In Vivo Prevalence of A360V
—The in vivo
prevalence of mutations in the connection domain of RT was studied in a large cohort of individuals from British Columbia, Canada, as a function of exposure to antiretroviral selection pressure. The populations studied included both treatment-experienced (n
= 2422) and treatment-naive (n
= 1606) individuals. Several positions in the connection domain, in particular Asn348
, showed large increases in prevalence of amino acid substitutions in the treatment-experienced population (). Substitutions at codon 348 increased in prevalence from 0.8% in the untreated population to over 12.1% in the treatment-experienced populations (26
), whereas those at position 360 increased in prevalence from 21.4% to over 28.8%, suggesting a significant selective advantage. The increase in prevalence at codon 360 was driven in part by a modest increase in the prevalence of a common polymorphism A360T, but predominantly by an increase in the A360V substitution. Like the N348I mutation, A360V was observed in less than 1% of the untreated population (n
= 15 of 1606) and increased to 8.5% after treatment (n
= 205 of 2422). Other mutations (A360I and A360S) were observed only rarely. Furthermore, A360V was highly correlated with various mutations considered to be part of the TAMs cluster; for example, of the samples with A360V, 35% were associated with M41L, 21% with D67N, 30% with K70R, 17% with L210W, 42% with T215Y/F, and 17% with K219Q/E. In contrast, less than 20% of samples with the A360T had TAMs. A360V usually appeared late in therapy, on average 2.5 years after the onset of antiretroviral treatment, and typically appeared after TAMs selection. The A360V mutation appeared in over 120 TAM contexts, but the most common TAM patterns in which it appeared consisted of M41L/215Y or M41L/L210W/T215Y.
Prevalence of mutations in the connection domain of RT in treated and untreated individuals
Connection Domain Mutations A360V and N348I Increase ATP-mediated Excision on DNA
—The combination of A360V and N348I, when present against a background of TAMs was shown to amplify resistance to AZT but not to d4T (33
). The crucial role played by TAMs suggests that the combination of TAMs and connection domain mutations further increase efficiency of ATP-mediated AZT-MP excision. To address this question directly, we monitored DNA synthesis in the presence of AZT-TP and ATP on a relatively long RNA template that allows polymerization of 185 nucleotides. The reaction covers incorporation and excision of AZT-MP as well as the ensuing rescues of DNA synthesis. The data show that enzymes containing TAMs/A360V and TAMs/N348I, respectively, increased full-length product formation when compared with TAMs (). The efficiency of rescue of DNA synthesis follows the order WT RT < TAMs < TAMs/A360V < TAMs/N348I < TAMs/A360V/N348I.
FIGURE 1. ATP-mediated excision of AZT-MP and rescue of DNA synthesis on a DNA·RNA substrate. The long DNA·RNA (PBS-28/PBS-250) substrate was employed to assess DNA synthesis rescue over time in the presence of AZT-MP and ATP for each of the RT (more ...) A360V and N348I Reduce RNase H Activity and Mediate the Accumulation of Truncated Substrates
—N348I-containing mutant enzymes were shown to reduce RNase H activity (26
). Here, time course experiments were employed to compare RNase H cleavage efficiencies in the context of each of the mutant enzymes described above. Reactions were monitored ona5′-end-labeled RNA template (). Deficits in RNase H activity are seen here in both delayed degradation of the full-length substrate and diminished formation of the shorter fragment. This finding suggests that connection mutations affect primary RNase H cuts as well as secondary cuts that occur when the RT enzyme moves in the 3′ to 5′ direction of the template (41
). The shortest fragment (i.e.
the cut at position -6) is only seen with WT RT and to a lesser degree with TAMs (). Overall, the efficiency of RNase H cleavage follows the order WT RT > TAMs > TAMs/A360V > TAMs/N348I > TAMs/A360V/N348I, which correlates inversely with the respective efficiencies of excision.
FIGURE 2. RNase H-mediated RNA template degradation. A, the 22-bp hybrid duplex consists of the DNA primer and the RNA template radiolabeled at the 5′-end (indicated by an asterisk). B, the DNA·RNA substrate was incubated with the RT enzyme (WT, (more ...)
Accumulation of Transiently Formed DNA
—As a consequence of the diminished RNase H activity with A360V and N348I, medium sized fragments (e.g.
fragments with RNase H cuts at positions -12, -9, and -8) accumulate when compared with WT RT that ultimately produces the -6 product (). We then wanted to determine whether the shorter RNA fragments can form stable DNA·RNA hybrids that serve as substrates for the excision reaction. Preformed RT complexes with a 21-bp DNA·RNA hybrid duplex were incubated with Mg2+
at different time points to monitor the effect of RNase H cleavage on product formation under nondenaturing conditions (). In this experiment, we compared WT RT with TAMs/A360V/N348I that showed a severe reduction in RNase H cleavage. Our results show an accumulation of transiently formed hybrids with the mutant enzyme (). The corresponding band co-migrates with an 11-bp DNA·RNA duplex that was derived from the original 21-bp substrate (). These data show that the mutant enzyme produces shorter hybrids that dissociate from the complex and accumulate in a short window of time. In contrast, such transiently formed hybrids are hardly seen with WT RT. In this case, RNase H cleavage appears to force the release of the primer at early time points, and the primer does not rebind to the enzyme under these conditions. The release of the primer is less efficient with the mutant enzyme, which provides experimental evidence for the hypothesis that connection domain mutations can delay the irreversible degradation of the DNA·RNA substrate (33
FIGURE 3. Accumulation of transiently formed hybrids with the TAMs RT containing connection domain mutations A360V and N348I. A, DNA·RNA·RT binary complex formation was monitored in a time course. Lane 1, control radiolabeled DNA primer alone. The (more ...)
This experiment also suggests that transiently formed hybrids dissociate from the RT complex before the template is completely degraded. However, the mutant shows an increased rate of excision, which requires rebinding of the shorter substrates. Excision can only occur when the 3′-end of the primer is located at the polymerase active site. In this conformation, the RNase H domain may only cleave the substrate when the hybrid region contains at least 18 or 19 base pairs, which defines the distance between polymerase and RNase H active sites (, top
). Shorter templates are not in contact with the RNase H domain. Thus, RNase H cleavage must occur in a different conformation, in which the polymerase active site has released the 3′-end of the primer (, bottom
). We therefore need to distinguish between polymerase- (or excision)-competent complexes and RNase H-competent complexes, respectively. These complexes are structurally distinct, and connection domain mutations may influence structure and function of RT depending on the particular conformation.
To analyze whether the shorter hybrids can serve as substrates for AZT-MP excision and rescue of DNA synthesis, we utilized an AZT-terminated primer that was hybridized with a short RNA fragment to form a 12-bp hybrid duplex (). The efficiency of excision showed the following order in a time course: WT RT < TAMs < TAMs/A360V/N348I (). Thus, connection domain mutations facilitate excision of AZT-MP on short DNA·RNA substrates. DNA·RNA hybrids with a duplex region that is shorter than 8 bp are not used for the combined excision/rescue reaction (supplemental Fig. S2).
FIGURE 4. Single-site AZT-MP excision and rescue of DNA synthesis. A, a single-site AZT-MP excision assay was performed on the 12-bp hybrid duplex, which was pre-chain-terminated with AZT (indicated by Z at the 3′-terminus of the DNA primer). ATP-mediated (more ...)
When the next complementary nucleotide occupies the active site of RT in the presence of a chain-terminated primer, a dead end complex is formed, and excision is no longer possible (19
). D4T-MP was shown to be sensitive to dead end complex formation, whereas AZT-MP compromises binding of the next nucleotide (47
). The same result was obtained with the short hybrid substrate, which helps to explain why connection domain mutations N348I and A360V do not amplify resistance to d4T (33
) (supplemental Fig. S3).
The Effect of Connection Domain Mutations on Substrate Binding—To determine the effects of connection domain mutations on substrate binding, we measured the equilibrium dissociation constant (Kd) in the context of both polymerase- and RNase H-competent complexes.
A recent study has shown that the connection domain mutation G333D can increase substrate binding under steady-state conditions (29
). Our experiments were performed under single turnover conditions to avoid multiple dissociation and reassociation events that complicate the interpretation of the data. We initially determined the minimum length of a DNA·RNA substrate that allowed us to measure Kd
values under single turnover conditions and found that RNase H activity could not be reliably quantified when the duplex region is shorter than 16 base pairs (supplemental Fig. S4). To determine the equilibrium dissociation constant of the RNase H-competent complex (Kd(RNase H)
), we monitored RNase H activity for each of the aforementioned mutant enzymes on the 16-bp hybrid substrate (). Substrate binding appears to be slightly compromised (~2-fold) with TAMs (Kd(RNase H)
of 188.1) when compared with WT RT, which showed a Kd(RNase H)
value of 104 nm
(). This effect is much more pronounced in the presence of connection mutations. TAMs/A360V showed a moderate 3.6-fold increase in Kd
values, TAMs/N348I showed an 8.8-fold increase, and RNase H activity is hardly detectable when both mutations were combined with TAMs.
FIGURE 5. Affinity of RT enzyme for the DNA·RNA substrate in the RNase H-competent complex. A, the 16-bp hybrid duplex consists of the DNA primer and the truncated RNA template radiolabeled at the 5′-end (indicated by an asterisk). B, RNA degradation (more ...)
A similar pattern is seen with a longer substrate that contains the 22-bp hybrid duplex region. However, substrate binding is generally increased when compared with the shorter 16-bp hybrid. Together, the data show that the loss of contacts with the polymerase domain cause severe reductions (5-10-fold) in substrate binding, and this effect is enhanced with the connection domain mutations.
These results raise the question of whether connection domain mutations may likewise affect binding in the context of polymerase- or excision-competent complexes. Diminished binding would compromise nucleotide incorporation and its excision, which is counterintuitive given the role of these mutations in AZT resistance. Excision of AZT-MP is generally inefficient and therefore difficult to measure under single turnover conditions. Thus, we made use of nucleotide incorporation events as a read-out to study binding in the polymerase/excision-competent complex. We utilized a 15-bp hybrid substrate that becomes extended to a 16-bp hybrid following nucleotide incorporation. WT and mutant RT enzymes show Kd(pol) values in a close range between 10 and 25 nm (). TAMs containing RT show a 2-fold increase in Kd(pol) when compared with WT RT. A360V appears to compensate for this deficit, whereas N348I shows no significant effect on substrate binding in this context. Kd(pol) values are at least an order of magnitude lower than Kd(RNase H) values, which confirms that the loss of contacts with the polymerase domain diminishes substrate binding in the RNase H-competent binding mode. Connection domain mutations appear to amplify these effects and cause substrate dissociation selectively from the RNase H-competent conformation.
Effect of N348I and A360V on HIV-1 RT Processivity—Differences in substrate binding in polymerase-competent complexes are generally low, although A360V appears to correct for the modest deficiency of TAMs. Such subtle differences in substrate binding, measured at a single primer-template position, may translate to differences in processive DNA synthesis that is monitored over multiple positions. To address this question, we compared DNA synthesis with the long RNA template under single turnover conditions in the absence of inhibitors. TAMs showed reduced full-length product formation under these conditions when compared with WT RT (). Processive DNA synthesis is then increased in the following order: TAMs/A360V < TAMs/N348I < TAMs/A360V/N348I. The latter mutant enzyme shows even higher levels of full-length product formation as compared with WT RT. Some pausing sites that occur toward the end of the template are more pronounced, with enzymes containing connection domain mutations. This observation shows that these enzymes produce more intermediate product; however, the complexes are likewise vulnerable to dissociation. To provide a measure for processive DNA synthesis, we have focused on the final product and compared the fraction of competent complexes between the different enzymes that have reached the end of the template ().
FIGURE 6. The effect of connection domain mutations on processive DNA synthesis. The long DNA·RNA substrate was incubated with each of the RT enzymes, and DNA elongation was initiated with the simultaneous addition of MgCl2 and the heparin trap. Product (more ...)
Thus, the subtle increases in substrate binding in the polymerase-competent mode translate into increases in processive DNA synthesis with A360V-containing enzymes. Unexpectedly, N348I-containing enzymes can likewise increase processive DNA synthesis, although these enzymes do not appear to increase nucleic acid binding in this context.
N348I Increases PPi-mediated Excision of AZT-MP
—At this point, our data show that both factors (i) reduced RNase H activity and (ii) increased processivity correlate with increases in ATP-dependent excision of AZT-MP and rescue of DNA synthesis when TAMs and connection domain mutations are combined. Reductions in RNase H cleavage and increases in processive DNA synthesis affect the excision reaction indirectly. Diminished RNase H activity provides more time for excision to occur, and increases in processive DNA synthesis can compensate for the low rates associated with this reaction. Thus, increases in excision and rescue of DNA synthesis may be seen independently of the nature of the PPi
donor. To test this hypothesis, we studied the effects of the various mutant enzymes on PPi
-mediated excision of AZT-MP and the ensuing rescue of DNA synthesis. PPi
was used at physiologically relevant concentrations of 50 μm
(). The RT enzymes containing TAMs alone or TAMs/A360V did not show significant differences in full-length product formation. However, marked increases in AZT-MP excision and rescue of DNA synthesis with TAMs/N348I and TAMs/A360V/N348I were observed. To test whether these effects can be seen independently of TAMs, which do not appear to improve binding and usage of PPi
), we conducted the same experiment with the single mutations A360V and N348I, respectively. Our data show that the N348I mutant confers increases in PPi
-mediated rescue of AZT-terminated DNA synthesis when compared with WT RT and TAMs (supplemental Fig. S5). The effects of A360V are by far not as pronounced as seen with N348I.
FIGURE 7. PPi-mediated multisite excision of AZT-MP on the DNA·RNA nucleic acid substrate. DNA synthesis rescue was monitored over time in the presence of AZT and PPi for each of the RT enzymes (WT, TAMs, TAMs/A360V, TAMs/N348I, and TAMs/A360V/N348I, respectively) (more ...) Connection Domain Mutations Can Increase AZT-MP Excision Independently of RNase H Activity
—Finally, we studied whether increases in processive DNA synthesis may be sufficient to increase levels of rescue of DNA synthesis. To address this, we introduced the RNase H-negative E478Q mutation (48
) against a background of TAMs and TAMs/A360V/N348I. The latter shows the strongest effects with regard to increases in processive DNA synthesis, increases in AZT-MP excision, and decreases in RNase H activity. When comparing TAMs with TAMs/E478Q, we observed increases in ATP-dependent rescue of AZT-terminated DNA synthesis (). We obtained essentially the same results with PPi
as the substrate for excision (supplemental Fig. S6). Also, the same effect is seen when comparing TAMs/A360V/N348I with TAMs/A360V/N348I/E478Q, which shows that the loss of RNase H activity enhances the rescue reaction. In addition, the comparison of TAMs/A360V/N348I/E478Q with TAMs/E478Q points to a strong RNase H-independent component. Formation of the full-length product is much higher with TAMs/A360V/N348I/E478Q, which shows that the contribution to increases in rescue of DNA synthesis is in part RNase H-independent.
FIGURE 8. RNase H-independent ATP-mediated AZT-MP excision on the DNA·RNA substrate. DNA synthesis and ATP-mediated AZT-MP excision were monitored with enzymes containing the E478Q mutation (TAMs, TAMs/E478Q, TAMs/A360V/N348I, and TAMs/A360V/N348I/E478Q, (more ...)