Related Experiment Video
Updated: May 30, 2026
13:07
Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells
Published on: January 30, 2019
Subunit-specific mutational analysis of residue N348 in HIV-1 reverse transcriptase
Jessica Radzio1, Nicolas Sluis-Cremer
1Department of Medicine, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Retrovirology
|August 24, 2011
Summary
The N348I mutation in HIV-1 reverse transcriptase (RT) confers resistance to zidovudine (AZT) and nevirapine. This resistance is specifically linked to alterations in the p51 subunit of RT, impacting enzyme function and drug susceptibility.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- The N348I mutation in HIV-1 reverse transcriptase (RT) is known to confer resistance to zidovudine (AZT) and nevirapine.
- Biochemical studies suggest N348I increases AZT resistance by reducing ribonuclease H (RNase H) cleavage and AZT-monophosphate (MP) excision.
- Mechanisms of nevirapine resistance associated with N348I are debated, with hypotheses including decreased inhibitor binding or RNase H phenotype alterations.
Purpose of the Study:
- To elucidate the structural mechanisms underlying N348I-mediated resistance to AZT and nevirapine.
- To investigate the role of the p51 subunit of RT in N348I-associated resistance phenotypes.
- To clarify the conflicting literature regarding nevirapine resistance mechanisms.
Main Methods:
- Utilized a novel modeled structure of RT complexed with an RNA/DNA template/primer (T/P).
- Performed site-directed mutagenesis to introduce N348I, N348L, N348A, and N348Q mutations into the p51 subunit of RT.
- Purified mutant RT enzymes and assessed their RNase H cleavage activity, AZT-MP excision efficiency, and susceptibility to nevirapine.
Main Results:
- A putative interaction between the p51 subunit's β14-β15 loop and the RNA template was identified; N348I and N348L abrogated this interaction.
- N348I and N348L mutations significantly decreased RNase H cleavage and increased AZT-MP excision, consistent with the p51 subunit mutation.
- All N348 mutant RTs showed reduced nevirapine susceptibility, with N348I and N348L conferring higher resistance, primarily due to the p51 subunit mutation.
Conclusions:
- N348I-mediated resistance to AZT and nevirapine is definitively linked to a mutation within the p51 subunit of HIV-1 RT.
- The structural interaction identified between the p51 subunit and the RNA template provides a mechanistic basis for the observed RNase H and drug resistance phenotypes.
- This study resolves discrepancies in the literature by pinpointing the p51 subunit as the key determinant of N348I resistance.
