The role of steric hindrance in 3TC resistance of human immunodeficiency virus type-1 reverse transcriptase 1 1Edited by A. R. Fersht

Abstract

Treating HIV infections with drugs that block viral replication selects for drug-resistant strains of the virus. Particular inhibitors select characteristic resistance mutations. In the case of the nucleoside analogs 3TC and FTC, resistant viruses are selected with mutations at amino acid residue 184 of reverse transcriptase (RT). The initial change is usually to M184I; this virus is rapidly replaced by a variant carrying the mutation M184V. 3TC and FTC are taken up by cells and converted into 3TCTP and FTCTP. The triphosphate forms of these nucleoside analogs are incorporated into DNA by HIV-1 RT and act as chain terminators. Both of the mutations, M184I and M184V, provide very high levels of resistance in vivo; purified HIV-1 RT carrying M184V and M184I also shows resistance to 3TCTP and FTCTP in in vitro polymerase assays. Amino acid M184 is part of the dNTP binding site of HIV-1 RT. Structural studies suggest that the mechanism of resistance of HIV-1 RTs carrying the M184V or M184I mutation involves steric hindrance, which could either completely block the binding of 3TCTP and FTCTP or allow binding of these nucleoside triphosphate molecules but only in a configuration that would prevent incorporation. The available kinetic data are ambiguous: one group has reported that the primary effect of the mutations is at the level of 3TCTP binding; another, at the level of incorporation. We have approached this problem using assays that monitor the ability of HIV-1 RT to undergo a conformational change upon binding a dNTP. These studies show that both wild-type RT and the drug-resistant variants can bind 3TCTP at the polymerase active site; however, the binding to M184V and M184I is somewhat weaker and is sensitive to salt. We propose that the drug-resistant variants bind 3TCTP in a strained configuration that is salt-sensitive and is not catalytically competent.

Selection and Characterization of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase Mutations

“…Such aphidicolin-resistant viruses exhibit altered sensitivities to other substrate analogues (17). Sequence analysis and marker transfer experiments located point mutations within the conserved regions of both HSV and vaccinia virus DNApols, which conferred resistance to aphidicolin (10,22,47 (4,16,35,36,38,45).The aim of this study was to select and characterize AcMNPV mutants resistant to antiviral compounds that target the viral DNApol enzyme. We hypothesized that "escape" mutants selected from the serial passage of the parental AcMNPV in the presence of increasing concentrations of these drugs would harbor mutation(s) in the AcMNPV genome, particularly within the DNA polymerase gene (dnapol).…”

“…Such aphidicolin-resistant viruses exhibit altered sensitivities to other substrate analogues (17). Sequence analysis and marker transfer experiments located point mutations within the conserved regions of both HSV and vaccinia virus DNApols, which conferred resistance to aphidicolin (10,22,47 (4,16,35,36,38,45).…”

Point Mutations Associated with HIV-1 Drug Resistance, Evasion of the Immune Response and AIDS Pathogenesis

“…The exclusion mechanism involves enhanced discrimination at the time the NRTI tri-phosphate is incorporated in the nascent DNA strand. The M184V/I point mutation (Figure 2 and Table 2), which selectively reduces incorporation of abacavir (ABC); emtricitabine (FTC) and lamivudine (3TC) NRTIs into a nascent DNA chain by steric hindrance (Gao et al, 2000;Sarafianos et al, 1999) is a common example of the exclusion mechanism. M184V point mutation causes a median 1.5-fold and 3.0-fold reduction in susceptibility to didanosine (ddI) and ABC, respectively in the PhenoSenseGT assay (Petropoulos et al, 2000;Rhee et al, 2004).…”

Probing the Mechanistic Consequences of 5-Fluorine Substitution on Cytidine Nucleotide Analogue Incorporation by HIV-1 Reverse Transcriptase

“…Both of these findings illustrate the large difference in steric and electrostatic constraints for the 2′-and 3′-positions in the D-versus L-conformation. These differences are apparent in structural models of L-analogues binding in the HIV-1 RT active site (Feng & Anderson, 1999a;Gao et al, 2000;Ray et al, 2002a). However, as discussed below, the steric contact with the 2′-and 3′-position of L-isomers may be increased by the M184V mutation causing marked changes in incorporation kinetics and antiviral activity (Tables 2 and 3).…”

“…A combination of kinetic studies and structural modeling have led to the hypothesis that 3TC resistance is caused by the addition of a β-branched amino acid at position 184, causing steric hindrance towards the binding of 3TCTP (Feng & Anderson, 1999b;Sarafianos et al, 1999;Gao et al, 2000). A recent transient kinetic report also shows that L-D4FCMP incorporation is selected against by the M184V/I mutation while D-D4FCTP is unaffected (Table 2) (Ray et al, 2002a).…”