Background: After the initial discovery of 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) and tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and thione (TIBO) derivatives, several other non-nucleoside reverse transcriptase (RT) inhibitors (NNRTI), including nevirapine (BI-RG-587), pyridinone derivatives (L-696,229 and L-697,661), delavirdine (U-90152), agr;-anilinophenylacetamides (agr;-APA) and various other classes of NNRTI have been described. The hallmark of NNRTI has been based on their ability to interact with a specific site (‚pocket‚) of HIV-1 RT.
Objective: To investigate whether, in addition to HIV-1, different strains of HIV-2 (ROD and EHO) and SIV (mac251, agm3 and mndGB1) are sensitive to a selection of NNRTI i.e. delavirdine, the HEPT derivative I-EBU (MKC-442), 8-chloro-TIBO (tivirapine), agr;-APA (loviride), nevirapine and the pyridinone derivative L-697,661.
Methods and results: The NNRTI tested inhibited the replication of the different strains of HIV-2 and SIV at micromolar concentrations. The inhibitory effects of the NNRTI on HIV-2-induced cytopathicity correlated well with their inhibitory effects on HIV-2 RT activity. Drug-resistant HIV-2 (EHO) variants containing the Ser102Leu and/or Glu219Asp mutations in their RT were selected after passaging the virus in MT-4 cells in the presence of increasing concentrations of delavirdine. The EHO virus mutants were at least 20-fold less susceptible to the antiviral effects of delavirdine. Some cross-resistance, depending on the mutant strain, was observed with the other NNRTI tested (i.e. MKC-442, tivirapine, loviride and pyridinone L-697,661).
Conclusions: Our data demonstrate that NNRTI are not exclusively specific for HIV-1 but are also inhibitory to different HIV-2 and SIV strains. These observations will have important implications for the development of new NNRTI with higher activity against both HIV-1 and HIV-2. Furthermore, in view of their anti-SIV activity, NNRTI could be evaluated further for their in vivo anti-retrovirus efficacy in non-human primate models.
From the Rega Institute for Medical Research, KULeuven, Leuven, Belgium.
Sponsorship: Supported in part by the Biomedical Research Programme of the European Commission (EC Biomed2 grant BMH4-C5-95-1634) and by grants from the Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen (FWO grant G.0140.98), the Geconcerteerde Onderzoeksacties (GOA 95/5) Vlaamse gemeenschap and the Janssen Research Foundation.
Requests for reprints to: M.Witvrouw, Rega Institute for Medical Research, KULeuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Received: 28 October 1998; revised: 7 May 1999; accepted: 12 May 1999.
The causative agent of AIDS, HIV-1, was discovered in 1983 . Three years later, a second human immunodeficiency virus (designated HIV-2) was isolated from west African patients with AIDS [2,3].
Although the biological properties of HIV-1 and HIV-2 are very similar, a genetic comparison of the two human viruses reveals significant sequence divergence. The predicted amino acid sequence similarity for the entire pol gene of HIV-1 and HIV-2 is approximately 60%. Hizi et al.  demonstrated that, despite the limited sequence similarity between HIV-1 and HIV-2 reverse transcriptase (RT), both proteins are quite similar in overall structure and functionality. This suggests that at least some of the drugs effective against HIV-1 RT could also be effective against HIV-2 RT. Whereas HIV-1 is closely related to a primate lentivirus isolated from wild chimpanzees (SIVcpz) [5-7], HIV-2 appears to be closely related to SIV strains isolated from different species of immunodeficient macaques (SIVmac)  or SIVsm from sooty mangabeys [9,10]. Besides these two genetically distinct lineages of HIV and SIV strains, another three distinct lineages of African SIV strains have been isolated: SIVagm from African green monkeys [11,12], SIVmnd from mandrills  and SIVsyk from Sykes‚ monkeys . SIVagm, SIVmnd and SIVsyk all appear to be equidistant from both HIV-1 and HIV-2 .
We have now studied different strains of HIV-2 and SIV for their susceptibility to inhibition by previously reported HIV-1-specific RT inhibitors, the non-nucleoside RT inhibitors (NNRTI) [16-27]. All of these compounds share a number of common features which distinguish them from the dideoxynucleoside type of RT inhibitors, e.g. zidovudine (3′-azido-2′,3′-dideoxythymidine), didanosine (2′,3′-dideoxyinosine), zalcitabine (2′,3′-dideoxycytidine), stavudine (2′,3′-didehydro-2′,3′-dideoxythymidine) and lamivudine [(-)-3′-thia-2′-deoxycytidine]. They do not require intracellular metabolization, and interact with the RT of HIV in a non-competitive manner with regard to the natural substrate (dNTP) at an allosteric (non-nucleoside) binding site . Not only are they independent of the phosphorylating enzyme machinery of the cells [which may be unsatisfactory (i.e., in resting cells such as monocytes/macrophages)], their action at the RT level cannot be outweighed by competing dNTP pool levels. For the last decade NNRTI have been reported to achieve a highly selective suppression of HIV-1 replication in cell culture, with little, if any cytotoxicity, and without affecting the replication of HIV-2. Use of NNRTI leads rapidly to the emergence of drug-resistant HIV-1 mutant strains , the mutations being clustered around the putative binding site of the NNRTI (non-nucleoside binding site; NNBS). Here we report on the activity of NNRTI against different strains of HIV-2 and SIV; their inhibitory activity against these viruses is probably mediated by interference with the reverse transcription process as shown previously for HIV-1. Mutant HIV-2 (EHO) virus strains were selected in cell culture in the presence of increasing concentrations of delavirdine.
Materials and methods
The HIV strains used were HIV-1 (IIIB)  and HIV-2 (ROD  and EHO ). The SIV (mac251) strain was originaly isolated by Daniel et al.  and was obtained from C. Bruck (Smith Kline-RIT, Rixensart, Belgium); SIV (agm3) [11,12] was a kind gift from R. Kurth, Paul Ehrlich Institut, Langen, Germany; SIV (mndGB1)  was from G. Hunsmann, Deutsches Primatenzentrum, Göttingen, Germany.
MT-4 cells and MOLT-4 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum, 2mM L-glutamine, 0.1% sodium bicarbonate, and 20μg/ml gentamicin.
8-Chloro TIBO R86183 (tivirapine) and agr;-APA R89439 (loviride) were obtained from the Janssen Research Foundation (Beerse, Belgium). U-90152 (delavirdine) was kindly provided by Pharmacia & Upjohn (Kalamazoo, Michigan, USA) by B. Bruce. BI-RG587 (nevirapine) was obtained from Boehringer Ingelheim (Ridgefield, Connecticut, USA). Pyridinone L-697,661 was provided by Merck Sharp & Dohme (West Point, Pennsylvania, USA).
Cell viability of MT-4 cells infected at 200× the CCID50 (50% cell culture infective dose) of HIV-1 (IIIB), HIV-2 (ROD), HIV-2 (EHO) or SIV (mac251) was determined by a tetrazolium colorimetric method (MTT), as described previously . This method was used to determine the 50% effective concentrations (EC50). Antiviral efficacy in MOLT-4 cells infected with SIV (agm3) or SIV (mndGB1) at 200× the CCID50, was evaluated microscopically by quantifying virus-induced cytopathicity.
Selection of delavirdine-resistant virus strains
HIV-2 (EHO) was subjected to passages in MT-4 cell cultures (4×105 cells/ml) in the presence of dose- escalating concentrations of delavirdine (see Fig. 1). Passages were performed every 3-5 days by adding 0.5ml of the infected cultures to 4.5ml of 4×105 uninfected MT-4 cells/ml. As soon as virus-induced syncytium formation became prominent in the cell culture, the supernatant was frozen in aliquots at -70°C and the RT gene of the virus was characterized.
Nucleic acid extraction: cDNA synthesis, amplification, and solid-phase sequencing
DNA from 1×106 cells was extracted using the QIAamp blood kit (Qiagen, Hilden, Germany) following the manufacturer‚s instructions. DNA was resuspended in 100μl water, and 10μl DNA solution was used in one PCR amplification. A 1504bp fragment from the HIV-2 RT gene was amplified by PCR using primers AV161 (sense, 5′-GCAATTACACCCC AAAAATAGTAG, nt 434-457 of EHO) and AV162 (antisense, 5′-CCCCTGTCTGTGACATATCCTG, nt 1917-1938 of EHO). The buffer used was 10mM Tris-HCl pH8.3, 50mM KCl, 3mM MgCl2, 200μM dNTPs (Amersham Pharmacia Biotech, Uppsala, Sweden), 0.2μM primers and 0.025U/μl AmpliTaq (Perkin-Elmer, Brussels, Belgium). The cycling conditions, in a thermocycler (model 9600, Perkin-Elmer), were 40 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 1 min. Then 4μl of the outer PCR product was used in a 100μl nested PCR with primers M13-USP-AV163 (sense, 5′-GTA AAA CGA CGG CCA GTC CCC AAT AAA CAT TTT TGG CAG, nt 552-573 of EHO) and M13-RSP-AV164 (antisense, 5′-biotin-CAG GAA ACA GCT ATG ACC TGA GCT GCC CAA TTT AAT ACT C, nt 1357-1379 of EHO) to sequence the 827 bp fragment representing the first part of HIV-2 (EHO) RT gene and primers M13-USP-AV183 (sense, 5′-GTA AAA CGA CGG CCA GTC CAT TCA AAT GGA TGG GGT ATG, nt 1255-1277 of EHO) and M13-RSP-AV184 (antisense, 5′-biotin-CAG GAA ACA GCT ATG ACC CTT TGT TAC AGG ATC CAT CTG TG, nt 1875-1898 of EHO) to sequence the 643bp fragment representing the last part of the HIV-2 (EHO) RT gene; the M13 sequencing tags are given in italics. The buffer used was 10mM Tris-HCl pH 8.3, 50mM KCl, 3mM MgCl2, 200μM dNTPs (Amersham Pharmacia Biotech), 0.2μM primers and 0.025U/μl AmpliTaq (Perkin Elmer). The cycling conditions on a thermocycler (Biometra; Westburg, Leusden, The Netherlands) were 30 cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 1 min. Sequencing was performed with a fluorescent-labelled M13 universal sequencing primer [fluorescein isothiocyanate (FITC)-M13-USP: 5′-GTAAAACG ACGGCCAGT] or fluorescent-labelled M13 reverse sequencing primer (FITC-M13-RSP: 5′-CAGGAAA CAGCTATGA) for the sequencing reaction. The inner PCR product (3μl) was analysed by polyacrylamide gel electrophoresis with ethidium bromide staining to check for the presence of a sufficient quantity of DNA and the absence of non-specific bands. Sequencing was performed on an Automated DNA Sequencer (ALF, Amersham Pharmacia Biotech) using the AutoRead Sequencing Kit (Amersham Pharmacia Biotech). The sequence alignments were done using the software GeneWorks 2.5.1 (IntelliGenetics Inc., Oxford, UK) as described previously .
The purified recombinant HIV-1 RT was prepared as described previously . Virion-derived HIV-2 (ROD, EHO, EHO/S102L, EHO/E219D and EHO/S102L/E219D) RT was obtained from the cell culture fluids of infected MT-4 cells. The cells were clarified by low-speed centrifugation (10 min, 140g). Supernatants were filtered through a 0.22μm Millex GV filter (Millipore, Molsheim, France). Virus particles were subsequently pelleted by centrifugation at 100000g for 2 h. Pellets were resuspended in a solution containing 5mM Tris-HCl pH 8.1, 1mM dithiothreitol, 0.1% (w/v) Triton X-100 and 0.5M KCl. Resuspended pellets were stored in aliquots at -70°C until use.
The RT reaction mixture (50μl) contained 50mM Tris-HCl pH 8.1, 10mM MgCl2, 100mM KCl, 2.2mM dithiothreitol and 0.05% (w/v) Triton X-100. The template/primer [poly(C)/oligo(dG)12-18] (Amersham Pharmacia Biotech) was used at a concentration of 65μg/ml. Tritium-labelled dGTP was from Amersham and used at a final concentration of 2.5μM. Specific activity was 11Ci/mmol (1Ci=37GBq). Following addition of the inhibitors at various concentrations and 10μl of the different enzyme preparations, the reaction mixture was incubated for 1 h at 37°C. The incorporation rate was determined by a standard trichloracetic acid precipitation procedure using Whatman GF/C glass fibre filters (Whatman, Maidstone, England) and liquid scintillation counting (Ready-Protein; Beckman, Fullerton, CA, USA).
MKC-442 (I-EBU, tivirapine and loviride were found to inhibit HIV-2 (strains ROD and EHO) and SIV (strains mac251, agm3 and mndGB1) replication in cell culture, MKC-442 being the most active with an EC50 of 0.3μM against SIV (agm3) (Table 1). This is at a 50-fold higher concentration than the concentration required to inhibit HIV-1 (IIIB) replication in MT-4 cells. MKC-442 inhibited the replication of HIV-2 (ROD and EHO) and SIV (mac251 and mndGB1) at EC50 values in the range 5.6-13.2μM. Tivirapine showed comparable antiviral activities against HIV-2 (ROD and EHO) and SIV (mac251, agm3 and mndGB1) (EC50 values in the range 6.2-15.5μM). Loviride was equally active against the replication of HIV-2 (EHO) and SIV (mac251) (EC50: 7.4 and 11.1μM, respectively) but five to tenfold less active than against the replication of HIV-2 (ROD) and SIV strains agm3 and mndGB1. Delavirdine and pyridinone L-697,661 inhibited the replication of HIV-2 (EHO) and SIV (agm3) at an EC50 in the range 2.1-28.4μM. However, HIV-2 (ROD), SIV (mac251) and SIV (mndGB1) were not sensitive to the antiviral effect of delavirdine and pyridinone L-697,661 at subtoxic concentrations. Nevirapine inhibited the replication of SIV (agm3) only at an EC50 of 18.8μM.
When HIV-2 (EHO)-infected MT-4 cell cultures were exposed to increasing concentrations of delavirdine, a delavirdine-resistant HIV-2 (EHO) strain emerged after six passages (Fig. 1a). The mutant HIV-2 (EHO/Ser102Leu) strain showed a single amino acid change (serine to leucine) at position 102 of its RT. The Ser102Leu RT mutation was still the only mutation seen after an additional five and 12 passages in the presence of 41μM delavirdine (Fig. 1a). When the selection procedure was repeated for resistant virus (Fig. 1b), the Glu219Asp mutation emerged after seven passages of HIV-2 (EHO) in MT-4 cells in the presence of delavirdine. After an additional 13 passages, Glu219Asp was still the only mutation present whereas an additional Ser102Leu mutation emerged after 11 more passages (31 passages in total) (Fig. 1b). The same two mutations were seen after an additional 12 passages (43 passages in total) in the presence of 41μM delavirdine. The mutant HIV-2 (EHO/Ser102Leu) strain showed more than 20-fold resistance to delavirdine, as compared with wild-type HIV-2 (EHO) (Table 2). From the other four NNRTI tested L-697,661 showed the highest cross-resistance (>sixfold), whereas tivirapine and loviride did not loose any activity against the Ser102Leu mutant EHO strain. The mutant EHO/Glu219Asp strain lost more than 20-fold sensitivity, in comparison with the wild-type HIV-1 (EHO), towards delavirdine, whereas no cross-resistance was observed to the other four NNRTI tested. The EHO/Ser102Leu/ Glu219Asp mutant strain was also >20-fold more resistant to delavirdine, whereas MKC-442 and pyridinone L-697,661 showed at least 6-23-fold more cross-resistance. The activity of tivirapine and loviride was reduced only slightly.
The inhibitory effects of the NNRTI on the cytopathicity induced by HIV-2 (ROD) correlated well with their respective inhibitory effects on virion-derived HIV-2 (ROD) RT activity (Table 3). As shown previously for HIV-1 , the compounds inhibited HIV-2 (ROD) RT activity at approximately 15-150-fold higher concentrations than the concentrations required to inhibit HIV-2 (ROD) replication in cell culture. The virion-derived HIV-2 (EHO) RT was sensitive only to delavirdine, the most active drug from the series tested against HIV-2 (EHO) in cell culture (EC50, 2.1μM). When the EHO/Ser102Leu and EHO/Ser102Leu/Glu219Asp virion-derived RT were tested, sensitivity to delavirdine was lost completely at concentrations up to 1028μM, whereas the EHO/Glu219Asp virion-derived RT was only half as sensitive to delavirdine as compared with the HIV-2 (EHO) wild-type virion-derived RT (IC50, 393μM).
Our data demonstrate that NNRTI, i.e. delavirdine, MKC-442, tivirapine, loviride, nevirapine and pyridinone L-697,661 are not exclusively specific for HIV-1 but are also inhibitory to different HIV-2 (ROD and EHO) and SIV strains (mac251, agm3 and mndGB1). MKC-442, tivirapine and loviride were able to inhibit the replication of all five non-HIV-1 viruses tested, whereas delavirdine and pyridinone L-697,661 were not able to inhibit the replication of HIV-2 (ROD) and SIV (mac251 and mndGB1) at subtoxic concentrations. Nevirapine inhibited the replication of SIV (agm3) only. Moreover, the concentrations of NNRTI required to inhibit HIV-2 or SIV replication had to be at least 50-fold the concentrations required to inhibit HIV-1(IIIB) replication in cell culture. This is probably the reason why this anti-HIV-2 and anti-SIV activity was not noticed before with less powerful NNRTI. For divergent strains, it is difficult to draw conclusions on drug sensitivity based on genetic information; nevertheless, we studied the amino acid alignment of the RT of the different HIV-2 and SIV strains (based on published EMBL accession numbers K03455, M15390, M27470, M30931 and U27200 L14545), but there was no indication that a particular amino acid change could be responsible for the observed differences in sensitivity between HIV-1 and HIV-2 or SIV.
Several observations point to RT as a target for the inhibitory effects of NNRTI on the replication of HIV-2 and SIV: (i) the positive correlation between the inhibitory effects of NNRTI on HIV-2- and SIV-induced cytopathicity; (ii) the inhibitory effects of NNRTI on HIV-2 and SIV RT activity (at higher concentrations than those required for inhibition of cytopathicity, an effect also seen in the activity of NNRTI to HIV-1 replication versus HIV-1 RT ); and (iii) the selection of a TCA (serine) to TTA (leucine) mutation at amino acid 102 and a GAA (glutamic acid) to GAT (aspartic acid) mutation at amino acid 219 in the RT gene of HIV-2 EHO in the presence of delavirdine.
Although active, the NNRTI tested, i.e. delavirdine, MKC-442, tivirapine, loviride, nevirapine and pyridinone L-697,661 interact less specifically with HIV-2 RT than with HIV-1 RT. The following arguments support this statement: (i) at least 50-fold higher concentrations were required to inhibit the replication of HIV-2 (strains ROD and EHO) and SIV (strains mac251, agm3 and mndGB1) in comparison with the concentrations required to inhibit the replication of HIV-1(IIIB) in cell culture; (ii) mutations in HIV-2 RT appear at positions Ser102 and Glu219. The homologous amino acid Lys102 (Fig. 2) is situated in the NNBS of HIV-1 RT, suggesting a similar putative binding site of the NNRTI to the HIV-2 RT. Treatment of HIV-1-infected patients with NNRTI led rapidly to the emergence of drug resistant HIV-1 mutant strains , the mutations being clustered around the NNBS. In contrast, amino acid Glu219 is most probably situated at the catalytic site of HIV-2 RT. Treatment of HIV-1-infected patients with nucleoside RT inhibitors such as zidovudine, led to the emergence of mutations at the catalytic site of HIV-1 RT including the homologous amino acid Lys219 . As mutations in HIV-2 RT in the presence of delavirdine occur at both positions 102 and 219 which cannot be part of a single binding pocket, we can only speculate on the interaction of delavirdine with HIV-2 RT: this could be to a similar NNRTI pocket as in the HIV-1 RT, but less specific. Possibly, this ‚duality‚ in the interaction of NNRTI with the HIV-2 RT results from the fact that delavirdine has not been optimized to fit perfectly the HIV-2 RT NNRTI binding pocket. Therefore, it might be possible to optimize the chemical structure of NNRTI to obtain compounds which interact more specifically with HIV-2.
In conclusion, NNRTI are active not only against HIV-1, but also against HIV-2 and SIV. These observations may have important implications for the development of new NNRTI that are active against both HIV-1 and HIV-2, the latter in view of the increasing incidence of HIV-2 infection in developed countries and India [37-39]. Furthermore, given their anti-SIV efficacy, these NNRTI could be evaluated for their activity in vivo in non-human primate models before being launched in the clinic.
The authors thank J. Balzarini, Z. Debyser and R. Esnouf for helpful discussions, K. Erven, C. Heens and B. Van Remoortel for excellent technical assistance, V. Fikkert for help with sequencing and I. Aerts for editorial help.
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