Our results show that AZT-resistant HIV-1 RTs that contain TAMs and have enhanced nucleotide excision activity also have increased ability to use dinucleoside tetraphosphates of the form Np4
ddN or ddNp4
ddN as substrates for DNA elongation and chain termination. By contrast, when ddNTPs were provided as substrates, the efficiency of chain termination was about the same for TAM-containing RTs and WT RT. The difference between WT and mutant RTs was greatest when MANT-Ap4
ddG was used as a substrate. Chain termination with this substrate by RTMDR
was 120-fold more efficient than by RTWT
. Chain termination with MANT-Ap4
ddG by RTAZT
was 19-fold more efficient than by RTWT
. Other dinucleoside tetraphosphate compounds showed smaller but still substantial differences between mutant and WT RT. Incorporation of ddNMP occurred without prior formation of a ddNTP intermediate, in agreement with a previous report (33
). Our results provide tools for further mechanistic studies and development of drugs that target this class of HIV-1 mutants.
Our results suggest that dinucleoside tetraphosphates have the ability to interact with two adjacent nucleoside binding surfaces on RT: one identified as the binding site for the incoming dNTP in the polymerase reaction and the second identified by the residues mutated in TAMs and lying just distal to the end of the phosphate chain of the incoming dNTP. The dNTP binding site is known from the crystal structure of RTWT
-P/T-dTTP ternary complex (10
), and the locations for the second nucleoside and additional phosphate residue have been inferred by modeling in an ATP molecule with the β and γ phosphates superimposed on the γ and β phosphates of the dTTP (3
). In the proposed structure, the adenine base forms π-π interactions with tyrosine or phenylalanine side chains at residue 215 in the mutant RT. Tests of excision substrate specificity also provide support for this model (24
). The enhanced rate of Np4
ddN-mediated chain termination by TAM-containing RTs can be explained by more favorable binding of Np4
ddN to the mutant enzyme, as observed with ddGp4
ddG (Fig. ). While these results support the ability of the tetraphosphates to interact with adjacent sites on RT, our data indicate that functional binding of ddGTP is favored over ddGp4
ddG for TAM-containing RTs such as RTAZT
. This suggests that binding of Np4
ddN compounds to the ddNTP site is severely restricted in RTWT
and that TAMs confer increased ability to bind these compounds, possibly by removing steric constraints or by contributing binding interactions that can compensate for limited accessibility of the tetraphosphate compounds to the binding site. The enhanced rate of nucleotide-dependent excision by TAM-containing RTs is largely due to effects on catalytic rate that occur after substrate binding since TAMs have little effect on the KD
for the nucleotide substrate (29
). It has been proposed that the T215Y or F mutations increase the rate of nucleophilic attack by controlling the alignment of the pyrophosphate component of the acceptor substrate with respect to the bond to be cleaved in the nascent DNA chain (8
). Similar considerations may explain the ability of these mutations to enhance the use of dinucleoside tetraphosphates as substrates for incorporation.
Incorporation of ddGMP or ddCMP using ddGp4ddG or ddCp4ddC as a substrate was more efficient than incorporation of ddAMP or ddTMP using the corresponding dinucleoside tetraphosphate substrates. This suggests that a G-C base pair was preferred during the reaction. The difference in ddGMP incorporation between RTMDR and RTWT was greatest when MANT-Ap4ddG was used as a substrate, suggesting that RTMDR is better able to accommodate a bulky substituent on the ribose than is RTWT. AppNHppddG was a poor substrate for chain termination with all of the enzymes tested, indicating that the precise structure of the phosphate chain is important.
These results suggest that it may be possible to identify bifunctional compounds that have high affinity for RT and preferentially bind to the TAM-containing enzymes. Dinucleoside tetraphosphate derivatives that contain hydrolyzable phosphate links have the advantage that they may be able to bypass the need for intracellular phosphorylation to lead to chain termination. This would expand the range of nucleoside structures that can be used for chain termination by HIV-1 since current NRTIs are limited to molecules that are phosphorylated by cellular nucleoside kinases. On the other hand, the presence of phosphate residues may limit the ability of these compounds to enter mammalian cells and increase their sensitivity to degradative enzymes such as phosphodiesterases. However, modification of the phosphate residues might improve bioavailability and stability of dinucleoside polyphosphates (2
The ability of dinucleoside tetraphosphates to serve as substrates for DNA chain elongation and termination by HIV-1 RT and the strong preference of these compounds for use by TAM-containing mutant enzymes provide a useful tool for analysis of the effects of these mutations on RT activities. In addition, our studies provide evidence for an interaction surface on the mutant enzymes that recognizes both nucleoside components of the Np4ddN structure and may be an attractive target for future drug development.