Yudong Quan

Compensation by the E138K Mutation in HIV-1 Reverse Transcriptase for Deficits in Viral Replication Capacity and Enzyme Processivity Associated with the M184I/V Mutations

ABSTRACT Recently, several phase 3 clinical trials (ECHO and THRIVE) showed that E138K and M184I were the most frequent mutations to emerge in patients who failed therapy with rilpivirine (RPV) together with two nucleos(t)ide reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir (TDF). To investigate the basis for the copresence of E138K and M184I, we generated recombinant mutated and wild-type (WT) reverse transcriptase (RT) enzymes and HIV-1NL4-3 infectious clones. Drug susceptibilities were determined in cord blood mononuclear cells (CBMCs). Structural modeling was performed to analyze any impact on deoxynucleoside triphosphate (dNTP) binding. The results of phenotyping showed that viruses containing both the E138K and M184V mutations were more resistant to each of FTC, 3TC, and ETR than viruses containing E138K and M184I. Viruses with E138K displayed only modest resistance to ETR, little resistance to efavirenz (EFV), and no resistance to either FTC or 3TC. E138K restored viral replication capacity (RC) in the presence of M184I/V, and this was confirmed in cell-free RT processivity assays. RT enzymes containing E138K, E138K/184I, or E138K/184V exhibited higher processivity than WT RT at low dNTP concentrations. Steady-state kinetic analysis demonstrated that the E138K mutation resulted in decreased K m s for dNTPs. In contrast, M184I/V resulted in an increased K m for dNTPs compared to those for WT RT. These results indicate that the E138K mutation compensates for both the deficit in dNTP usage and impairment in replication capacity by M184I/V. Structural modeling shows that the addition of E138K to M184I/V promotes tighter dNTP binding.

Mechanistic Studies of Early Pausing Events during Initiation of HIV-1 Reverse Transcription

We have investigated the role of sequences that surround the primer binding site (PBS) in the reverse transcriptase-mediated initiation of (−) strand DNA synthesis in human immunodeficiency virus type 1. In comparisons of reverse transcription initiated from either the cognate primer tRNALys.3 or a DNA primerd-Lys.3, bound to PBS sequences, we observed that a +3 pausing site occurred in both circumstances. However, the initiation reaction with tRNALys.3 was also characterized by a pausing event after incorporation of the first nucleotide. Alteration of sequences at the 5′-end instead of the 3′-end of the PBS resulted in elimination of the +3 pausing site, suggesting that this site was template sequence-dependent. In contrast, the pausing event at the +1 nucleotide position was still present in experiments that employed either of these mutated RNA templates. The mutations at the 5′-end of the PBS also caused a severely diminished rate of initiation and the strong arrest of reactions at the +1 stage when tRNALys.3 was used as primer. Therefore, we propose that the +1 pausing event is an initiation-specific event in regard to reactions primed by tRNALys.3 and that sequences at the 5′-end of the PBS may facilitate the release of reverse transcription from initiation to elongation.

Drug resistance profiles of recombinant reverse transcriptases from human immunodeficiency virus type 1 subtypes A/E, B, and C.

We have expressed purified recombinant reverse transcriptase (RT) from clinical isolates of human immunodeficiency virus subtypes B, C, and A/E in Escherichia coli. The drug sensitivities of these RTs were then determined for both nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs) in cell-free RT assays. Although A/E and C viruses contained numerous polymorphisms relative to subtype B (i.e., naturally occurring variations unrelated to drug resistance), the wild-type enzymes prepared from these or subtype A/E clinical isolates displayed <2-fold differences in drug sensitivities with regard to the active triphosphate active forms of NRTIs, as compared with RT expressed from BH-10 recombinant virus. Recombinant RTs from clinical isolates of subtypes B, C, and A/E that contained multiple resistance-associated mutations displayed expected variations in levels of resistance to the intracellular active forms of 3TC, ddI, ddC, and PMPA, that is, 3TCTP, ddATP, ddCTP, and PMPApp, respectively. Subtype A/E and C RT enzymes contained only minor NNRTI polymorphisms that distinguished them from wild-type subtype B enzymes and wild-type RTs from these various subtypes showed only 1- to 4-fold variability in IC(50) values for each of nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), and calanolide A. In contrast, RT enzymes from subtype B and C viruses harboring specific NNRTI mutations were highly resistant to all four tested NNRTIs. Subtype C variants containing the novel V106M resistance codon showed cross-resistance to all approved NNRTIs in cell-free RT assays.

Multidrug-Resistant Variants of HIV Type 1 (HIV-1) Can Exist in Cells as Defective Quasispecies and Be Rescued by Superinfection with Other Defective HIV-1 Variants

A tissue culture cell line infected with multidrug-resistant (MDR) human immunodeficiency virus type 1 (HIV-1) produced only noninfectious particles because of a lethal mutation in env. The defective MDR provirus was rescued by superinfection with either wild-type HIV-1 or a second replication-defective virus lethally mutated in capsid. Drug-resistance phenotyping revealed that the MDR viruses dominated if even single reverse-transcriptase inhibitors were present, reflecting linkage of the various drug resistance mutations on a single viral nucleic acid backbone. These results are most likely attributable to recombination during second rounds of infection and suggest that defective HIV-1 variants may nonetheless constitute part of the HIV-1 reservoir.

Effect of Mutations at Position E138 in HIV-1 Reverse Transcriptase and Their Interactions with the M184I Mutation on Defining Patterns of Resistance to Nonnucleoside Reverse Transcriptase Inhibitors Rilpivirine and Etravirine

ABSTRACT Impacts of mutations at position E138 (A/G/K/Q/R/V) alone or in combination with M184I in HIV-1 reverse transcriptase (RT) were investigated. We also determined why E138K is the most prevalent nonnucleoside reverse transcriptase inhibitor mutation in patients failing rilpivirine (RPV) therapy. Recombinant RT enzymes and viruses containing each of the above-mentioned mutations were generated, and drug susceptibility was assayed. Each of the E138A/G/K/Q/R mutations, alone or in combination with M184I, resulted in decreased susceptibility to RPV and etravirine (ETR). The maximum decrease in susceptibility to RPV was observed for E138/R/Q/G by both recombinant RT assay and cell-based assays. E138Q/R-containing enzymes and viruses also showed the most marked decrease in susceptibility to ETR by both assays. The addition of M184I to the E138 mutations did not significantly change the levels of diminution in drug susceptibility. These findings indicate that E138R caused the highest level of loss of susceptibility to both RPV and ETR, and, accordingly, E138R should be recognized as an ETR resistance-associated mutation. The E138K/Q/R mutations can compensate for M184I in regard to both enzymatic fitness and viral replication capacity. The favored emergence of E138K over other mutations at position E138, together with M184I, is not due to an advantage in either the level of drug resistance or viral replication capacity but may reflect the fact that E138R and E138Q require two distinct mutations to occur, one of which is a disfavorable G-to-C mutation, whereas E138K requires only a single favorable G-to-A hypermutation. Of course, other factors may also affect the concept of barrier to resistance.

Lamivudine Can Exert a Modest Antiviral Effect against Human Immunodeficiency Virus Type 1 Containing the M184V Mutation

ABSTRACT The M184V mutation in human immunodeficiency virus (HIV) reverse transcriptase is associated with high-level resistance to both (−)2′,3′-dideoxy-3′-thiacytidine (3TC) and (−)2′,3′-dideoxy-5-fluoro-3′-thiacytidine as well as low-level resistance to 2′,3′-dideoxyinosine, 2′,3′-dideoxycytidine, and abacavir. This mutation is also associated with diminished HIV replicative fitness as well as several functional changes in enzyme activity, including diminutions in polymerase processivity, pyrophosphorylysis, and nucleotide primer unblocking. Despite the fact that M184V encodes up to 1,000-fold resistance to 3TC, we asked whether this drug might still display some antiviral effect in regard to viruses containing this mutation. Cell-free assays revealed that high concentrations of 3TC triphosphate (i.e., >100 μM) could affect chain termination and/or inhibit purified reverse transcriptase containing the M184V substitution. This effect became more pronounced with elongation of reverse transcriptase products. In newly infected cells (i.e., peripheral blood mononuclear cells), we found that the amount of full-length reverse transcriptase product was diminished in the presence of 2 to 10 μM 3TC, although no decrease in the first product of the reverse transcriptase reaction, i.e., minus strong-stop DNA, was observed. In the presence of two other HIV inhibitors, e.g., nevirapine and indinavir, 3TC exerted additive effects in tissue culture at concentrations only marginally higher than the 50% inhibitory concentration (IC50). Reverse transcriptases cloned from clinical isolates harboring M184V in the context of multidrug resistance had similar IC50 values for 3TC triphosphate compared to reverse transcriptase containing only the M184V mutation. These results suggest that viruses containing M184V can retain a higher degree of sensitivity to 3TC than previously assumed.

The M230L nonnucleoside reverse transcriptase inhibitor resistance mutation in HIV-1 reverse transcriptase impairs enzymatic function and viral replicative capacity.

ABSTRACT The M230L mutation in HIV-1 reverse transcriptase (RT) is associated with resistance to first-generation nonnucleoside reverse transcriptase inhibitors (NNRTIs). The present study was designed to determine the effects of M230L on enzyme function, viral replication capacity (RC), and the extent to which M230L might confer resistance to the second-generation NNRTI etravirine (ETR) as well as to the first-generation NNRTIs efavirenz (EFV) and nevirapine (NVP). Phenotyping assays with TZM-bl cells confirmed that M230L conferred various degrees of resistance to each of the NNRTIs tested. Recombinant viruses containing M230L displayed an 8-fold decrease in RC compared to that of the parental wild-type (WT) virus. Recombinant HIV-1 WT and M230L mutant RT enzymes were purified; and both biochemical and cell-based phenotypic assays confirmed that M230L conferred resistance to each of EFV, NVP, and ETR. RT that contained M230L was also deficient in regard to each of minus-strand DNA synthesis, both DNA- and RNA-dependent polymerase activities, processivity, and RNase H activity, suggesting that this mutation contributes to diminished viral replication kinetics.

Dominance of the E89G Substitution in HIV-1 Reverse Transcriptase in Regard to Increased Polymerase Processivity and Patterns of Pausing

The substitution of a glycine for glutamic acid at position 89 in human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) (E89G) confers resistance to several nucleoside and non-nucleoside inhibitors of RT. As residue 89 contacts the template strand, it has been suggested that this mutation may modulate the conformation of the RT·template/primer complex. In addition, certain mutations in RT that confer resistance to nucleoside analogs, such as M184V, are located near the polymerase active site. To characterize further these substitutions, we performed processivity assays alongside an analysis of pausing profiles with wild-type (wt) RT and recombinant RTs containing substitutions at E89G, M184V, or both. We now show that E89G RT has higher processivity than wt enzyme as well as a different pattern of pausing sites. Similar findings were obtained with the doubly mutated RT, although enzyme containing only the M184V mutation had lower processivity than wt. Consistent with these observations, and from a mechanistic standpoint, both E89G-containing as well as doubly mutated RT had decreased dissociation constants from a complex consisting of RT and template-primer, in comparison with either wt RT or M184V-containing RT. No significant differences were observed among the various enzymes in regard to K m values for the heteropolymeric RNA template used in these studies. Viruses containing the E89G mutation synthesized longer strand DNA products than either wt viruses or viruses containing only the M184V mutation in endogenous RT assays. Thus, the E89G substitution is a dominant determinant in regard to each of the k offvalues from an RT·template/primer complex, RT processivity, and specific patterns of pausing during DNA polymerization.

Molecular Mechanism of Antagonism between the Y181C and E138K Mutations in HIV-1 Reverse Transcriptase

ABSTRACT Etravirine (ETR) is an expanded-spectrum nonnucleoside reverse transcriptase inhibitor (NNRTI) approved for use as an antiretroviral agent in treatment-experienced patients. Y181C and E138K in HIV-1 RT are among 20 different drug resistance mutations associated with ETR. However, E138K can be consistently selected by ETR when wild-type viruses but not viruses containing Y181C are grown in tissue culture. This study was carried out to evaluate any possible mechanisms that might explain antagonism between the Y181C and E138K mutations. Accordingly, we performed tissue culture studies to investigate the evolutionary dynamics of E138K in both a wild-type (WT) and a Y181C background. We also generated recombinant enzymes containing Y181C and E138K alone or in combination in order to study enzyme processivity, rates of processive DNA synthesis, enzyme kinetics, and susceptibility to ETR. We now show that the presence of the Y181C mutation prevented the emergence of E138K in cell culture and that the simultaneous presence of E138K and Y181C impaired each of enzyme activity, processivity, rate of processive DNA synthesis, and deoxynucleoside triphosphate (dNTP) affinity. The addition of E138K to Y181C also decreased the level of resistance to ETR compared to that obtained with Y181C alone.

Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors

Background: The viral RNA-dependent RNA polymerase (RdRp) enzymes of the Flaviviridae family are essential for viral replication and are logically important targets for development of antiviral therapeutic agents. Zika virus (ZIKV) is a rapidly re-emerging human pathogen for which no vaccine or antiviral agent is currently available. Methods: To facilitate development of ZIKV RdRp inhibitors, we have established an RdRp assay using purified recombinant ZIKV NS5 polymerase. Results: We have shown that both the hepatitis C virus (HCV) nucleoside inhibitor sofosbuvir triphosphate and a pyridoxine-derived non-nucleoside small-molecule inhibitor, DMB213, can act against ZIKV RdRp activity at IC50s of 7.3 and 5.2 &mgr;M, respectively, in RNA synthesis reactions catalysed by recombinant ZIKV NS5 polymerase. Cell-based assays confirmed the anti-ZIKV activity of sofosbuvir and DMB213 with 50% effective concentrations (EC50s) of 8.3 and 4.6 &mgr;M, respectively. Control studies showed that DMB213 did not inhibit recombinant HIV-1 reverse transcriptase and showed only very weak inhibition of HIV-1 integrase strand-transfer activity. The S604T substitution in motif B of the ZIKV RdRp, which corresponds to the S282T substitution in motif B of HCV RdRp, which confers resistance to nucleotide inhibitors, also conferred resistance to sofosbuvir triphosphate, but not to DMB213. Enzyme assays showed that DMB213 appears to be competitive with natural nucleoside triphosphate (NTP) substrates. Conclusions: Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.