scholarly journals Gln145Met/Leu Changes in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confer Resistance to Nucleoside and Nonnucleoside Analogs and Impair Virus Replication

2004 ◽  
Vol 48 (12) ◽  
pp. 4611-4617 ◽  
Author(s):  
Stefania Paolucci ◽  
Fausto Baldanti ◽  
Giovanni Maga ◽  
Reynel Cancio ◽  
Maurizio Zazzi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Multidrug Resistance ◽  
Amino Acid ◽  
Reverse Transcriptase ◽  
Virus Replication ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The frequencies of multidrug resistance-associated mutations at codons 145, 151, and 69 of the human immunodeficiency virus (HIV) reverse transcriptase (RT) gene in strains from a group of 3,595 highly active antiretroviral therapy (HAART)-experienced patients were 0.22, 2.36, and 0.86%, respectively. Several amino acid substitutions different from the recently reported Gln145Met change (S. Paolucci, F. Baldanti, M. Tinelli, G. Maga, and G. Gerna, AIDS 17:924-927, 2003) were detected at position 145. Thus, amino acid substitutions selected at position 145 were introduced into the wild-type HIV type 1 (HIV-1) RT gene by site-directed mutagenesis, and recombinant HIV strains were assayed for their drug susceptibilities. Only Met and Leu substitutions at position 145 of the HIV-1 RT conferred multidrug resistance, while other amino acid changes did not. Lower levels of replication of the Gln145Met recombinant strain compared with those of both Gln151Met and wild-type recombinant strains were observed. In in vitro inhibition assays, expression and purification of the recombinant Gln145Met HIV-1 RT revealed a strong loss of catalytic efficiency of the mutated enzyme, as well as significant resistance to both zidovudine and efavirenz. Specific amino acid substitutions in the HIV RT nucleotide-binding pocket might affect both antiretroviral drug recognition and binding and decrease the level of virus replication, possibly by interfering with the enzyme activity. This finding may explain the lower frequency of Gln145Met/Leu mutations observed compared with the frequencies of Gln151Met/Leu mutations and the insertion at position 69 in HAART-experienced patients.

scholarly journals Effects of the Δ67 Complex of Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Nucleoside Analog Excision

2004 ◽  
Vol 78 (18) ◽  
pp. 9987-9997 ◽  
Author(s):  
Paul L. Boyer ◽  
Tomozumi Imamichi ◽  
Stefan G. Sarafianos ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Coding Region ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Long-term use of combination therapy against human immunodeficiency virus type (HIV-1) provides strong selective pressure on the virus, and HIV-1 variants that are resistant to multiple inhibitors have been isolated. HIV-1 variants containing amino acid substitutions within the coding region of HIV-1 reverse transcriptase (RT), such as the 3′-azido-3′-deoxythymidine (AZT)-resistant variant AZT-R (M41L/D67N/K70R/T215Y/K219Q) and a variant containing an insertion in the fingers domain (S69SGR70/T215Y), are resistant to the nucleoside RT inhibitor (NRTI) AZT because of an increase in the level of excision of AZT monophosphate (AZTMP) from the primer. While rare, variants have also been isolated which contain deletions in the RT coding region. One such virus, described by Imamichi et al. (J. Virol 74:10958-10964, 2000; J. Virol. 74:1023-1028, 2000; J. Virol. 75:3988-3992, 2001), contains numerous amino acid substitutions and a deletion of codon 67, which we have designated the Δ67 complex of mutations. We have expressed and purified HIV-1 RT containing these mutations. We compared the polymerase and pyrophosphorolysis (excision) activity of an RT with the Δ67 complex of mutations to wild-type RT and the two other AZT-resistant variants described above. All of the AZT-resistant variants we tested excise AZTMP and 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA [tenofovir]) from the end of a primer more efficiently than wild-type RT. Although the variant RTs excised d4TMP less efficiently than AZTMP and PMPA, they were able to excise d4TMP more efficiently than wild-type RT. HIV-1 RT containing the Δ67 complex of mutations was not able to excise as broad a range of NRTIs as the fingers insertion variant SSGR/T215Y, but it was able to polymerize efficiently with low concentrations of deoxynucleoside triphosphates and seems to be able to excise AZTMP and PMPA at lower ATP concentrations than AZT-R or SSGR/T215Y, suggesting that a virus containing the Δ67 complex of mutations would replicate reasonably well in quiescent cells, even in the presence of AZT.

S-1153 Inhibits Replication of Known Drug-Resistant Strains of Human Immunodeficiency Virus Type 1

1998 ◽  
Vol 42 (6) ◽  
pp. 1340-1345 ◽  
Author(s):  
Tamio Fujiwara ◽  
Akihiko Sato ◽  
Mohamed El-Farrash ◽  
Shigeru Miki ◽  
Kenji Abe ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Reverse Transcriptase ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Reverse Transcriptase Inhibitors ◽  
Human T Cell ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT S-1153 is a new imidazole compound that inhibits human immunodeficiency virus (HIV) type 1 (HIV-1) replication by acting as a nonnucleoside reverse transcriptase inhibitor (NNRTI). This compound inhibits replication of HIV-1 strains that are resistant to nucleoside and nonnucleoside reverse transcriptase inhibitors. S-1153 has a 50% effective concentration in the range of 0.3 to 7 ng/ml for strains with single amino acid substitutions that cause NNRTI resistance, including the Y181C mutant, and also has potent activity against clinical isolates. The emergence of S-1153-resistant variants is slower than that for nevirapine, and S-1153-resistant variants contained at least two amino acid substitutions, including F227L or L234I. S-1153-resistant variants are still sensitive to the nucleoside reverse transcriptase inhibitors zidovudine (AZT) and lamivudine. In a mouse and MT-4 (human T-cell line) in vivo HIV replication model, S-1153 and AZT administered orally showed a marked synergy for the inhibition of HIV-1 replication. S-1153 shows a significant accumulation in lymph nodes, where most HIV-1 infection is thought to occur. S-1153 may be an appropriate candidate for two- to three-drug combination therapy for HIV infection.

scholarly journals YADD Mutants of Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Reverse Transcriptase Are Resistant to Lamivudine Triphosphate (3TCTP) In Vitro

2001 ◽  
Vol 75 (14) ◽  
pp. 6321-6328 ◽  
Author(s):  
Paul L. Boyer ◽  
Hong-Qiang Gao ◽  
Patrick K. Clark ◽  
Stefan G. Sarafianos ◽  
Edward Arnold ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT When human immunodeficiency virus type 1 (HIV-1) is selected for resistance to 3TC, the methionine normally present at position 184 is replaced by valine or isoleucine. Position 184 is the X of the conserved YXDD motif; positions 185 and 186 form part of the triad of aspartic acids at the polymerase active site. Structural and biochemical analysis of 3TC-resistant HIV-1 reverse transcriptase (RT) led to a model in which a β-branched amino acid at position 184 would act as a steric gate. Normal deoxynucleoside triphosphates (dNTPs) could still be incorporated; the oxathiolane ring of 3TCTP would clash with the β branch of the amino acid at position 184. This model can also explain 3TC resistance in feline immunodeficiency virus and human hepatitis B virus. However, it has been reported (14) that murine leukemia viruses (MLVs) with valine (the amino acid present in the wild type), isoleucine, alanine, serine, or methionine at the X position of the YXDD motif are all resistant to 3TC. We prepared purified wild-type MLV RT and mutant MLV RTs with methionine, isoleucine, and alanine at the X position. The behavior of these RTs was compared to those of wild-type HIV-1 RT and of HIV-1 RT with alanine at the X position. If alanine is present at the X position, both MLV RT and HIV-1 RT are relatively resistant to 3TCTP in vitro. However, the mutant enzymes were impaired relative to their wild-type counterparts; there appears to be steric hindrance for both 3TCTP and normal dNTPs.

scholarly journals Amino Acid Insertions near Gag Cleavage Sites Restore the Otherwise Compromised Replication of Human Immunodeficiency Virus Type 1 Variants Resistant to Protease Inhibitors

2004 ◽  
Vol 78 (21) ◽  
pp. 12030-12040 ◽  
Author(s):  
Sadahiro Tamiya ◽  
Sek Mardy ◽  
Mark F. Kavlick ◽  
Kazuhisa Yoshimura ◽  
Hiroaki Mistuya
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Amino Acid Substitutions ◽  
Cleavage Sites ◽  
Wild Type ◽  
Gag Proteins ◽  
Infectious Clones ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A variety of amino acid substitutions in the protease and Gag proteins have been reported to contribute to the development of human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors. In the present study, full-length molecular infectious HIV-1 clones were generated by using HIV-1 variants isolated from heavily drug-experienced and therapy-failed AIDS patients. Of six full-length infectious clones generated, four were found to have unique insertions (TGNS, SQVN, AQQA, SRPE, APP, and/or PTAPPA) near the p17/p24 and p1/p6 Gag cleavage sites, in addition to the known resistance-related multiple amino acid substitutions within the protease. The addition of such Gag inserts mostly compromised the replication of wild-type HIV-1, whereas the primary multidrug-resistant HIV infectious clones containing inserts replicated significantly better than those modified to lack the inserts. Western blot analyses revealed that the processing of Gag proteins by wild-type protease was impaired by the presence of the inserts, whereas that by mutant protease was substantially improved. The present study represents the first report clearly demonstrating that the inserts seen in the proximity of the Gag cleavage sites in highly multi-PI resistant HIV-1 variants restore the otherwise compromised enzymatic activity of mutant protease, enabling the multi-PI-resistant HIV-1 variants to remain replication competent.

A Mutation at Position 190 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Interacts with Mutations at Positions 74 and 75 via the Template Primer

1998 ◽  
Vol 42 (2) ◽  
pp. 447-452 ◽  
Author(s):  
Paul L. Boyer ◽  
Hong-Qiang Gao ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Polymerase Activity ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have analyzed amino acid substitutions at position G190 in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The mutation G190E, which is responsible for resistance to certain nonnucleoside inhibitors, results in RT that has significantly less polymerase activity and that is less processive than wild-type RT. Its kinetic profile with respect to dGTP and poly(rC) · oligo(dG) is significantly altered compared to that of wild-type RT. The combination of either of the mutations L74V or V75I with the G190E mutation appears to be compensatory and mitigates many of the deleterious effects of the G190E mutation.

scholarly journals Switch to Unusual Amino Acids at Codon 215 of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Gene in Seroconvertors Infected with Zidovudine-Resistant Variants

1998 ◽  
Vol 72 (5) ◽  
pp. 3520-3523 ◽  
Author(s):  
Sabine Yerly ◽  
Abdelrahim Rakik ◽  
Sabine Kinloch De Loes ◽  
Bernard Hirschel ◽  
Diane Descamps ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Reverse Transcriptase ◽  
Wild Type ◽  
Unusual Amino Acids ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Over Time

ABSTRACT Sequences of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) domain were determined by direct sequencing of HIV-1 RNA in successive plasma samples from eight seroconverting patients infected with virus bearing the T215Y/F amino acid substitution associated with zidovudine (ZDV) resistance. At baseline, additional mutations associated with ZDV resistance were detected. Three patients had the M41L amino acid change, which persisted. Two patients had both the D67N and the K70R amino acid substitutions; reversion to the wild type was seen at both positions in one of these patients and at codon 70 in the other one. Reversion to the wild type at codon 215 was observed in only one of eight patients. Unusual amino acids, such as aspartic acid (D) and cysteine (C), appeared at position 215 in four patients during follow-up. These variants isolated by coculturing were sensitive to ZDV. Overgrowth of these variants suggests that they have better fitness than the original T215Y variant. Intraindividual nucleoside substitutions over time were 10 times more frequent in codons associated with ZDV resistance (41, 67, 70, 215, and 219) than in other codons of the RT domain. The predominance of nonsynonymous substitutions observed over time suggests that most changes reflect adaptation of the RT function. The variance in sequence evolution observed among patients, in particular at codon 215, supports a role for chance in the evolution of the RT domain.

scholarly journals Mutational Analysis of the Hydrophobic Tail of the Human Immunodeficiency Virus Type 1 p6Gag Protein Produces a Mutant That Fails To Package Its Envelope Protein

1999 ◽  
Vol 73 (1) ◽  
pp. 19-28 ◽  
Author(s):  
David E. Ott ◽  
Elena N. Chertova ◽  
Laura K. Busch ◽  
Lori V. Coren ◽  
Tracy D. Gagliardi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Human Immunodeficiency Virus Type ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hydrophobic Tail ◽  
Carboxyl Terminal ◽  
Hiv 1

ABSTRACT The p6Gag protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6Gag between Y36 and P37, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6Gag proteins in HIV-1MN. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6Gag, site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6Gag, Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41TM cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6Gag mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.

scholarly journals The Thiocarboxanilide Nonnucleoside Inhibitor UC781 Restores Antiviral Activity of 3′-Azido-3′-Deoxythymidine (AZT) against AZT-Resistant Human Immunodeficiency Virus Type 1

1999 ◽  
Vol 43 (2) ◽  
pp. 259-263 ◽  
Author(s):  
Gadi Borkow ◽  
Dominique Arion ◽  
Mark A. Wainberg ◽  
Michael A. Parniak
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT N-[4-Chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furancarbothioamide (UC781) is an exceptionally potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. We found that a 1:1 molar combination of UC781 and 3′-azido-3′-deoxythymidine (AZT) showed high-level synergy in inhibiting the replication of AZT-resistant virus, implying that UC781 can restore antiviral activity to AZT against AZT-resistant HIV-1. Neither the nevirapine plus AZT nor the 2′,5′-bis-O-(t-butyldimethylsilyl)-3′-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide plus AZT combinations had this effect. Studies with purified HIV-1 reverse transcriptase (from a wild type and an AZT-resistant mutant) showed that UC781 was a potent inhibitor of the pyrophosphorolytic cleavage of nucleotides from the 3′ end of the DNA polymerization primer, a process that we have proposed to be critical for the phenotypic expression of AZT resistance. Combinations of UC781 plus AZT did not act in synergy to inhibit the replication of either wild-type virus or UC781-resistant HIV-1. Importantly, the time to the development of viral resistance to combinations of UC781 plus AZT is significantly delayed compared to the time to the development of resistance to either drug alone.

scholarly journals High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutants Is Due to Selective Targeting of Different Mechanistic Forms of the Enzyme

2005 ◽  
Vol 49 (11) ◽  
pp. 4546-4554 ◽  
Author(s):  
Reynel Cancio ◽  
Romano Silvestri ◽  
Rino Ragno ◽  
Marino Artico ◽  
Gabriella De Martino ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mechanism Of Action ◽  
Drug Resistant ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation.

scholarly journals Identification of novel thiocarboxanilide derivatives that suppress a variety of drug-resistant mutant human immunodeficiency virus type 1 strains at a potency similar to that for wild-type virus.

1996 ◽  
Vol 40 (6) ◽  
pp. 1454-1466 ◽  
Author(s):  
J Balzarini ◽  
W G Brouwer ◽  
D C Dao ◽  
E M Osika ◽  
E De Clercq
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Effective Concentration ◽  
Mutant Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Virus Strains ◽  
Hiv 1

A large variety of carboxanilide and thiocarboxanilide derivatives in which the original oxathiin or aliphatic moieties present in the prototype compounds UC84 and UC38 were replaced by an (un) substituted furanyl, thienyl, phenyl, or pyrrole entity have been evaluated for activity against wild-type human immunodeficiency virus type 1 strain IIIB [HIV-1 (IIIB)] and a series of mutant virus strains derived thereof. The mutant viruses contained either the Leu-100-->Ile, Lys-103-->Asn, Val-106-->Ala, Glu-138-->Lys, Tyr-181-->Cys, or Tyr-188-->Leu mutation in their reverse transcriptase. Several 3-(2-methylfuranyl)- and 3-(2-methylthienyl)-thiocarboxanilide ester, (thio)ether, and oxime ether derivatives showed exquisitely potent antiviral activity against wild-type HIV-1 (50% effective concentration, 0.009 to 0.021 microM). The pentenylethers of the 2-methylfuranyl and 2-methylthienyl derivatives (i.e., 313, N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]- 2-methyl-3-furancarbothioamide or UC-781, and 314, N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl] -2-methyl-3-thiophenecarbothioamide or UC-82) proved virtually equally inhibitory for wild-type and the Ile-100, Ala-106, and Lys-138 mutant virus strains (50% effective concentration, 0.015 to 0.021 microM). Their inhibitory effect against the Asn-103 and Cys-181 reverse transcriptase mutant virus strains was decreased only four- to sevenfold compared with wildtype virus. UC-781 and UC-82 should be considered potential candidate drugs for the treatment of HIV-1-infected individuals.

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