Impact of Residues in the Nonnucleoside Reverse Transcriptase Inhibitor Binding Pocket on HIV-1 Reverse Transcriptase Heterodimer Stability

Abstract

Previous studies have demonstrated that nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) act as chemical enhancers of human immunodeficiency virus type 1 (HIV-1) RT dimerization. In the current study, we sought to define the role of key residues (101, 103, 108, 181, 188, 190, 225 and 318) in the NNRTI-binding pocket on HIV-1 RT heterodimer stability. Thirteen mutant RTs were constructed and evaluated for p66/p51 RT heterodimer formation using the well-established yeast two-hybrid assay. We found that the mutations K101A, P225H, Y318F and Y318W decreased RT heterodimer stability whereas K103N, V108I, V108W, Y181C, Y188L, G190A, G190E, G190W and P225W increased RT heterodimer stability. While these results demonstrate that residues that comprise the NNRTI-binding pocket contribute to the stability of p66/p51 HIV-1 RT, they did not suggest any obvious correlation between RT dimer stability and the extent of NNRTI resistance. Remarkably, mutations at residue G190 (A, E, W) in the p66 RT subunit were found to dramatically increase heterodimer stability. Notably, the G190W mutation increased RT dimer stability almost to the same extent as did 5 microM efavirenz. In light of these findings, we characterized the in vitro activity of recombinant RT expressing mutations at G190 in the p66 subunit only and compared them with a wild-type enzyme complexed with efavirenz. We found that while mutations at G190 had a significant effect on both the DNA polymerase and ribonuclease H activity of the enzyme, their phenotypic effects did not mirror those induced by efavirenz-binding to RT.

A Novel Molecular Mechanism of Dual Resistance to Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors

“…It has been proposed that T369I and N348I mutants confer dual NRTI and NNRTI resistance by affecting the RT heterodimer stability (23). While a recent study did not reveal any obvious correlation between RT dimer stability and NNRTI resistance (18), further studies are needed to determine the relationship between RT dimerization, RNase H activity, the balance between polymerase and RNase H activities, and NRTI/NNRTI drug resistance.…”

The Connection Domain Mutation N348I in HIV-1 Reverse Transcriptase Enhances Resistance to Etravirine and Rilpivirine but Restricts the Emergence of the E138K Resistance Mutation by Diminishing Viral Replication Capacity

“…Substitutions at certain residues in HIV-1 RT can impair RNase H activity and contribute to reductions in HIV-1 replication fitness (69,70). In addition, some NNRTI resistance mutations are associated with impaired RNase H activity (69,(71)(72)(73)(74)(75), and N348I has been shown to reduce the rate of RNA template degradation (8,10,24,76). Furthermore, reduced RNase H activity may be associated with enhanced NNRTI resistance (77).…”

Effects of the W153L Substitution in HIV Reverse Transcriptase on Viral Replication and Drug Resistance to Multiple Categories of Reverse Transcriptase Inhibitors

“…Substitutions at certain residues in HIV-1 RT can impair RNase H activity and contribute to reductions in HIV-1 replication fitness (44,45). In addition, some NNRTI resistance substitutions are associated with impaired RNase H activity (44,(46)(47)(48)(49)(50), and reduced RNase H activity may also be associated with enhanced resistance to both NRTIs and NNRTIs (51,52). To investigate the effect of W153L, alone or in a background of K65R, M184I, K101E, K103N, E138K, or Y181C, on RNase H activity, we monitored multicycle RNase H-mediated RNA cleavage in time course experiments using WT RT and the W153L, W153L/K65R, W153L/M184I, W153L/K101E, W153L/ K103N, W153L/E138K, and W153L/Y181C variants.…”