Related Experiment Video
Updated: Jun 13, 2026
08:33
Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors
Published on: January 19, 2015
Nucleotide-dependent conformational change governs specificity and analog discrimination by HIV reverse transcriptase
Matthew W Kellinger1, Kenneth A Johnson
1Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.
Summary
Nucleoside analogs like lamivudine triphosphate (3TC-TP) do not bind HIV reverse transcriptase tighter than normal nucleotides (dCTP). Instead, slower incorporation rates explain differences in HIV drug efficacy.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Nucleoside analogs are crucial in antiviral therapies, including for HIV.
- Previous studies on HIV reverse transcriptase (HIV RT) kinetics yielded conflicting results regarding nucleotide analog binding affinity.
- Rational structural predictions did not align with observed binding affinities of nucleoside analogs to HIV RT.
Purpose of the Study:
- To resolve controversies surrounding the binding affinities of nucleoside analogs versus normal nucleotides to HIV RT.
- To investigate the kinetic mechanisms governing nucleotide incorporation by HIV RT.
- To elucidate the structural and kinetic basis for the efficacy of nucleoside analog drugs.
Main Methods:
- Utilized single turnover studies to monitor enzyme- kinetics.
- Measured nucleotide-induced changes in HIV reverse transcriptase enzyme structure.
- Analyzed the kinetics of dCTP (deoxycytidine triphosphate) and 3TC-TP (lamivudine triphosphate) incorporation.
Main Results:
- The specificity constant for normal nucleotide (dCTP) incorporation is limited by the binding and isomerization rate, which commits the substrate to reaction.
- Nucleoside analog (3TC-TP) incorporation is slow, allowing conformational changes to reach equilibrium and revealing similar binding affinities to dCTP.
- Previously reported tight binding of analogs was an artifact of incomplete kinetic measurements.
Conclusions:
- dCTP and 3TC-TP exhibit nearly equal binding affinities to HIV RT.
- HIV RT discriminates against 3TC-TP due to its slower incorporation rate, caused by substrate/residue misalignment.
- This kinetic discrimination, not differential binding affinity, underlies the therapeutic effectiveness of nucleoside analogs.
More Related Videos
13:07Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells
Published on: January 30, 2019
10:29Quantitative Structure-Activity Relationship, Activity Prediction, and Molecular Dynamics of Non-nucleotide Reverse Transcriptase Inhibitors
Published on: May 9, 2025
Related Concept Videos
01:26Size and Structure of Viral Genomes
Viral genomes exhibit remarkable diversity in size, structure, and composition, influencing their replication strategies and interactions with host cells. These genomes consist of either DNA or RNA and may be linear or circular. Additionally, they can be single-stranded or double-stranded, with each configuration affecting how the virus propagates within a host. RNA viruses, for instance, generally have smaller genomes than DNA viruses, a factor that contributes to their high mutation rates and...
02:27DNA Base Pairing
Erwin Chargaff’s rules on DNA equivalence paved the way for the discovery of base pairing in DNA. Chargaff’s rules state that in a double-stranded DNA molecule,
01:19Inhibitors of Virion Maturation and Assembly
As part of their replication cycle, certain viruses synthesize long precursor proteins called polyproteins within infected host cells. In human immunodeficiency virus (HIV), two major polyproteins are produced: Gag and Gag-Pol. The Gag polyprotein supplies the structural components of the virus, while Gag-Pol includes essential viral enzymes such as reverse transcriptase, integrase, and protease. After synthesis, these polyproteins move to the host cell membrane, where they assemble into an...
01:29Viruses with RNA Genomes
RNA viruses are categorized into positive-strand, negative-strand, or double-stranded groups based on their genomic structure and replication mechanisms. This classification dictates how they exploit host cellular machinery for protein synthesis and replication. Some RNA viruses also utilize reverse transcription as part of their life cycle, further diversifying their replication strategies.Positive-Strand RNA VirusesPositive-strand RNA viruses have genomes that function directly as messenger...
01:22Antiviral Nucleoside Inhibitors
Antiviral Nucleoside InhibitorsAntiviral nucleoside inhibitors are structural analogs of natural nucleosides that interfere with viral DNA or RNA synthesis. These compounds selectively target viral polymerases due to their resemblance to host nucleosides, thereby disrupting viral genome replication.Mechanism of Acyclovir ActionAcyclovir is a guanosine analog with a three-carbon acyclic side chain. It selectively targets herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2),...
01:10Retrovirus Life Cycles
Retroviruses have a single-stranded RNA genome that undergoes a special form of replication. Once the retrovirus has entered the host cell, an enzyme called reverse transcriptase synthesizes double-stranded DNA from the retroviral RNA genome. This DNA copy of the genome is then integrated into the host’s genome inside the nucleus via an enzyme called integrase. Consequently, the retroviral genome is transcribed into RNA whenever the host’s genome is transcribed, allowing the retrovirus to...