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Updated: Aug 14, 2026
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Published on: March 30, 2014
Drug-resistant HIV-1 proteases identify enzyme residues important for substrate selection and catalytic rate
T W Ridky1, A Kikonyogo, J Leis
1Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Abstract:
A series of mutations, first identified in protease inhibitor-resistant HIV-1 viral isolates, were introduced into HIV-1 PR as individual substitutions. Mutants containing R8K, V32I, V82T, I84V, G48V/L90M, or V82T/I84V substitutions were analyzed for differences in substrate preference and catalytic efficiency using a set of single amino acid substituted HIV-1 CA-NCa cleavage site peptides. All mutants exhibited wild-type preference for large hydrophobic residues, especially Phe, in the P1' substrate position. Only the R8K and V32I mutants showed significant differences in subsite selection compared to wild-type enzyme. In a parallel study, the individual mutations R10K, L12V, I44V, A60M, I71V, and I108V were introduced into RSV PR. These amino acid positions are structurally equivalent to Arg8, Leu10, Val32, Met46, Ile54, and Ile84 in HIV-1 PR, respectively, which mutate in drug-resistance. The RSV R10K substitution significantly altered substrate specificity and catalytic rate, compared to wild-type, in a manner similar to that of the HIV-1 R8K mutant. Crystal structures of the RSV PR R10K, I44V, I71V, and Il08V mutant enzymes presented here indicate that each of these substitutions has little effect on the overall structure of the respective enzymes. Taken together, these data provide an explanation for the reported in vivo predilection for selection of large hydrophobic residues in the P1' substrate position of second locus mutations in the Gag polyprotein PR cleavage sites. The data also suggest that the selection of resistant enzymes is not simply limited to loss of binding to inhibitor but affects other steps in proteolysis.
Insights
Mutations in HIV-1 protease (PR) affect substrate preference and catalytic efficiency. These changes in drug-resistant HIV-1 explain in vivo selection of mutations in Gag polyprotein cleavage sites.
Area of Science:
- Biochemistry
- Virology
- Structural Biology
Background:
- Protease inhibitor (PI) resistance in Human Immunodeficiency Virus type 1 (HIV-1) is a significant clinical challenge.
- HIV-1 protease (PR) plays a crucial role in viral maturation by cleaving viral polyproteins.
- Mutations conferring drug resistance can alter the enzyme's substrate specificity and catalytic activity.
Purpose of the Study:
- To investigate the impact of specific mutations, identified in drug-resistant HIV-1 isolates, on the substrate preference and catalytic efficiency of HIV-1 PR.
- To compare the effects of structurally equivalent mutations in RSV PR to understand conserved mechanisms.
- To elucidate the structural basis for altered substrate recognition in resistant HIV-1 PR mutants.
Main Methods:
- Introduction of individual amino acid substitutions into HIV-1 PR and RSV PR.
- Analysis of substrate preference using synthetic HIV-1 CA-NCa cleavage site peptides.
- Determination of catalytic efficiency for wild-type and mutant enzymes.
- X-ray crystallography of selected RSV PR mutants to assess structural impact.
Main Results:
- HIV-1 PR mutants generally retained preference for large hydrophobic residues (e.g., Phe) at the P1' position.
- Mutations R8K and V32I in HIV-1 PR significantly altered subsite selection.
- RSV PR R10K substitution mimicked the effects of HIV-1 R8K, altering substrate specificity and catalytic rate.
- Crystal structures revealed minimal structural changes in RSV PR mutants (R10K, I44V, I71V, I108V).
Conclusions:
- The studied mutations provide mechanistic insight into the in vivo selection of mutations in Gag polyprotein cleavage sites.
- Altered substrate specificity and catalytic efficiency contribute to HIV-1 drug resistance beyond simple inhibitor binding.
- Structural and functional analyses of viral proteases offer targets for novel antiviral strategies.
