Novel mutations in reverse transcriptase of human immunodeficiency virus type 1 reduce susceptibility to foscarnet in laboratory and clinical isolates

Mellors, John W.; Bazmi, Hengameh Z.; Schinazi, Raymond F.; Roy, Bidyut; Hsiou, Y.; Arnold, Edward; Weir, Jerry P.; Mayers, Douglas L.

doi:10.1128/aac.39.5.1087

Cited by 68 publications

(88 citation statements)

References 33 publications

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“…With few exceptions, many of these mutations are too far away from the active site to affect the interaction with the dNTP or nucleic acid substrate directly (8). The W88G change is perhaps the most prevalent mutation that is found in clinical isolates (7). Crystallographic data suggest that a change at the adjacent position, i.e.…”

Section: Resultsmentioning

confidence: 99%

“…In this context it is also important to note that template position ϩ5 is associated with intrinsic pausing of RT at limiting dNTP concentrations, which is in keeping with the observation that the stability of the post-translocated complex is likewise diminished. The selective pressure of PFA can cause the emergence of resistance conferring mutations in the RT gene in vitro and in vivo (7,9). With few exceptions, many of these mutations are too far away from the active site to affect the interaction with the dNTP or nucleic acid substrate directly (8).…”

Section: Resultsmentioning

confidence: 99%

“…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 NNRTIs do not affect susceptibility to PFA (7)(8)(9). Most importantly, mutations associated with PFA resistance were shown to increase susceptibility to the nucleoside analogue 3Ј-azido-3Ј-deoxythymidine (zidovudine or AZT) (10,11).…”

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

Marchand

Tchesnokov

Götte

2007

Journal of Biological Chemistry

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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) 4 of the human immunodeficiency virus type 1 (HIV-1) have been developed (1). 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 (2, 3). Here we studied the mechanism of action of the pyrophosphate (PP i ) 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 (4). 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 (5). Toxic side effects and its poor bioavailability limit its clini...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Marchand

Tchesnokov

Götte

2007

Journal of Biological Chemistry

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“…Previous studies (8) demonstrated that mutations that confer resistance to dideoxynucleoside analogues, including L74--V, apparently lead to changes at the active site through changing the conformation or position of the template-primer. Analyses of foscarnet-resistant HIV-1 RT also suggest that mutations across the DNA-binding cleft may lead to alterations in binding of foscarnet at the polymerase active site, again "communicating" from the site of mutation through the template-primer (26). We therefore propose a connection between quinoxaline-specific mutations that impair polymerase activity and the NRTI mutations that occur in the fingers subdomain.…”

Section: Analysis Of the Hiv-1 Rt Gene During In Vitro Selection Formentioning

confidence: 99%

Selective pressure of a quinoxaline nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on HIV-1 replication results in the emergence of nucleoside RT-inhibitor-specific (RT Leu-74-->Val or Ile and Val-75-->Leu or Ile) HIV-1 mutants.

Kleim¹,

Rösner²,

Winkler³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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The quinoxaline nonnucleoside RT inhibitor (NNRTI) (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4-dihydroquinoxaline-2(1H)-thione (HBY 097) was used to select for drug-resistant HIV-1 variants in vitro. The viruses first developed mutations affecting the NNRTIbinding pocket, and five of six strains displayed the RT G190--E substitution, which is characteristic for HIV-1 resistance against quinoxalines. In one variant, a new mutant (G190->Q) most likely evolved from preexisting G190->E mutants. The negative charge introduced by the G190->E substitution was maintained at that site of the pocket by simultaneous selection for V179--D together with G190--Q. After continued exposure to the drug, mutations at positions so far known to be specific for resistance against nucleoside RT inhibitors (NRTIs) (L74-*V/I and V75->L/I) were consistently detected in all cultures. The inhibitory activities of the cellular conversion product of 2',3'-dideoxyinosine (ddl, didanosine), 2',3'-dideoxyadenosine (ddA) and of 2',3'-didehydro-3'-deoxythymidine (d4T, stavudine) against these late-passage viruses were shown to be enhanced with the L74->V/I RT mutant virus as compared with the wild-type (wt) HIV-1MN isolate. Clonal analysis proved linkage of the codon 74 and codon 75 mutations to the NNRTI-specific mutations in all RT gene fragments. The nonnucleoside-and nucleoside-resistance mutation sites are separated by approximately 35 A. We propose that the two sites "communicate" through the template-primer which is situated in the DNAbinding cleft between these two sites. Quinoxalines cause high selective pressure on HIV-1 replication in vitro; however, the implication of these findings for the treatment of infection has yet to be determined. Viral resistance against both nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs) of human immunodeficiency virus type 1 (HIV-1) replication develops in vitro (cell culture) and in vivo (patients). NRTIs select for RT amino acid substitutions at positions that can be allocated to different structural elements of the protein in the range of residue numbers 41-219 (1-5). A molecular mechanism for some alterations can be assumed on the basis of the RT crystal structure (6-8). In contrast, NNRTI-specific substitutions map exclusively to those protein secondary structure elements that together form a lipophilic pocket within the palm domain of the p66 RT subunit. All NNRTIs are believed to bind to this region, and mutations selected for by these drugs usuallyThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. occur in the segments composed of RT amino acids 98-108, 179-190, and 230-236 (9-16). Especially the Y181-*C RT mutant appears with many chemically distinct compounds, including different NNRTI combinations (12).6-Chloro-3,3-dimethyl-4-(isopropenyloxycarbonyl)-3,4-dihydroqu...

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“…The low fidelity of retroviral replication is a major force for generation of high levels of variation in retroviral populations (8), which allows them to adapt quickly to changes in their environment. One important outcome of this high adaptability is the rapid development of resistance to antiretroviral drugs and escape from host immune responses (9). Mutations that occur in retroviruses during replication include substitutions, frameshifts, deletions, duplications, and hypermutations (10,11).…”

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confidence: 99%

Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine–thymine tracts

Zhang

Svarovskaia²,

Barr³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Using in vivo fidelity assays in which bacterial ␤-galactosidase or green fluorescent protein genes served as reporters of mutations, we have identified a murine leukemia virus (MLV) RNase H mutant (Y586F) that exhibited an increase in the retroviral mutation rate Ϸ5-fold in a single replication cycle. DNA-sequencing analysis indicated that the Y586F mutation increased the frequency of substitution mutations 17-fold within 18 nt of adenine-thymine tracts (AAAA, TTTT, or AATT), which are known to induce DNA bending. Sequence alignments indicate that MLV Y586 is equivalent to HIV-1 Y501, a component of the recently described RNase H primer grip domain, which contacts and positions the DNA primer strand near the RNase H active site. The results suggest that wild-type reverse transcriptase (RT) facilitates a specific conformation of the template-primer duplex at the polymerase active site that is important for accuracy of DNA synthesis; when an adeninethymine tract is within 18 nt of the polymerase active site, the Y586F mutant RT cannot facilitate this specific template-primer conformation, leading to an increase in the frequency of substitution mutations. These findings indicate that the RNase H primer grip can affect the template-primer conformation at the polymerase active site and that the MLV Y586 residue and template-primer conformation are important determinants of RT fidelity. R everse transcriptase (RT), which copies retroviral genomic RNA into double-stranded DNA during reverse transcription, contains two major enzymatic activities: a DNA polymerase activity that uses either RNA or DNA as a template and an RNase H activity that can degrade RNA in an RNA⅐DNA hybrid (1). The crystal structure of HIV-1 RT in complex with a template-primer and a dNTP substrate has shown that the polymerase active site and the RNase H cleavage site are separated by 18 nt of the template-primer (2). In the case of murine leukemia virus (MLV) RT, the DNA polymerase and RNase H activities reside in physically separable domains of a single monomeric protein (3).The RNase H activity of all retroviral RTs performs several functions that are essential for the viral life cycle (4), which include degradation of the RNA template after synthesis of the minusstrand DNA, generation of a specific polypurine primer from which plus-strand DNA synthesis initiates, subsequent removal of the polypurine primer to complete synthesis of the viral doublestranded DNA, and removal of the minus-strand tRNA primer (5). During minus-strand DNA synthesis, RT positions the RNase H active site and cleaves the RNA Ϸ18 nt 3Ј of the site of DNA synthesis (6).All retroviral RTs carry out error-prone DNA synthesis, in part because they lack a proofreading activity and possess a low affinity for the template (7). The low fidelity of retroviral replication is a major force for generation of high levels of variation in retroviral populations (8), which allows them to adapt quickly to changes in their environment. One important outcome of this high adaptability is t...

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Novel mutations in reverse transcriptase of human immunodeficiency virus type 1 reduce susceptibility to foscarnet in laboratory and clinical isolates

Cited by 68 publications

References 33 publications

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

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

Selective pressure of a quinoxaline nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on HIV-1 replication results in the emergence of nucleoside RT-inhibitor-specific (RT Leu-74-->Val or Ile and Val-75-->Leu or Ile) HIV-1 mutants.

Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine–thymine tracts

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