scholarly journals Mutational analysis of Lys65 of HIV-1 reverse transcriptase

2000 ◽  
Vol 348 (1) ◽  
pp. 77-82 ◽  
Author(s):  
Nicolas SLUIS-CREMER ◽  
Dominique ARION ◽  
Neerja KAUSHIK ◽  
Henry LIM ◽  
Michael A. PARNIAK
Keyword(s):  
Amino Acid ◽  
Reverse Transcriptase ◽  
Mutational Analysis ◽  
Catalytic Efficiency ◽  
Salt Bridge ◽  
Polymerase Activity ◽  
Ph Optimum ◽  
Modelling Studies ◽  
Dntp Binding ◽  
Hiv 1

Amino acid Lys65 is part of the highly flexible β3-β4 loop in the fingers domain of the 66 kDa subunit of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Recent crystal data show that the ϵ-amino group of Lys65 interacts with the γ-phosphate of the bound deoxynucleoside triphosphate (‘dNTP’) substrate [Huang, Chopra, Verdine and Harrison (1998) Science 282, 1669-1675]. In order to biochemically define the function of RT Lys65, we have used site-specific mutagenesis to generate RT with a variety of substitutions at this position, including K65E, K65Q, K65A and K65R. Kinetic analyses demonstrate that if Lys65 in RT is substituted with an amino acid other than arginine the enzyme exhibits dramatic decreases in the binding affinity (Km) for all dNTP substrates, in RT catalytic efficiency (kcat/Km) and in the mutant enzyme's ability to carry out pyrophosphorolysis, the reverse reaction of DNA synthesis. The pH optimum for the DNA polymerase activity of K65E RT was 6.5, compared to 7.5 for the wild-type enzyme, and 8.0 for the K65R, K65A and K65Q mutants. Molecular modelling studies show that mutations of Lys65 do not affect the geometry of the loop's α-carbon backbone, but rather lead to changes in positioning of the side chains of residues Lys70 and Arg72. In particular, Glu in K65E can form a salt bridge with Arg72, leading to the diminution of the latter residue's interaction with the α-phosphate of the dNTP residue. This alteration in dNTP-binding may explain the large pH-dependent changes in both dNTP-binding and catalytic efficiency noted with the enzyme. Furthermore, the K65A, K65Q and K65E mutant enzymes are 100-fold less sensitive to all dideoxynucleoside triphosphate (‘ddNTP’) inhibitors, whereas the K65R mutation results in a selective 10-fold decrease in binding of ddCTP and ddATP only. This implies that mutations at position 65 in HIV-1 RT influence the nucleotide-binding specificity of the enzyme.

Disruption of a Salt Bridge between Asp 488 and Lys 465 in HIV-1 Reverse Transcriptase Alters Its Proteolytic Processing and Polymerase Activity

Virology ◽  
1993 ◽  
Vol 196 (2) ◽  
pp. 731-738 ◽  
Author(s):  
Laura Goobar-Larsson ◽  
Kristina Bäckbro ◽  
Torsten Unge ◽  
Ramagauri Bhikhabhai ◽  
Lotta Vrang ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Salt Bridge ◽  
Polymerase Activity ◽  
Proteolytic Processing ◽  
Hiv 1

scholarly journals Functional Analysis of Amino Acid Residues Constituting the dNTP Binding Pocket of HIV-1 Reverse Transcriptase

1998 ◽  
Vol 273 (50) ◽  
pp. 33624-33634 ◽  
Author(s):  
Dylan Harris ◽  
Neerja Kaushik ◽  
Pradeep K. Pandey ◽  
Prem N. S. Yadav ◽  
Virendra N. Pandey
Keyword(s):  
Functional Analysis ◽  
Amino Acid ◽  
Reverse Transcriptase ◽  
Binding Pocket ◽  
Amino Acid Residues ◽  
Dntp Binding ◽  
Hiv 1

Synergistic Inhibition of HIV-1 Reverse Transcriptase DNA Polymerase Activity and Virus Replication in Vitro by Combinations of Carboxanilide Nonnucleoside Compounds

Biochemistry ◽  
1995 ◽  
Vol 34 (32) ◽  
pp. 10106-10112 ◽  
Author(s):  
Ronald S. Fletcher ◽  
Dominique Arion ◽  
Gadi Borkow ◽  
Mark A. Wainberg ◽  
Gary I. Dmitrienko ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Virus Replication ◽  
Polymerase Activity ◽  
Synergistic Inhibition ◽  
Hiv 1

scholarly journals A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

2015 ◽  
Vol 89 (16) ◽  
pp. 8119-8129 ◽  
Author(s):  
Eytan Herzig ◽  
Nickolay Voronin ◽  
Nataly Kucherenko ◽  
Amnon Hizi
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

scholarly journals Efficacy of Dideoxynucleosides against Human Foamy Virus and Relationship to Its Reverse Transcriptase Amino Acid Sequence and Structure

2001 ◽  
Vol 75 (15) ◽  
pp. 7184-7187 ◽  
Author(s):  
Anne Yvon-Groussin ◽  
Pierre Mugnier ◽  
Philippe Bertin ◽  
Marc Grandadam ◽  
Henri Agut ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reverse Transcriptase ◽  
Foamy Virus ◽  
Three Dimensional ◽  
Retroviral Vectors ◽  
Dimensional Structure ◽  
Human Foamy Virus ◽  
Amino Acid Residues ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human foamy virus (HFV), a retrovirus of simian origin which occasionally infects humans, is the basis of retroviral vectors in development for gene therapy. Clinical considerations of how to treat patients developing an uncontrolled infection by either HFV or HFV-based vectors need to be raised. We determined the susceptibility of the HFV to dideoxynucleosides and found that only zidovudine was equally efficient against the replication of human immunodeficiency virus type 1 (HIV-1) and HFV. By contrast, zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), and didanosine (ddI) were 3-, 3-, 30-, and 46-fold less efficient against HFV than against HIV-1, respectively. Some amino acid residues known to be involved in HIV-1 resistance to ddC, 3TC, d4T, and ddI were found at homologous positions of HFV reverse transcriptase (RT). These critical amino acids are located at the same positions in the three-dimensional structure of HIV-1 and HFV RT, suggesting that both enzymes share common patterns of inhibition.

scholarly journals Nucleotide specificity of HIV-1 reverse transcriptases with amino acid substitutions affecting Ala-114

2005 ◽  
Vol 387 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Clara E. CASES-GONZÁLEZ ◽  
Luis MENÉNDEZ-ARIAS
Keyword(s):  
Catalytic Efficiency ◽  
Fold Increase ◽  
Polymerase Activity ◽  
Site Directed Mutagenesis ◽  
Van Der Waals Interactions ◽  
Reverse Transcriptases ◽  
Nucleotide Incorporation ◽  
Steady State Kinetic ◽  
Nucleotide Specificity ◽  
Hiv 1

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3′-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent Km values for dNTP. Discrimination between AZTTP (3′-azido-3′-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5–6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G→A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.

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.

scholarly journals Thymidine Analogue Excision and Discrimination Modulated by Mutational Complexes Including Single Amino Acid Deletions of Asp-67 or Thr-69 in HIV-1 Reverse Transcriptase

2011 ◽  
Vol 286 (23) ◽  
pp. 20615-20624 ◽  
Author(s):  
Mónica Kisic ◽  
Tania Matamoros ◽  
María Nevot ◽  
Jesús Mendieta ◽  
Javier Martinez-Picado ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reverse Transcriptase ◽  
Single Amino Acid ◽  
Thymidine Analogue ◽  
Hiv 1

scholarly journals Nonnucleoside Inhibitor Binding Affects the Interactions of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with DNA

2004 ◽  
Vol 78 (7) ◽  
pp. 3387-3397 ◽  
Author(s):  
Elena N. Peletskaya ◽  
Alex A. Kogon ◽  
Steven Tuske ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Cross Linking ◽  
Immunodeficiency Virus ◽  
Dntp Binding ◽  
Hiv 1

ABSTRACT Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the “open” configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.

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