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

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.

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Crystal Structure of a Retroviral Polyprotein: Prototype Foamy Virus Protease-Reverse Transcriptase (PR-RT)

Viruses ◽

10.3390/v13081495 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1495

Author(s):

Jerry Joe E. K. Harrison ◽

Steve Tuske ◽

Kalyan Das ◽

Francesc X. Ruiz ◽

Joseph D. Bauman ◽

...

Keyword(s):

Crystal Structure ◽

Reverse Transcriptase ◽

Foamy Virus ◽

Polymerase Activity ◽

Rnase H ◽

Proteolytic Processing ◽

Nucleic Acid Binding ◽

Foamy Viruses ◽

Polyprotein Precursor ◽

Hiv 1

In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs) express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) that can carry out both proteolytic processing and reverse transcription but is in a configuration not competent for proteolytic or polymerase activity. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins.

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

Biochemistry ◽

10.1021/bi00032a002 ◽

1995 ◽

Vol 34 (32) ◽

pp. 10106-10112 ◽

Cited By ~ 17

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

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

Journal of Virology ◽

10.1128/jvi.00809-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8119-8129 ◽

Cited By ~ 4

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.

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Efavirenz enhances the proteolytic processing of an HIV-1 pol polyprotein precursor and reverse transcriptase homodimer formation

FEBS Letters ◽

10.1016/j.febslet.2004.11.099 ◽

2004 ◽

Vol 579 (2) ◽

pp. 379-384 ◽

Cited By ~ 30

Author(s):

Gilda Tachedjian ◽

Katie L. Moore ◽

Stephen P. Goff ◽

Nicolas Sluis-Cremer

Keyword(s):

Reverse Transcriptase ◽

Proteolytic Processing ◽

Polyprotein Precursor ◽

Hiv 1

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HIV-1 Reverse Transcriptase Still Remains a New Drug Target: Structure, Function, Classical Inhibitors, and New Inhibitors with Innovative Mechanisms of Actions

Molecular Biology International ◽

10.1155/2012/586401 ◽

2012 ◽

Vol 2012 ◽

pp. 1-23 ◽

Cited By ~ 52

Author(s):

Francesca Esposito ◽

Angela Corona ◽

Enzo Tramontano

Keyword(s):

Reverse Transcriptase ◽

Drug Target ◽

Polymerase Activity ◽

Rnase H ◽

Primary Target ◽

Lethal Mutagenesis ◽

Rt Inhibitors ◽

Process Characteristic ◽

Hiv 1 ◽

Chain Terminators

During the retrotranscription process, characteristic of all retroviruses, the viral ssRNA genome is converted into integration-competent dsDNA. This process is accomplished by the virus-coded reverse transcriptase (RT) protein, which is a primary target in the current treatments for HIV-1 infection. In particular, in the approved therapeutic regimens two classes of drugs target RT, namely, nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). Both classes inhibit the RT-associated polymerase activity: the NRTIs compete with the natural dNTP substrate and act as chain terminators, while the NNRTIs bind to an allosteric pocket and inhibit polymerization noncompetitively. In addition to these two classes, other RT inhibitors (RTIs) that target RT by distinct mechanisms have been identified and are currently under development. These include translocation-defective RTIs, delayed chain terminators RTIs, lethal mutagenesis RTIs, dinucleotide tetraphosphates, nucleotide-competing RTIs, pyrophosphate analogs, RT-associated RNase H function inhibitors, and dual activities inhibitors. This paper describes the HIV-1 RT function and molecular structure, illustrates the currently approved RTIs, and focuses on the mechanisms of action of the newer classes of RTIs.

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Analysis of the Contribution of Reverse Transcriptase and Integrase Proteins to Retroviral RNA Dimer Conformation

Journal of Virology ◽

10.1128/jvi.79.10.6338-6348.2005 ◽

2005 ◽

Vol 79 (10) ◽

pp. 6338-6348 ◽

Cited By ~ 14

Author(s):

Penelope Buxton ◽

Gilda Tachedjian ◽

Johnson Mak

Keyword(s):

Reverse Transcriptase ◽

Leukemia Virus ◽

Proteolytic Processing ◽

Moloney Murine Leukemia Virus ◽

Stable Conformation ◽

Rna Dimerization ◽

Immunodeficiency Virus ◽

Rna Dimer ◽

Hiv 1

ABSTRACT All retroviruses contain two copies of genomic RNA that are linked noncovalently. The dimeric RNA of human immunodeficiency virus type 1 (HIV-1) undergoes rearrangement during virion maturation, whereby the dimeric RNA genome assumes a more stable conformation. Previously, we have shown that the packaging of the HIV-1 polymerase (Pol) proteins reverse transcriptase (RT) and integrase (IN) is essential for the generation of the mature RNA dimer conformation. Analysis of HIV-1 mutants that are defective in processing of Pol showed that these mutant virions contained altered dimeric RNA conformation, indicating that the mature RNA dimer conformation in HIV-1 requires the correct proteolytic processing of Pol. The HIV-1 Pol proteins are multimeric in their mature enzymatically active forms; RT forms a heterodimer, and IN appears to form a homotetramer. Using RT and IN multimerization defective mutants, we have found that dimeric RNA from these mutant virions has the same stability and conformation as wild-type RNA dimers, showing that the mature enzymatically active RT and IN proteins are dispensable for the generation of mature RNA dimer conformation. This also indicated that formation of the mature RNA dimer structure occurs prior to RT or IN maturation. We have also investigated the requirement of Pol for RNA dimerization in both Mason-Pfizer monkey virus (M-PMV) and Moloney murine leukemia virus (MoMuLV) and found that in contrast to HIV-1, Pol is dispensable for RNA dimer maturation in M-PMV and MoMuLV, demonstrating that the requirement of Pol in retroviral RNA dimer maturation is not conserved among all retroviruses.

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