNA-dependent
DNA polymerase activity. With respect to the first factor, it appears
that the fraction of transformants capable of complementation can be
adjusted by controlling the level of HIV RT expression. Thus, we
observed that the fraction of transformants that form colonies at 37
°C decreased from 12 to 7% when IPTG was deleted from the selection
plates (data not shown). By reducing the level of expression, we
presumably limited recovery to mutants with relatively high specific
activity. In the experiments described here where use of 1 mM IPTG gave 12% positive transformants, one-third (i.e. 4%)
yielded colonies of wild-type size, suggestive of an intracellular HIV
RT activity comparable to wild-type. That higher levels of HIV RT
expression can be lethal to the host (Kim and Loeb, 1995a) indicates
that there is an upper limit of activity compatible with growth. A
second factor affecting the fraction of positive transformants is the
degree of randomness in the targeted region, with greater randomness
and more amino acid substitutions mitigating against levels of activity
that promote growth and colony formation. In the present experiments,
the target sequence was replaced with 12% random nucleotides at each
position, and as a result the wild-type sequence should have occurred
at a frequency of 1%, corresponding to 120 of the 12,000 cells plated.
Assuming that the predicted 120 wild-type sequences were included in
the 1400 positive transformants we recovered, we should have found 9
wild-type isolates (120/1400) among the 109 that we sequenced. That we
found 6 wild-type sequences among the 109, approximately the expected
occurrence, substantiates the designated composition of the library.
Third, it is likely that the importance of the targeted amino acids for
DNA polymerase activity affects the fraction of transformants selected,
with substitution of residues having crucial functions mitigating
against ability to complement. The high frequency of positive
transformants that we recovered by targeting the
3-
4 flexible
loop is consistent with the nonessential nature of the individual amino
acids.
The amino acid replacement we observed among mutants that
complement the Pol I phenotype (Fig. 2) indicates
that Trp-71, Arg-72, and Arg-78 are relatively intolerant of
substitution compared with other residues in our 12-amino acid target.
This pattern is consistent with substitutions recovered in HIV isolates
and with sequence conservation among various viral RTs. Within the
target region, substitution at two positions, S68G and D76N, occurs in
natural HIV RT variants, and substitution at 6 positions (Asp-67,
Thr-69, Lys-70, Leu-74, Val-75, and Phe-77) occurs in drug-resistant
viral isolates. Of the 4 residues (Trp-71, Arg-72, Lys-73, and Arg-78)
not included among viral mutations, Trp-71, Arg-72, and Arg-78 yielded
no substitutions in the single mutants we sequenced (Fig. 2A); moreover, mutants with multiple
substitutions including one or more of these three positions showed low
complementation efficiency (Fig. 2C). These data
indicate that Trp-71, Arg-72, and Arg-78 are relatively rarely mutable
and suggest that they may be important in DNA-dependent DNA polymerase
activity. In fact, a recent study showed that two mutations at Arg-72,
including R72K, result in greatly decreased activity due to reduction
in translocation (Sarafinos et al., 1995). Notably, sequence
comparison among viral RTs reveals that Trp-71, Arg-72, and Arg-78, as
well as Leu-74 and Asp-76 are highly conserved (Barber et al.,
1990). Taken together, available data indicate that mutations at Arg-72
may be incompatible with viral replication in the natural host.
Among the mutants we analyzed, the number of amino acid substitutions is inversely correlated with colony size and with DNA polymerase activity on a gapped DNA template (Table 2). These observations suggest that ability to complement in the E. coli system reflects the DNA-dependent DNA polymerase activity of HIV RT, with mutants having 10% of wild-type DNA-dependent DNA polymerase activity being selectable. It has been reported that an HIV RT mutant with 37% of wild-type activity, L74M, supports production of viable virions in cultured human cells, whereas another mutant, L74A, exhibiting 30% of wild-type activity, does not (Lacey and Larder, 1994). Thus, selection for mutants that support bacterial growth is apparently less stringent than selection for virion production in cell culture systems. In accord, the HIV RT mutants we selected that have significantly reduced DNA polymerase activity (Table 2) are unlikely to be identified in viral populations because they presumably cannot support viral replication.
Six amino acids within the segment we targeted (Arg-67, Thr-69, Lys-70, Leu-74, Val-75, and Phe-77) have been found to be mutated in variants resistant to nucleoside analogs (Tantillo et al., 1994). We analyzed some of the mutations we isolated at these positions for AZTTP resistance. As illustrated in Fig. 3A, F77Y and F77V conferred an 18- and 16-fold decrease in inhibition by AZTTP, respectively. Although these substitutions have not been reported in viral isolates from patients, it was recently reported that individuals receiving therapy with a combination of nucleoside analogs developed a F77L mutation subsequent to a Q151M mutation. Thus, F77L is encompassed in the evolution of a set of five mutations, beginning with Q151M, followed by F77L and F116Y and later by A62V and V75I, that confers multiple drug resistance (Shirasaka et al., 1995). The L74I mutation we isolated exhibited an 8-fold reduction in inhibition by AZTTP; Fig. 3B). A similar mutation, L74V, has been isolated from patients after ddI treatment (St. Clair et al., 1991) and exhibited reduced sensitivity to ddGTP (Lacey et al., 1992). Thus Leu-74 is likely to be involved directly or indirectly in substrate selection. Interestingly, L74V displayed wild-type sensitivity to AZTTP in combination with V75L. The mutation D67N, in combination with additional mutations (K70R, T215Y, and K219Q) exhibits a more than 100-fold increased resistance to AZT in a cell culture system (Larder and Kemp, 1989). However, a purified HIV RT mutant containing all four mutations failed to show resistance to AZTTP in vitro (Skalka and Goff, 1993). In our study, D67N did not affect sensitivity to AZTTP, consistent with the observation that virions containing D67N as a single mutation did not show increased resistance to AZT in vivo (Larder et al., 1991; Larder and Kemp, 1989). However, we did find that both D67G and D67N/K73T exhibited increased resistance to AZTTP (Fig. 3C).
Many mutations in HIV RT have been observed in virions that are resistant to nucleoside analogs in patients and in cultured cells. However, purified HIV RT bearing the same mutations frequently does not exhibit resistance to the corresponding nucleoside triphosphates when assayed in vitro. Conversely, we isolated both single and multiple substitutions that render HIV RT resistant to AZTTP, but have not yet been identified in drug-resistant viral isolates. Considering the exceptionally high mutation rate of the virus (Preston et al., 1988; Roberts et al., 1989), the high error rate of the reverse transcriptase in vitro (Preston et al., 1988) and recent evidence for rapid viral replication during the course of HIV infection (Delwart et al., 1993), it seems likely that all single base substitutions and many multiple substitutions would have arisen and been ``tested'' for resistance during AZT therapy in patients. If viruses containing the mutations we identified are in fact resistant to AZT, why have they not been selected? Some may not have a high enough DNA-dependent DNA polymerase activity to compete successfully (e.g. F77V, Table 2). However, some (e.g. F77Y and L74I which may have wild-type levels of this activity) might interfere with additional activities or properties of HIV RT which are essential for production of virions, such as strand transfer/displacement, processivity, or replication fidelity, and might be inadequate for viral replication in the natural host.
Several conclusions can be drawn from our initial experiments on random sequence substitution of HIV RT and selection by genetic complementation. First, our positive genetic selection system can identify active HIV RT mutants. The mutants we characterized exhibit from 5 to 100% of the DNA-dependent DNA polymerase activity of the wild-type enzyme and include a majority of substitutions located within the target region that have been observed as natural variants and drug-resistant mutations. The ability to select active mutations and to characterize their phenotypes in an alternate bacterial host provides a powerful means for assessing the consequences of specific amino acid substitutions, and combinations of substitutions, on DNA polymerase activity. Second, random mutagenesis of HIV RT enables us to assess the mutability of each amino acid residue in the enzyme. Rarely mutable or immutable residues can be identified by this approach, and such residues can serve as potential targets for more efficacious anti-HIV drug therapy that precludes or delays emergence of resistant variants. Since random mutagenesis can be applied to large targets in HIV RT, and can also survey all possible combinations of amino acid substitutions within the target, the approach offers different parameters for identifying essential amino acids than does inactivation by substitution with alanine (Richardson and Richardson, 1990). Although substitutions within the flexible region we targeted in this study did not reveal immutable sites, it remains to be determined if other more structured domains contain such immutable (essential) residues. Finally, random mutagenesis creates HIV RT mutants which are unlikely to be recovered from the natural host because they cannot support viral replication. Phenotypic and biochemical analysis of such mutants, many of which will have new or altered biochemical properties, can provide insight into the involvement of individual amino acid residues in catalysis and into structure-function relationships within the enzyme.