The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

The pyrophosphate (PPi) analogue phosphonoformic acid (PFA or foscarnet) inhibits the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1); however, the mechanisms of drug action and resistance remain elusive. Here we studied the effects of the translocational status of HIV-1 RT on drug binding and inhibition of DNA synthesis. We identified “hot spots” for inhibition during active elongation. Site-specific footprinting analyses revealed that the corresponding complexes exist predominantly in the pre-translocational state. The sensitivity to PFA is significantly reduced with sequences that show a bias toward the post-translocational state. Binding studies showed that PFA stabilizes selectively the complex in the pre-translocated configuration. These findings are consistent with a Brownian ratchet model of polymerase translocation. The enzyme can rapidly shuttle between pre- and post-translocated states. The bound inhibitor acts like a pawl of a ratchet and prevents the forward motion of HIV-1 RT, whereas the bound nucleotide binds to the post-translocated complex and prevents the reverse motion. The proposed mechanisms of RT translocation and drug action are consistent with the PFA-resistant phenotypes. We show that certain sequences and the PFA-resistant E89K mutant diminishes the stability of the pre-translocated complex. In these cases, the enzyme is seen at multiple positions around the 3′ end of the primer, which provides a novel mechanism for resistance. These findings validate the pre-translocated complex as a target for the development of novel, perhaps less toxic and more potent inhibitors that block HIV-1 RT translocation. The pyrophosphate (PPi) analogue phosphonoformic acid (PFA or foscarnet) inhibits the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1); however, the mechanisms of drug action and resistance remain elusive. Here we studied the effects of the translocational status of HIV-1 RT on drug binding and inhibition of DNA synthesis. We identified “hot spots” for inhibition during active elongation. Site-specific footprinting analyses revealed that the corresponding complexes exist predominantly in the pre-translocational state. The sensitivity to PFA is significantly reduced with sequences that show a bias toward the post-translocational state. Binding studies showed that PFA stabilizes selectively the complex in the pre-translocated configuration. These findings are consistent with a Brownian ratchet model of polymerase translocation. The enzyme can rapidly shuttle between pre- and post-translocated states. The bound inhibitor acts like a pawl of a ratchet and prevents the forward motion of HIV-1 RT, whereas the bound nucleotide binds to the post-translocated complex and prevents the reverse motion. The proposed mechanisms of RT translocation and drug action are consistent with the PFA-resistant phenotypes. We show that certain sequences and the PFA-resistant E89K mutant diminishes the stability of the pre-translocated complex. In these cases, the enzyme is seen at multiple positions around the 3′ end of the primer, which provides a novel mechanism for resistance. These findings validate the pre-translocated complex as a target for the development of novel, perhaps less toxic and more potent inhibitors that block HIV-1 RT translocation. Different classes of inhibitors that target the reverse transcriptase (RT) 4The abbreviations used are: RT, reverse transcriptase; HIV, human immunodeficiency virus; NRTI, nucleoside analogue reverse transcriptase inhibitor; NNRTI, non-nucleoside analogue RT inhibitor; PFA, phosphonoformic acid; PPT, polypurine tract; AZT, 3′-azido-3′-deoxythymidine. 4The abbreviations used are: RT, reverse transcriptase; HIV, human immunodeficiency virus; NRTI, nucleoside analogue reverse transcriptase inhibitor; NNRTI, non-nucleoside analogue RT inhibitor; PFA, phosphonoformic acid; PPT, polypurine tract; AZT, 3′-azido-3′-deoxythymidine. of the human immunodeficiency virus type 1 (HIV-1) have been developed (1De Clercq E. Expert Opin. Emerg. Drugs. 2005; 10: 241-273Crossref PubMed Scopus (142) Google Scholar). Nucleoside analogue reverse transcriptase inhibitors (NRTIs) are major components in drug regimens that are currently used in the clinic. Two different NRTIs are usually combined with a non-nucleoside analogue RT inhibitor (NNRTI) or a protease inhibitor. The triphosphate forms of NRTIs compete with natural nucleotide pools for incorporation, and, once incorporated, the monophosphate acts as a chain terminator. NNRTIs bind to a hydrophobic pocket in the vicinity but not at the active site of HIV-1 RT. Previous studies have suggested that these compounds interfere with the chemical step, whereas nucleotide binding does not appear to be largely affected (2Spence R.A. Anderson K.S. Johnson K.A. Biochemistry. 1996; 35: 1054-1063Crossref PubMed Scopus (79) Google Scholar, 3Rittinger K. Divita G. Goody R.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8046-8049Crossref PubMed Scopus (188) Google Scholar). Here we studied the mechanism of action of the pyrophosphate (PPi) analogue phosphonoformic acid (PFA or foscarnet), which represents a third class of RT inhibitors. PFA shows a broad spectrum of antiviral activities against various members of the Herpesviridae and Retroviridae (4Oberg B. Pharmacol. Ther. 1982; 19: 387-415Crossref PubMed Scopus (179) Google Scholar). The inhibitor is used in the clinic to treat infection with herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), the human cytomegalovirus, and other related herpesviruses when firstline agents have failed (5De Clercq E. J. Clin. Virol. 2004; 30: 115-133Crossref PubMed Scopus (832) Google Scholar). Toxic side effects and its poor bioavailability limit its clinical utility as a component in drug regimens for HIV treatment. Despite these problems, PFA is sometimes used in patients infected with multidrug-resistant HIV-1 variants when no other treatment options are available (6Mathiesen S. Roge B.T. Weis N. Lundgren J.D. Obel N. Gerstoft J. Aids. 2004; 18: 1076-1078Crossref PubMed Scopus (14) Google Scholar). The beneficial effects of this drug have been primarily associated with its resistance profile. With few exceptions, mutations that confer decreased susceptibility to NRTIs and NNR-TIs do not affect susceptibility to PFA (7Mellors J.W. Bazmi H.Z. Schinazi R.F. Roy B.M. Hsiou Y. Arnold E. Weir J. Mayers D.L. Antimicrob. Agents Chemother. 1995; 39: 1087-1092Crossref PubMed Scopus (75) Google Scholar, 8Meyer P.R. Matsuura S.E. Zonarich D. Chopra R.R. Pendarvis E. Bazmi H.Z. Mellors J.W. Scott W.A. J. Virol. 2003; 77: 6127-6137Crossref PubMed Scopus (62) Google Scholar, 9Tachedjian G. Hooker D.J. Gurusinghe A.D. Bazmi H. Deacon N.J. Mellors J. Birch C. Mills J. Virology. 1995; 212: 58-68Crossref PubMed Scopus (39) Google Scholar). Most importantly, mutations associated with PFA resistance were shown to increase susceptibility to the nucleoside analogue 3′-azido-3′-deoxythymidine (zidovudine or AZT) (10Tachedjian G. Mellors J. Bazmi H. Birch C. Mills J. J. Virol. 1996; 70: 7171-7181Crossref PubMed Google Scholar, 11Hammond J.L. Koontz D.L. Bazmi H.Z. Beadle J.R. Hostetler S.E. Kini G.D. Aldern K.A. Richman D.D. Hostetler K.Y. Mellors J.W. Antimicrob. Agents Chemother. 2001; 45: 1621-1628Crossref PubMed Scopus (43) Google Scholar). Biochemical data have shown that PFA-associated resistance mutations diminish the phosphorolytic excision of incorporated chain terminators such as AZT-monophosphate (8Meyer P.R. Matsuura S.E. Zonarich D. Chopra R.R. Pendarvis E. Bazmi H.Z. Mellors J.W. Scott W.A. J. Virol. 2003; 77: 6127-6137Crossref PubMed Scopus (62) Google Scholar, 12Arion D. Sluis-Cremer N. Parniak M.A. J. Biol. Chem. 2000; 275: 9251-9255Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Wild type HIV-1 RT is capable of removing AZT-monophosphate in the presence of pyrophosphate (PPi) (13Rigourd M. Lanchy B. C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Google or a such as P.R. Matsuura S.E. Scott W.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of the excision are significantly with RT resistance which provides mechanism for resistance to this class of compounds P.R. Matsuura S.E. Scott W.A. Full Text Full Text PDF PubMed Scopus Google Scholar). The mechanisms in PFA drug action and drug resistance remain to be with RT and DNA of herpes simplex virus or a of inhibition of DNA with to the triphosphate (4Oberg B. Pharmacol. Ther. 1982; 19: 387-415Crossref PubMed Scopus (179) Google Scholar, J. 92: Full Text PDF PubMed Scopus Google Scholar, C. G. M. J. Virol. PubMed Scopus Google Scholar). In PFA and which to binding for PFA and D. Y. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar). HIV-1 RT been in various forms in the presence of and with a bound J. K. Hsiou Y. A.D. A. C. Arnold E. J. Biol. PubMed Scopus Google Scholar, H. Chopra PubMed Scopus Google however, of complexes with PFA or are not In a of nucleotide incorporation, is the of a Biochemistry. 30: PubMed Scopus Google Scholar, S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Johnson K.A. Anderson K.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Binding of the nucleotide the of this complex. The enzyme a to the nucleotide binding site motion to the acid the 3′ end of the to the or site A.D. K. S. Arnold E. J. PubMed Scopus Google Scholar, 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The is to as the post-translocational state. revealed that or the excision can in the pre-translocational A.D. S. K. D. H. Arnold E. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus (75) Google Scholar, B. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). for the of inhibition been suggested that PFA bind to the pre-translocated complex (8Meyer P.R. Matsuura S.E. Zonarich D. Chopra R.R. Pendarvis E. Bazmi H.Z. Mellors J.W. Scott W.A. J. Virol. 2003; 77: 6127-6137Crossref PubMed Scopus (62) Google Scholar). of PFA appear to incorporated which provides for this C. E. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to be seen the pre-translocational is during active DNA synthesis. is the We have developed footprinting that the between pre- and post-translocated B. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Here we that the of the acid the of pre- and post-translocational at a inhibition of DNA is to sequences that the of the pre-translocational state. These findings that pre- and post-translocated complexes nucleotide The analogue the pre-translocated which prevents binding of the PFA is a inhibitor. The of inhibition is a of the stability of the which diminishes the The of this validate the pre-translocated complex as a target for drug and development and reverse transcriptase and as J. PubMed Scopus Google Scholar). were the to the used in this were and The sequences were used as The sequences were used as PPT, and were at and phosphonoformic acid DNA of the with the The with of HIV-1 RT in a and in the or presence of PFA and of the The to at for and the of of and The DNA in of of and The were on Site-specific of with of on of to the and with of 2 were at In of the footprinting the 3′ with the The with of RT in a and in a of Different of PFA were to the of at The complex with of with of HIV-1 RT in a and in a of The DNA chain with the to of the PFA or the at different to the of The complexes were for at and the were on The were as the of for of the to a for site binding as Matsuura S. Scott W.A. Biochemistry. PubMed Scopus Google Scholar). and for nucleotide in the presence or of PFA were M. M. N. M.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We used different and that the of nucleotide The of the nucleotide at a in the presence of of the were in the presence of of were in the presence of of and of active site RT. the of to a of and were to for at positions and and at to that the of the were the The of the RT and the of and the of nucleotide The were as nucleotide as the of the DNA to 1 The of nucleotide The data were to the of to and as of nucleotide as the of at which the of nucleotide to the were and as a of the of of PFA in a of were the of PFA of PFA in decreased a of were the of PFA and for a nucleotide We that were to effects associated with inhibition of DNA synthesis. The of this to and complexes that increase or the effects of PFA to the between the of inhibition and the translocation status of HIV-1 RT. for of DNA a and the of DNA in the presence of of PFA and The the polypurine of HIV-1 as M. M. N. M.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). with PFA show at positions and of the that DNA to positions and of the inhibitor less can be which to data shows that inhibition of DNA in the presence of PFA is chain terminators that with a is not with to that are a as with the with PFA and that are in the presence of are seen at positions and as with major of inhibition with PFA that appear at and of the reverse the in the that and PFA can bind to the the of PFA to block DNA are significantly as with the of at the in the pre-translocated is that sequences associated with for or PFA binding the pre-translocated state. this we footprinting that to the of HIV-1 RT on its at nucleotide of the of HIV-1 RT on PFA to the complexes that are associated with of These are to as and we have for complexes at positions and With the of the effects of PFA are less at the at is seen at which of of these complexes with the chemical which is a of shown to the site at positions to the nucleotide that is with the nucleotide of the B. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). at is for the post-translocated whereas at is for the state. The is in the which is in the vicinity of the we the footprinting in the and presence of of PFA to in the can affect the of complexes that exist pre- or and the inhibitor or the other configuration. The data show that complexes associated with PFA exist predominantly in the pre-translocated at in the of inhibitor. of PFA do not a in the In complex which is not a PFA as a of pre- and post-translocated with toward at of PFA a toward which provides that the inhibitor binds to and stabilizes the pre-translocated state. is seen with the other and and and and and inhibition and these complexes in the pre-translocated in the of inhibitor. The other complexes exist as The of pre- and post-translocated on and the of PFA that is to the complex in the pre-translocated configuration. of PFA are at positions and that are to PFA of PFA are at positions and and these complexes are associated with of inhibition with These findings to a between the of inhibition and to the pre-translocational which is of with PFA or the of the translocation status of HIV-1 RT with to the mechanism of PFA we the increase in of the pre-translocational with binding of the inhibitor. have revealed that complexes of HIV-1 RT and are when with a Matsuura S. Scott W.A. Biochemistry. PubMed Scopus Google Scholar). the presence of can the which prevents in the presence of These findings are consistent with footprinting that the nucleotide the complex in the post-translocated B. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, B. M. J. Biol. 2004; PubMed Scopus Google Scholar). Here we used complexes as for a pre-translocated and as for a post-translocated to at the stability of complexes with PFA and the We that is with of PFA, whereas a complex is not with The to which the nucleotide stabilizes the complexes the The for at complex is significantly as with complex The data are consistent with and to the that PFA and the nucleotide bind to pre- and post-translocated that a complex predominantly in or the other a in this PFA binds at or to the binding site in the pre-translocated we in the and presence of of PFA are to the complex in the presence of which is in the of The for complex with when with data that PFA binding to the pre-translocated in to the binding at the active site of HIV-1 RT. bind to a however, be in the of binding of and PFA the in the and the of PFA to as these findings that binding of PFA and the of the pre-translocated limit to the post-translocated state. The bind to the pre-translocated the 3′ end of the and the bound PFA the nucleotide binding model that the inhibition of DNA with PFA is in the of the translocation status on for a nucleotide and its inhibition with PFA, we a complex that is to PFA inhibition and a complex that is less to PFA inhibition We have complex as for a that is not associated with PFA inhibition The sequences to limit DNA to nucleotide in The are shown in 1 and with to and are shown in The of nucleotide the These findings are consistent with the that sequences that the post-translocated nucleotide whereas sequences that exist predominantly diminish the of nucleotide The different of the and is that can affect the of the inhibitor is in and the of PFA inhibition the These findings are consistent with the that sequences that the pre-translocated PFA binding whereas sequences that exist predominantly diminish PFA of nucleotide and its inhibition in a with the complex is associated with in whereas to a of which is when the multiple on or (4Oberg B. Pharmacol. Ther. 1982; 19: 387-415Crossref PubMed Scopus (179) Google Scholar). In inhibition of the complex is in whereas to a of in this binding of PFA and the nucleotide is The and the are in with and these data that PFA is a inhibitor that acts at sequences that the pre-translocated state. The of inhibition seen with of inhibition The of the complex diminishes enzyme and its to the which the is to PFA, which that binding of the inhibitor to the and the complex is to PFA mechanisms associated with resistance to PFA, we footprinting with complexes at positions and The that exist predominantly which nucleotide binding and prevents binding of PFA In associated with at multiple which that the PFA can bind in this The presence of PFA the pre-translocated at binding of PFA to complexes and which to the resistance at these In this is to that is associated with of RT at which is in with the that the stability of the post-translocated complex is The of PFA can the of resistance mutations in the RT in and in (7Mellors J.W. Bazmi H.Z. Schinazi R.F. Roy B.M. Hsiou Y. Arnold E. Weir J. Mayers D.L. Antimicrob. Agents Chemother. 1995; 39: 1087-1092Crossref PubMed Scopus (75) Google Scholar, 9Tachedjian G. Hooker D.J. Gurusinghe A.D. Bazmi H. Deacon N.J. Mellors J. Birch C. Mills J. Virology. 1995; 212: 58-68Crossref PubMed Scopus (39) Google Scholar). With few exceptions, of these mutations are the active site to affect the with the or acid (8Meyer P.R. Matsuura S.E. Zonarich D. Chopra R.R. Pendarvis E. Bazmi H.Z. Mellors J.W. Scott W.A. J. Virol. 2003; 77: 6127-6137Crossref PubMed Scopus (62) Google Scholar). The is perhaps the that is in clinical (7Mellors J.W. Bazmi H.Z. Schinazi R.F. Roy B.M. Hsiou Y. Arnold E. Weir J. Mayers D.L. Antimicrob. Agents Chemother. 1995; 39: 1087-1092Crossref PubMed Scopus (75) Google Scholar). data that a at the E89K that is of PFA, affect the with at H. Chopra PubMed Scopus Google Scholar). the with to we and that the mutant does not diminish the to the is not in of the the We footprinting with complexes and to effects of the mutant on the translocation status The with that exist predominantly is of the data with In the of PFA, the type enzyme a at whereas the of the mutant enzyme is not are between and the of PFA the complex in the state. the of PFA that is to the pre-translocated complex is with the mutant enzyme when with type RT. the footprinting with the mutant enzyme that the E89K diminishes to the which provides a mechanism for PFA resistance The of nucleotide is when with RT, which is in with that the mutant enzyme can be at multiple positions around the active The data in this show that the analogue PFA the pre-translocational of HIV-1 RT. These findings have for the mechanism of action of this inhibitor and the mechanism of HIV-1 RT translocation. The proposed mechanisms for drug action and resistance have been developed on the of a model for polymerase translocation. of HIV-1 RT studies with polymerase the mechanism of translocation during as active that nucleotide or as a that of nucleotide The to as model 2004; Full Text Full Text PDF PubMed Scopus Google developed on the of of complexes with and bound and The model that the chemical nucleotide is that polymerase translocation or with the of model that nucleotide as for polymerase translocation been proposed for HIV-1 RT on the of of the polymerase in pre- and post-translocated A.D. K. S. Arnold E. J. PubMed Scopus Google Scholar, A. J. C. A.D. Arnold E. Biochemistry. 1995; PubMed Scopus Google Scholar). The model that the enzyme to the which to the pre-translocated during active DNA synthesis. findings with HIV-1 RT, that PFA the pre-translocated these are with this model of polymerase to as Brownian ratchet or model J. Biol. PubMed Scopus Google that the polymerase can rapidly between pre- and post-translocated and is to the forward and reverse motion. The nucleotide acts like a pawl of a ratchet that the complex in its post-translocational which prevents the reverse motion. more complex of this model been proposed for S. B. E. J. S. A.D. C. A. J. S. Arnold E. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, G. A. E. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). footprinting with HIV-1 RT complexes revealed that the presence of the nucleotide can and the post-translocated complex B. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The nucleotide the of a complex that and the excision of chain P.R. Matsuura S.E. Scott W.A. Full Text Full Text PDF PubMed Scopus Google Scholar, Matsuura S. Scott W.A. Biochemistry. PubMed Scopus Google Scholar). these are consistent with a Brownian ratchet model for polymerase translocation M. PubMed Scopus Google Scholar). been shown that the pre-translocated complex can be in the of nucleotide is to that the enzyme can shuttle between Here we to show that PFA the pre-translocated complex. the bound PFA can be as a pawl of a ratchet that prevents the forward motion. of the on the We identified complexes that exist predominantly pre- or or as of the The presence of or in footprinting that pre- and post-translocated are to be the however, the to in the stability between the is for the mechanism of PFA drug for inhibition are seen at sequences that the of the pre-translocated state. In the of PFA or these complexes exist predominantly in the pre-translocated state. that exist predominantly are less or to PFA These that the translocational is rapidly the of novel and the of studies with polymerase that and complexes are to J. Biol. PubMed Scopus Google Scholar). and chemical footprinting studies with this enzyme have to nucleotide of the when the were in the presence of and J. Biol. PubMed Scopus Google Scholar, M. J. Biol. PubMed Scopus Google Scholar, J. J. Biol. 2000; PubMed Scopus Google Scholar). studies with E. polymerase revealed during J.W. 2005; PubMed Scopus Google Scholar). The Brownian ratchet model for polymerase translocation is consistent with of the with E. and with the DNA polymerase HIV-1 RT. The that affect the translocational remain to be The RT enzyme forms with the bound acid and of these affect polymerase translocation J. K. Hsiou Y. A.D. A. C. Arnold E. J. Biol. PubMed Scopus Google Scholar). at that confer resistance to PFA affect the of the enzyme be to sequences that the pre- or the post-translocational state. The pre-translocated is associated with a at the 3′ end of the however, other have been identified in this which the that the active site can affect polymerase translocation. Binding of of PFA to the pre-translocational that the binding for and its analogue at which is that the presence of increase the stability of the complex data have shown that binding is associated with a of the of HIV-1 RT H. Chopra PubMed Scopus Google Scholar). is this that stabilizes the complex as with its Matsuura S. Scott W.A. Biochemistry. PubMed Scopus Google Scholar). Binding of PFA to the pre-translocated complex a The between and PFA and with HIV-1 RT be at this state. The to DNA with are of as with is in the of We for PFA in the whereas for are in the between and S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). PFA the of the in to its natural that to be the complex. The that have been identified in this and exist predominantly to HIV-1 RT in the complex with PFA to these data show that binds to the pre-translocated to the as the and of nucleotide in the post-translocated 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of with to the mechanism of drug action of the complex to nucleotide binding and predominantly in its pre-translocated state. PFA binds and the complex in this which in prevents binding of the These in a of binding of the nucleotide is in the of complexes that and pools of HIV-1 RT at these positions not binding of PFA and the to the complex in its pre-translocated is of translocation of in which nucleotide binding can pre- and at J.W. 2005; PubMed Scopus Google Scholar, Y. H. H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). We a to the of inhibition in studies (4Oberg B. Pharmacol. Ther. 1982; 19: 387-415Crossref PubMed Scopus (179) Google Scholar, J. 92: Full Text PDF PubMed Scopus Google Scholar). the of PFA, the nucleotide is incorporated and a translocational is at the bias toward at this binding of PFA, which stabilizes the complex. the of inhibition is a of a reduced which that multiple nucleotide to a of to PFA inhibition is seen in the of sequences that binding of PFA to the to the complex of a nucleotide The complexes exist predominantly or the stability of is and can be the these data that the enzyme is not in the pre- or post-translocated the of footprinting that the the of the PFA-resistant E89K mutant diminishes the stability of the which provides a novel mechanism for resistance to The mechanism to the of the excision in the of the E89K The of pre- and post-translocated the of the The of DNA appear to which is the for the development of resistance G. Mellors J.W. Bazmi H. Mills J. PubMed Scopus Google Scholar). data in this for a Brownian ratchet model of HIV-1 RT translocation. The of a translocational nucleotide to the pre-translocated can be and during active DNA synthesis. These findings with to the of this and in the of pre- and post-translocational affect nucleotide binding and incorporation, and with other RT inhibitors. Most importantly, the of this validate the pre-translocated complex as a target for the development of novel, more potent and less toxic compounds that block the translocation The identified in this the for inhibitors that block polymerase translocation. We for

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