We report here the kinetic parameters associated with DNA polymerization and mutation synthesis by MuLV RT on an RNA template. By concomitantly relating these findings to the HIV-1 RT pre-steady-state kinetic results, we provide a comprehensive mechanistic comparison of the HIV-1 and MuLV RTs. Firstly, we examined the kinetics for the incorporation of four different dNTPs (correct) by MuLV and HIV-1 RTs. In this experiment, we observed that whereas the kpol
values of HIV-1 and MuLV RTs for dNTPs are very similar, MuLV RT binds nucleotide substrates with 6.5–121.6-fold lower affinity than HIV-1 RT. We reasoned that the lower binding affinity of MuLV RT to correct dNTPs may contribute to its higher steady-state Km
values compared with HIV-1 RT (7
). As shown in our multiple nucleotide incorporation reaction (), MuLV RT clearly displayed a great reduction of the DNA synthesis at low dNTP concentrations. It is possible that, when the dNTP availability is low, the dNTP binding step of MuLV RT becomes rate-limiting, which reduces its capability of executing multiple nucleotide incorporations that normally occur during viral DNA synthesis. We recently demonstrated that MuLV RT also shows lower dNTP incorporation efficiency than HIV-1 RT under a single round of processive DNA synthesis at low dNTP concentrations (i.e.
). In contrast, due to the tight dNTP binding affinity of HIV-1 RT, this RT protein is still able to retain synthesis activity, even at low dNTP concentrations. This is also consistent with the low steady-state Km
values of HIV-1 RT, compared with those of MuLV RT (6
). The same multiple nucleotide incorporation difference seen between HIV-1 and MuLV RTs () was also observed between the wild type and two dNTP binding mutants (Q151N and V148I) of HIV-1 RT (6
). Models suggested that these two mutations disrupt the interaction between the RT active site and the 3′ OH of the incoming dNTP substrate, resulting in an increase of the Kd
values without affecting the kpol
). Basically, these two dNTP binding HIV-1 RT mutant proteins kinetically mimic MuLV RT in DNA polymerization.
Here, using a series of virological and cellular aspects related with these two different retroviruses, MuLV and HIV-1, we envision possible significances of the dNTP binding difference between these two RTs. Firstly, HIV-1, which is a lentivirus, uniquely infects not only dividing cells (i.e.
activated CD4+ T cells) but also nondividing/terminally differentiated cells (mature macrophages), whereas MuLV, an oncoretrovirus, requires proliferating cells (4
). Macrophage infection, which is observed in the early asymptomatic phase of HIV-1 infection, is a hallmark phenotype of HIV-1 pathogenesis. Secondly, numerous studies have reported that nondividing cells have much lower cellular dNTP concentrations than dividing cells (5
). Using a novel dNTP assay, we recently demonstrated that the dNTP concentration of human mature macrophages is ~40 nm
, which is ~100-fold lower than that of activated CD4+ T cells (2–5 μm
). More importantly, the steady-state Km
values of HIV-1 and MuLV RTs lie near the cellular dNTP concentrations found in macrophages and activated T cells, respectively. Considering all these findings, we can speculate that the tight binding of the HIV-1 active site to the incoming dNTP substrate may contribute to the unique capability of HIV-1 to infect nondividing cells containing low cellular dNTP concentrations. Conversely, the active site of MuLV RT may have adapted to the high dNTP concentration environments found in the dividing cells that MuLV normally infects. This possibility is further supported by our recent observation that HIV-1 variants harboring RT mutants (V148I and Q151N), which kinetically mimic MuLV RT due to their reduced dNTP binding affinity, failed to infect macrophages even though these mutant viruses normally infect dividing cells (i.e.
activated CD4+ T cells and transformed cell lines) (6
Pre-steady-state kinetic study on the M184V 3TC-resistant HIV-1 RT mutant demonstrated that the M184V mutation, which lies near the active site containing the conserved YX
DD sequence, slightly increases Kd
values (2–6-fold) to dCTP (41
), which may explain low infectivity in cells with low dNTP contents (51
). This mutant RT also has reduced processivity (53
), compared with wild-type HIV-1 RT, which could be responsible for lower infectivity of this mutant virus in primary cells (53
). Pre-steady-state kinetic analysis on M184I, which only transiently appears during the 3TC treatment, has not been reported. Interestingly, unlike M184V, M184I has noticeable decreases in the DNA synthesis at low dNTP concentrations,2
supporting that M184I RT, which has a longer β-branched side chain than the Val mutation, may also affect the binding of dNTP to the active site as observed in Q151N and V148I. Our recent pre-steady-state kinetic analysis showed that RT of simian immunodeficiency virus, another lentivirus infecting nondividing cells, also has lower Kd
values, like HIV-1 RT (49
). Additionally, we observed that RT of feline leukemia virus (an oncoretrovirus) has higher Km
values than RT of feline immunodeficiency virus (a lentivirus).2
These combined findings support the idea that the dNTP utilization efficiency and dNTP binding affinity of RTs contribute to the host cell specificity of retroviruses. The generality of this idea needs to be further explored by analyzing more RT proteins isolated from different retroviruses. Furthermore, note that, in order to complete viral replication in nondividing cells, HIV-1 also requires a viral accessory protein, viral protein R (Vpr), which enables the pre-integration complex containing the synthesized proviral DNA to enter the nucleus through the nuclear membrane that remains intact in nondividing cells (55
). In contrast, other retroviruses, which infect dividing cells, do not require this transport mechanism because the pre-integration complex of these viruses can access chromosomes during mitosis where the nuclear membrane barrier disintegrates.
Both MuLV and HIV-1 RTs displayed similar characteristics in dNTP binding and catalysis during misinsertion events. Whereas the rate (kpol) at which MuLV RT incorporates incorrect dNTPs is comparable with that observed with HIV-1 RT, the binding affinity (Kd) of MuLV RT for incorrect dNTPs is 13.2–85.4-fold lower than that of HIV-1 RT. However, because MuLV RT has diminished binding affinity (Kd) for both correct and incorrect dNTPs, there is a simultaneous reduction in its efficiency (kpol/Kd) of correct and incorrect dNTP incorporation. The net effect is that HIV-1 and MuLV RTs have similar misinsertion fidelities ().
The differences between these two RTs became apparent when we measured the kinetics of mismatch extension for MuLV RT. When incorporating correct dNTP onto a mismatched T/P, this polymerase is altered in its ability to bind (Kd) and catalyze the incorporation (kpol) of the nucleotide substrate, even though the primary difference between these RTs is the rate (kpol) at which they carry out mismatch extension. HIV-1 RT incorporates dTTP onto a mismatched T/P at a maximum rate of 0.45 s−1, which is 43.8-fold faster than the rate of mismatch extension observed with the MuLV RT. The changes in dNTP binding and chemical catalysis translate into a 137.9-fold reduction in mismatch extension efficiency (kpol/Kd) for MuLV RT in comparison with HIV-1 RT. The fact that MuLV RT is so inefficient at carrying out mismatch extension supports the likelihood that the polymerase stalls and falls off the mismatched T/P substrate. In this scenario, it is necessary to assess the capability of MuLV RT to rebind the mismatched T/P. We show that MuLV RT has a 3.8-fold lower binding affinity for mismatched T/P (KD) than HIV-1 RT. This result indicates that the overall higher fidelity of MuLV RT relative to HIV-1 RT is due to multiple effects of 1) a 3.1-fold higher mismatch extension fidelity and 2) a 3.8-fold reduced ability to rebind mismatched T/P when RT disassociates from the T/P substrate. Due to a 137.9-fold less efficient mismatch extension and poor binding to mismatched T/P, MuLV RT may more likely release from the mismatched T/P after misinsertion. This could create an additional mechanistic barrier that could slow down the mismatch extension step as observed in and overall mutation synthesis by MuLV RT. In contrast, due to the tighter binding affinity to the mismatched primer and highly efficient capability to extend the mismatched primer, relative to MuLV RT, HIV-1 RT may be able to continue the mismatch extension without falling off from the mismatched T/P, which can lead to high success in the completion of the mutation synthesis by HIV-1 RT.
Using fingerprinting and sequencing analysis of proviral DNA, Monk et al.
) reported that MuLV infection of a clonal cell line yielded a mutation rate of 2 × 10−5
bases per replication cycle. Using the cell-free M13 lac
Zα forward mutation assay, Roberts et al.
) reported that the MuLV RT had an error rate of 1/30,000, which can account for the estimated in vivo
MuLV mutation rate published in the aforementioned study. Nevertheless, Roberts et al.
) did report that the error rate of MuLV RT is ~15-fold higher than that of HIV-1 RT, and our findings suggest that the mechanism responsible for this fidelity difference is their differing abilities to complete the process of mismatch extension. The idea that mismatch extension plays an important role in determining the overall fidelity of retroviral RTs was established in 1989 when Perrino et al.
) reported that HIV-1 RT is 50-fold more efficient than DNA polymerase α at synthesizing DNA from a mismatched T/P. This property was characterized in a more quantitative manner when Bakhanashvili and Hizi (20
) measured the steady-state kinetics of mismatch extension for both HIV-1 and MuLV RTs. When they measured the mismatch extension capabilities of these two polymerases with three different mismatched T/Ps, the authors reported that the relative mismatch extension frequencies of MuLV RT were, on average, 2–3-fold lower than those seen with HIV-1 RT.
The fact that HIV-1 RT is very efficient (kpol
) at carrying out mismatch extension emphasizes the underlying differences between HIV-1 RT and other retroviral polymerases. The viral polymerase of HIV-1 must have evolved so that its active site is more permissive to introducing mutations into the viral genome. Interestingly, our laboratory has recently identified a molecular interaction within the HIV-1 RT active site that influences how efficiently this polymerase is able to complete the second step of mutation synthesis (39
). The Gln151
residue is part of the highly conserved LPQG motif found in all retroviral RTs. Previous studies have shown that the interaction between this residue and the dNTP substrate is a determinant for HIV-1 RT fidelity (26
). When we performed a comprehensive analysis on the Q151N mutant RT in order to assess its mutation synthesis capabilities, we discovered that alterations in the Gln151
residue reduced the efficiency with which HIV-1 RT incorporated correct dNTP into both matched and mismatched T/Ps (39
). More specifically, Q151N has a higher Kd
value and a lower kpol
value than wild-type HIV-1 RT during mismatch extension, as observed in this study with MuLV RT. In addition, whereas Q151N has a reduced Kd
(but not kpol
) value for both correct and incorrect dNTPs compared with wild-type HIV-1 RT, Q151N does not alter the binding affinity to matched or mismatched primers. This finding suggested that the interactions between Gln151
and the dNTP substrate within the HIV-1 RT active site appear to influence how efficiently this polymerase is able to complete mismatch extension. Interestingly, however, these mechanistic differences between wild-type and Q151N HIV-1 RT proteins are very similar to those observed between HIV-1 and MuLV RTs. This indicates that the active site of the Q151N dNTP binding mutant HIV-1 RT kinetically functions more like the active site of MuLV RT. A similar interaction may occur within the MuLV RT active site, given that it encodes an equivalent residue, Gln190
. Biochemical data showing that alterations (Q190N) in this residue can increase the fidelity of the MuLV RT suggest that this residue has the same functional role in both the HIV-1 and MuLV RTs (28
). However, given the dramatic difference in the mismatch extension fidelities between the wild-type proteins of these two RTs, it is plausible that there exist interactions (in addition to those with Gln151
) between HIV-1 RT and the mismatch extension intermediates that are not present in the MuLV RT.
In addition to Q151N and V148I, the fidelity of the M184V HIV-1 RT, which renders 3TC resistance, has been characterized by pre-steady-state kinetic analysis (41
). The M184V HIV-1 RT, which showed an ~2-fold increase fidelity in M13 lac
Zα forward mutation assay (62
), showed a maximum 2.4× higher fidelity than wild-type RT in the kinetic analysis. Interestingly, the Kd
increase contributes to the high fidelity nature of M184V. In addition, M184V HIV-1 RT also showed reduced mismatch primer extension capability (63
). M184I HIV-1 RT showed slightly higher fidelity than M184V (62
). However, the pre-steady-state kinetic analysis has not been reported. A number of other HIV-1 RT mutations altering enzyme fidelity were isolated: D76V (64
), R78A (65
), E89G (63
), and the residues in the minor groove binding track (66
). Structural models on D76V, R78A, and E89G suggested that these mutations may affect the RT interaction with template, whereas the minor groove binding track residues interact with the minor groove of the T/P, where the transition from A to B DNA and bending occur. Our pre-steady-state kinetic study shows that D76V and R78A do not show any altered Kd
which is consistent with the previous report that these mutations mainly affect RT binding to the single-stranded part of the primer (64
). Mutations in the minor groove binding track region (i.e.
) mainly affect replication frameshift mutation and processivity through altering the RT interaction with the minor groove of the double-stranded T/P. Basically, these studies with the RT mutants with altered fidelity suggest that the RT interaction with substrates, dNTP and T/P, is a key determinant for RT fidelity.
In summary, this is the first time that a comprehensive pre-steady-state kinetic analysis of dNTP incorporation and mutation synthesis by MuLV RT has been reported. The mechanistic analysis of HIV-1 and MuLV RTs clearly sheds lights on the functional and evolutionary relatedness between the dNTP interactions within the RT active site and the virological characteristics of retroviruses such as cell type specificity and genomic mutagenesis.