Fluorometric determination of 2'-beta-fluoro-2',3'-dideoxyadenosine 5'-triphosphate, the active metabolite of a new anti-human immunodeficiency virus drug, in human lymphocytes.

A sensitive precolumn derivatization method has been developed to measure the 5'-triphosphate of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA, lodenosine), a new anti-HIV drug, in human lymphocytes by HPLC using fluorescence detection. Reaction of chloroacetaldehyde with F-ddA triphosphate in extracts from human lymphocytes produces a highly fluorescent etheno adduct. This derivative is then separated and quantitated by reverse-phase paired-ion chromatography. Degradation of natural nucleic acid ribosides, such as ATP, using periodate oxidation simplifies the chromatogram and minimizes interference with detection of the target analyte. This method, modeled using cultured MOLT-4 T-lymphocytes, achieves a linear detector response for peak area measurements over the range 2.5 to 22.5 pmol (50-450 nM using 50 microl sample). Analyte recovery is greater than 90%, and the method achieves a limit of detection and limit of quantitation of 1.4 and 2.5 pmol per HPLC injection (50 microl sample containing cellular extract from 2.5 x 10(6) cells), respectively. Application of this method to measure F-ddATP in peripheral blood mononuclear cells from HIV-infected patients treated with F-ddA at 3.2 mg/kg twice daily for 22 days shows F-ddATP levels which range from 1.5 to 3.5 pmol/10(6) cells.

Mutations in the HIV type 1 integrase of patients receiving long-term dideoxynucleoside therapy do not confer resistance to zidovudine.

Metabolites of AZT can inhibit HIV-1 integrase in vitro (Mazumder A, et al., Proc Natl Acad Sci USA 1994;91:5771-5775). To determine if long-term dideoxynucleoside therapy can lead to the emergence of HIV-1 AZT-resistant variants containing mutations in the integrase, we have sequenced the proviral DNA encoding the HIV-1 integrase of nine HIV-1-infected patients at different time points during treatment. Four of the nine patients developed mutations during the course of treatment. Although most mutations occurred at nonconserved amino acids, one patient developed a mutation at codon (R166T), a residue that is conserved among all integrases from known HIV-1 isolates. This mutation was introduced in the recombinant HIV-1 integrase protein to determine if it could confer resistance to AZT in vitro. We show that the R166T integrase mutant is still proficient at carrying 3'-processing and 3' end-joining but that the enzyme is not resistant to AZT-TP. Our results suggest that it is unlikely that integrase inhibition contributes to the antiviral activity of AZT.

Combination therapy with amprenavir, abacavir, and efavirenz in human immunodeficiency virus (HIV)-infected patients failing a protease-inhibitor regimen: pharmacokinetic drug interactions and antiviral activity.

Patients with plasma viral RNA >50,000 copies/mL, despite a protease-inhibitor regimen, received abacavir, amprenavir, and efavirenz to assess efavirenz-amprenavir drug interactions and to evaluate safety and antiviral response. Patients first received amprenavir with abacavir and other nucleoside analogs. Amprenavir levels were measured before and after adding efavirenz. Patients then received a second protease inhibitor. There was evidence of genotypic and phenotypic resistance at study entry. No patient had study drugs discontinued because of toxicity. Efavirenz decreased the steady-state area under the curve, maximum plasma concentration, and minimum plasma concentration of amprenavir by 24%, 33%, and 43%, respectively. Three of 10 patients had >1.5 log10 viral response to abacavir and amprenavir. All 8 patients who added efavirenz had >0.5 log10 decline in viral load, and this response lasted >24 weeks for 3 of the patients. A combination regimen that included abacavir, amprenavir, and efavirenz was well tolerated and had sustained activity in some patients. Concomitant efavirenz therapy decreases amprenavir concentrations.

In vitro induction of human immunodeficiency virus type 1 variants resistant to phosphoralaninate prodrugs of Z-methylenecyclopropane nucleoside analogues.

Two methylenecyclopropane nucleoside analogues with a phenylphosphoralaninate moiety, QYL-685 and QYL-609, exert potent and specific activities against human immunodeficiency virus type 1 strain LAI (HIV-1(LAI)) and HIV-2 in vitro. In this study, we induced HIV-1 variants resistant to QYL-685 by exposing HIV-1(LAI) to increasing concentrations of QYL-685. After 16 passages, the virus (HIV-1(P16)) was less sensitive to QYL-685 (104-fold), QYL-609 (>41-fold), and (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC) (>1, 100-fold) than was HIV-1(LAI) and contained an M184I mutation. Two infectious clones, HIV-1(M184I) and HIV-1(M184V), were resistant to QYL-685, QYL-609, and 3TC, confirming that the M184I mutation was responsible for the observed resistance. Viral-fitness analyses (competitive HIV-1 replication assays) revealed that in the absence of drugs, M184I and M184V conferred a replication disadvantage on the virus compared to the replication efficiency of the wild-type infectious clone (HIV-1(wt)). However, in the presence of QYL-685 (4 microM), HIV-1(M184I) and HIV-1(M184V) showed greater fitness than HIV-1(wt). These data may provide structural and virological relevance with regard to the emergence of M184I and M184V substitutions in HIV-1.

JE-2147: a dipeptide protease inhibitor (PI) that potently inhibits multi-PI-resistant HIV-1.

We designed, synthesized, and identified JE-2147, an allophenylnorstatine-containing dipeptide HIV protease inhibitor (PI), which is potent against a wide spectrum of HIV-1, HIV-2, simian immunodeficiency virus, and various clinical HIV-1 strains in vitro. Drug-resistant clinical HIV-1 strains, isolated from seven patients who had failed 9-11 different anti-HIV therapeutics after 32-83 months, had a variety of drug-resistance-related amino acid substitutions and were highly and invariably resistant to all of the currently available anti-HIV agents. JE-2147 was, however, extremely potent against all such drug-resistant strains, with IC(50) values ranging from 13-41 nM (<2-fold changes in IC(50) compared with that of wild-type HIV-1). The emergence of JE-2147-resistant HIV-1 variants in vitro was substantially delayed compared with that of HIV-1 resistant to another allophenylnorstatine-containing compound, KNI-272, and other related PIs. Structural analysis revealed that the presence of a flexible P2' moiety is important for the potency of JE-2147 toward wild-type and mutant viruses. These data suggest that the use of flexible components may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1. Further development of JE-2147 for treating patients harboring multi-PI-resistant HIV-1 is warranted.

Comparative fitness of multi-dideoxynucleoside-resistant human immunodeficiency virus type 1 (HIV-1) in an In vitro competitive HIV-1 replication assay.

We examined whether human immunodeficiency virus type 1 (HIV-1) fitness was altered upon the acquisition of a set or subset of five mutations (A62V, V75I, F77L, F116Y, and Q151M) in the pol gene, which confers resistance to multiple dideoxynucleosides (MDR), as well as the zidovudine resistance-associated mutation T215Y, using a competitive HIV-1 replication assay in a setting of an HXB2D genetic background. Target H9 cells were exposed to a 50:50 mixture of paired infectious molecular clones, and HIV-1 in the culture supernatant was transmitted to new cultures every 7 to 10 days. The polymerase-encoding region of the virus was sequenced at various time points, and the relative proportion of the two viral populations was determined. In the absence of drugs, the comparative order for replicative fitness was HIV-162/75/77/116/151 > HIV-177/116/151 > HIV-1151 > wild-type HIV-1 (HIV-1wt) > HIV-175/77/116/151 > HIV-1151/215 > HIV-1215. In the presence of zidovudine or didanosine, the order was HIV-162/75/77/116/151 > HIV-177/116/151 > HIV-175/77/116/151 > HIV-1151 > HIV-1215. HIV-1215S(TCC), a putative intermediate infectious clone for HIV-1215, replicated comparably to HIV-1wt, while two putative intermediates for HIV-1151 [HIV-1151L(CTG) and HIV-1151K(AAG)] replicated much less efficiently than HIV-1wt and HIV-1151, suggesting that for HIV-1151 to develop, two base substitutions are likely to occur concurrently or within a short interval. These data may illustrate the molecular basis by which HIV-1151 emerges much less frequently than HIV-1215. The present data also demonstrate that several MDR HIV-1 variants are more fit than HIV-1wt in the absence of drugs and that resistance-associated mutations and drug pressure are critical variates for HIV-1 fitness.

Emergence of multi-dideoxynucleoside-resistant human immunodeficiency virus type 1 variants, viral sequence variation, and disease progression in patients receiving antiretroviral chemotherapy.

A set of five reverse transcriptase mutations, which include Q151M, is known to confer multi-dideoxynucleoside resistance (MDR) in human immunodeficiency virus type 1 (HIV-1). MDR mutations were found in 6 (17%) HIV-1 isolates from 36 patients, most of whom were receiving long-term combination therapy. Q151M was among the first of the substitutions to appear. Additional substitutions were observed, although none were common among all 6 patients. Certain zidovudine-related mutations were not observed together with the MDR mutations, indicating possible enzymatic constraint. During chemotherapy, the HIV-1 RNA levels in the 6 patients initially decreased and then rose. Initially, CD4 cell counts also responded favorably but were near or below baseline beyond 40 months of therapy. Such loss of clinical benefits appeared to coincide with the appearance of the MDR mutations. A common background genotype was not observed among HIV-1 isolates with or without MDR.

In vitro induction of human immunodeficiency virus type 1 variants resistant to 2'-beta-Fluoro-2',3'-dideoxyadenosine.

2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA) is an acid-stable purine dideoxynucleoside analog active against a wide spectrum of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains in vitro. F-ddA is presently undergoing a phase I clinical trial at the National Cancer Institute. We induced HIV-1 variants resistant to F-ddA by exposing wild-type HIV-1 (HIV-1LAI) to increasing concentrations of F-ddA in vitro. After 18 passages, the virus was fourfold less sensitive to F-ddA than HIV-1LAI. Sequence analyses of the passage 18 virus revealed changes in three amino acids in the reverse transcriptase (RT)-encoding region of the pol gene: P to S at codon 119 (P119S; present in 3 of 13 and 28 of 28 molecular clones before and after F-ddA exposure, respectively), V179D (0 of 13 and 9 of 28, respectively), and L214F (9 of 13 and 28 of 28, respectively). Drug sensitivity assays using recombinant infectious clones confirmed that P119S was directly responsible for the reduced sensitivity of HIV-1 to F-ddA. Various infectious clones with single or multiple amino acid substitutions conferring viral resistance against nucleoside RT inhibitors, including HIV-1 variants with multi-dideoxynucleoside resistance, were generally sensitive to F-ddA. The moderate level of resistance of HIV-1 to F-ddA, together with the lack of conferment of significant cross-resistance by the F-ddA-associated amino acid substitutions, warrants further investigation of F-ddA as a potential antiviral agent for use in treatment of HIV-1 infection.

HIV-1 acquires resistance to two classes of antiviral drugs through homologous recombination.

Genetic recombination contributes to the genomic heterogeneity of human immunodeficiency virus type 1 (HIV-1). In the present study, we demonstrate that HIV-1 readily develops resistance to two classes of anti-HIV-1 drugs through in vitro genetic recombination involving large segments of the viral genome. Co-transfection of COS-7 cells with an HIV-1 plasmid (pSUM13) carrying five mutations in the reverse transcriptase (RT)-encoding region (A62V, V75I, F77L, F116Y, Q151M), conferring resistance to multiple dideoxynucleoside analogs (ddNs), and another HIV-1 plasmid (pSUM431) carrying five mutations in the protease-encoding region (V321, L33F, K451, 184V, L89M), conferring resistance to protease inhibitors such as KNI-272, readily produced HIV-1 carrying both sets of mutations when propagated in MT-2 cells in the presence of azidothymidine (AZT) and KNI-272. The resultant HIV-1 variant was highly resistant to both ddNs and KNI-272. Co-infection of MT-2 cells with HIV-1SUM13 carrying the RT mutations and HIV-1SUM431 carrying the mutations in the protease also generated HIV-1 with both sets of mutations when cultured with AZT and KNI-272. We also report here that the problematic artifactual recombination occurring during genetic analyses of heterogeneous nucleic acid sequences using polymerase chain reaction can be successfully obviated.

Genotypic and phenotypic characterization of HIV-1 isolated from patients receiving (--)-2',3'-dideoxy-3'-thiacytidine.

We attempted to determine whether HIV-1 developed resistance to (--)-2',3'-dideoxy-3'-thiacytidine ((--)-3TC or 3TC, lamivudine) in patients with advanced human immunodeficiency virus type 1 (HIV-1) infection during therapy with 3TC. Genotypic analysis of HIV-1 strains isolated from 6 patients receiving 3TC revealed that as early as 2 months of therapy, HIV-1 developed a Met to Val amino acid substitution at codon 184 (Met184-->Val) in the reverse transcriptase-coding region of the pol gene. A detailed study of a series of HIV-1 strains isolated from a patient demonstrated that Met at codon 184 was first substituted with Ile by 2 weeks of 3TC therapy, followed by the substitution with Val by 8 weeks. All HIV-1 strains with the Met184-->Val substitution were profoundly less susceptible to 3TC (1800- to 5500-fold decreased sensitivity) as compared to pretherapy virus strains. These strains were also moderately less sensitive to 2',3'-dideoxycytidine (4.5- to 9-fold), but more sensitive to 3'-azido-2',3'-dideoxythymidine (2- to 14-fold). A decrease in viremia levels and an increase in CD4 counts were observed early in therapy; however, these changes were only transient. Our data suggest that reversal of such beneficial changes is associated with the Met184-->Val substitution of the pol gene of HIV-1. The data also suggest that 3TC, as a single agent, may induce virologic and immunologic improvement in patients with advanced HIV-1 infection, but only transiently.

Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides.

A set of mutations [Ala-62-->Val(A62V), V75I, F77L, F116Y, and Q151M] in the polymerase domain of reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) confers on the virus a reduced sensitivity to multiple antiretroviral dideoxynucleosides and has been seen in HIV-1 variants isolated from patients receiving combination chemotherapy with 3'-azido-3'-deoxythymidine (AZT) plus 2',3'-dideoxycytidine (ddC) or 2',3'-dideoxyinosine (ddI). The IC50 values of AZT, ddC, ddI, 2',3'-dideoxyguanosine, and 2',3'-didehydro-3'-deoxythymidine against an infectious clone constructed to include the five mutations were significantly higher than those of a wild-type infectious clone. The K1 value for AZT 5'-triphosphate determined for the virus-associated RT from a posttherapy strain was 35-fold higher than that of RT from a pretherapy strain. Detailed analysis of HIV-1 strains isolated at various times during therapy showed that the Q151M mutation developed first in vivo, at the time when the viremia level suddenly increased, followed by the F116Y and F77L mutations. All five mutations ultimately developed, and the viremia level rose even further. Analyses based on the three-dimensional structure of HIV-1 RT suggest that the positions where at least several of the five mutations occur are located in close proximity to the proposed dNTP-binding site of RT and the first nucleotide position of the single-stranded template.

Inhibition of RNase H activity and viral replication by single mutations in the 3' region of Moloney murine leukemia virus reverse transcriptase.

Selected conserved amino acids in the putative RNase H domain of reverse transcriptase (RT) were modified in a molecularly cloned infectious provirus and in a Moloney murine leukemia virus RT expression vector by site-directed mutagenesis. Substitution of either of two conserved aspartic acid residues in proviral DNA prevented production of infectious particles in transfected NIH 3T3 cells, and the same modifications depressed RT-associated RNase H activity by more than 25-fold with little or no effect on polymerase activity.