Mutations in Both Env and Gag Genes Are Required for HIV-1 Resistance to the Polysulfonic Dendrimer SPL2923, As Corroborated by Chimeric Virus Technology

Home , Gp41

Antiviral Chemistry & Chemotherapy 16:253–266 Mutations in both env and gag genes are required for HIV-1 resistance to the polysulfonic dendrimer SPL2923, as corroborated by chimeric virus technology

Anke Hantson1,†, Valery Fikkert1,†, Barbara Van Remoortel1, Chistophe Pannecouque3, Peter Cherepanov1, Barry Matthews2, George Holan2, Erik De Clercq3, Anne-Mieke Vandamme3, Zeger Debyser1 and Myriam Witvrouw1*

1Laboratory for Molecular Virology, Molecular Medicine, Katholieke Universiteit Leuven and KULAK, Flanders, Belgium 2Starpharma Limited, Melbourne, Victoria, Australia 3Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium

*Corresponding author: Tel: +32 1633 2170; Fax: +32 1633 6336; E-mail: [email protected]

†Anke Hantson and Valery Fikkert contributed equally to this paper

A drug-resistant NL4.3/SPL2923 strain has previ- NL4.3/SPL2923 were sufficient to reproduce the ously been generated by in vitro selection of HIV- cross resistance to enfuvirtide. Unexpectedly, the 1(NL4.3) in the presence of the polysulfonic reduced sensitivity towards SPL2923 was not fully dendrimer SPL2923 and mutations were reported reproduced after gp160-recombination. The search in its gp120 gene (Witvrouw et al., 2000). Here, we for mutations in NL4.3/SPL2923 in viral genes further analysed the (cross) resistance profile of other than env revealed several mutations in the NL4.3/SPL2923. NL4.3/SPL2923 was found to gene encoding the HIV p17 matrix protein (MA) contain additional mutations in gp41 and showed and one mutation in the gene encoding the p24 reduced susceptibility to SPL2923, dextran sulfate capsid protein (CA). In order to analyse the impact (DS) and enfuvirtide. To delineate to what extent of the gag mutations alone and in combination the mutations in each env gene were accountable with the mutations in env on the phenotypic resis- for the phenotypic (cross) resistance of tance towards SPL2923, we developed a novel NL4.3/SPL2923, the gp120-, gp41- and gp160- p17- and p17/gp160-CVT. Phenotypic analysis of sequences derived from this strain were placed the NL4.3/SPL2923 p17- and p17/gp160-recom- into a wild-type background using env chimeric bined strains indicated that the mutations in both virus technology (CVT). The cross resistance of env and gag have to be present to fully reproduce NL4.3/SPL2923 towards DS was fully reproduced the resistance of NL4.3/SPL2923 towards SPL2923. following gp160-recombination, while it was only partially reproduced following gp120- or gp41- Key words: HIV, entry, bicyclams, gag, env, recombination. The mutations in gp41 of resistance

Introduction

Current treatments for human immunodeficiency virus entry into its target cells involves the interaction of the viral type 1 (HIV-1) infection are based on drug combination protein gp120 with the CD4 receptor on the cell surface regimens, consisting of drugs that target reverse transcrip- and the successive interaction of gp120 with the coreceptor. tase (RT) and protease (PR) (Richman, 2001). Due to toxi- This results in a conformational change to the prehairpin city and the emergence of virus strains resistant to the intermediate, in which the fusion peptide of gp41 pene- antiretrovirals, the development of drugs preferentially trates the cell membrane. The prehairpin intermediate acting on new targets in the HIV replication cycle remains resolves to the fusion-active hairpin structure, resulting in crucial (Vandamme et al., 1999). An attractive new target the apposition of the viral envelope and the plasma for anti-HIV therapy is entry, since blocking entry should membrane and is followed by the fusion of the envelope lead to suppression of infectivity and replication. HIV with the cellular lipid bilayer (Chan & Kim, 1998).

Numerous entry inhibitors acting at different stages of peptidic agents, for example, the polyphemusin T22, T134 the entry process have been reported, of which a growing and T140 (Arakaki et al., 1999; Murakami et al., 1997; number has recently been introduced into clinical trials or Tamamura et al., 1998a; Tamamura et al., 1998b), and the is in preclinical development. Typical polyanionic Tat protein analogues ALX40-4C (Doranz et al., 1997) compounds, like dextran sulfate (DS) inhibit HIV binding and CGP64222 (Daelemans et al., 2000). Also, low molec- to its target cells (Baba et al., 1988). They exert their anti- ular weight CXCR4 antagonists are the highly potent and HIV activity by shielding off the positively charged sites in selective bicyclams (De Clercq et al., 1992; De Vreese et al., the V3 loop of gp120, thereby blocking the attachment to 1996; Schols et al., 1997), represented by AMD3100, cell surface heparan sulfate (Witvrouw & De Clercq, which has been evaluated in Phase II clinical trials (Schols 1997). The dendrimer SPL2923 shares an analogous mode D, Claes S, De Clercq E, Hendrix C, Bridger G, Calandra of action due to its polysulfonated periphery. Dendrimers G, Henson GW, Fransen S, Huang W, Whitcomb JM & are highly branched macromolecules that are built up in Petropoulos CJ [2002] AMD3100 HIV study group. generations from a reactive core group by the use of AMD3100, a CXCR4 antagonist reduced HIV viral load branched building blocks to give spherical molecules. and X4 virus levels in humans. 9th Conference on SPL2923 consists of a fourth generation polyamidoamine Retroviruses & Opportunistic Infections. Seattle, WA, USA, (PAMAM) dendrimer scaffold built from an ammonia 24–28 February 2002. Oral Abstract 2.) but has not been core, which is fully capped on the surface with 24 naph- further pursued for the treatment of HIV infections thyldisulfonic acids (Witvrouw et al., 2000). PRO 542, an because of the lack of oral bioavailability. antibody-like fusion protein of the D1D2 domains of CD4 From the group of HIV entry inhibitors that interfere and IgG2, is a specific CD4 attachment inhibitor (Allaway with the hairpin formation and membrane fusion, enfuvir- et al., 1995; Jacobson et al., 2000). The conserved CD4- tide (T20), has recently been approved to be included in binding pocket on gp120 is a target for BMS-806, another anti-HIV combination regimens (Lalezari et al., 2003; HIV binding inhibitor. Preclinical development of this Lazzarin, 2003). T20 is a synthetic 36-amino acid peptide inhibitor is ongoing (Guo et al., 2003; Lin et al., 2003; segment corresponding to residues 127-162 of the Wang et al., 2003). ectodomain of gp41, that is, the envelope HR2 domain. In addition, co-receptor antagonists can inhibit replica- T20 binds to the N-terminal fusion peptide of the pre- tion. SCH-C is an orally bioavailable CCR5 antagonist hairpin intermediate and prevents the subsequent forma- (Strizki et al., 2001) which demonstrates good pharmaco- tion of the fusion-active hairpin conformation (Eckert & kinetics, safety and tolerability besides a clear decrease in Kin, 2001). A second generation fusion inhibitor, T1249, HIV RNA in Phase I/II clinical trials (Reynes J, Rouzier has already been identified (Sista P, Melby T, Dhingra U, R, Kanouni T, Baillet V, Baroudy B, Keung A, Hogan C, Cammack N, McKenna P, Dehertogh P & Matthews T Malowitz M & Laughlin [2002] SCH C. safety and [2001] The fusion inhibitors T20 and T1249 demonstrate antiviral effects of a CCR5 receptor antagonist in HIV-1 potent in vitro antiviral activity against clade B HIV-1 infected subjects. 9th Conference on Retroviruses & isolates resistant to reverse transcriptase and protease Opportunistic Infections. Seattle, WA, USA, 24–28 February inhibitors and non-B clades. 5th International Workshop on 2002. Oral Abstract 1.). SCH-D acts in a similar mode, but HIV Drug resistance & Treatment Strategies. Scottsdale, shows higher antiviral potency (Chen Z, Hu B, Huang W, Arizona, USA, 4–8 June 2001. Abstract 2.) and demon- He T, Huang Y, Strizki J, Xu S, Wojcik L, Whitcom J, strated a strong dose response in patients with multi-drug Zhang L, Petropoulos C, Baroudy B & Ho DD [2002] resistant strains (Miralles GD, DeMasi R, Sista P,Melby T, HIV-1 mutants less susceptible to SCH-D a novel small Duff F, Matthews T & the T1249-101 study group [2001] antagonist of CCR5. 9th Conference on Retroviruses & Baseline genotype and prior antiretroviral history do not Opportunistic Infections. 24–28 February, Seattle, WA, affect virologic response to T1249. 5th International USA. Abstract 396-T). PRO 140, an anti-CCR5 mono- Workshop on HIV Drug resistance & Treatment Strategies. clonal antibody, displayed anti-HIV activity in the thera- Scottsdale, Arizona, USA, 4–8 June 2001. Abstract 3.). peutic hu-PBMC-SCID mouse model (Franti M, O’Neill Most likely, more HIV entry inhibitors will follow. This Maddon P,Burton D, Poignard P & Olson W [2002] PRO makes the study of resistance towards inhibitors of viral 542 (CD4IgG2) has a profound impact on HIV-1 replica- entry and the development of phenotypic assays to evaluate tion in the Hu-PBL-SCID mouse model. 9th Conference on the susceptibility of clinical isolates towards HIV entry Retroviruses & Opportunistic Infections. Seattle, USA, 24–28 inhibitors mandatory. February 2002. Abstract 396-T.). A number of polycationic We previously reported the anti-HIV activity of polyan- molecules were found to interact electrostatically with the ionic dendrimers inhibiting the replication of HIV-1 in MT- negatively charged amino acid residues of CXCR4. The 4 cells in the nanomolar range (Witvrouw et al., 2000). In a majority of these compounds are high molecular weight time-of-addition experiment SPL2923 had to be present at

the moment of viral entry. However, at much higher concen- (Bethesda, MD, USA). NL4.3/SPL2923 has previously trations it was found to exert its anti-HIV activity by inter- been selected in our laboratory by serial passage of HIV-1 fering at a later stage in the replicative cycle, that is, at a step (NL4.3) in the presence of increasing concentrations of coinciding with the RT and/or integrase process. In view of SPL2923 as described in Witvrouw et al. (2000) (here the observation that SPL2923 was capable of penetrating the referred to as NL4.3/SPL2923B). After 30 passages, cells and that the compound showed inhibitory activity in NL4.3/SPL2923B was able to grow at a compound isolated RT and integrase enzymatic assays, the RT or inte- concentration of 20 µg/ml, a concentration that is 60-fold grase steps could not be excluded as antiviral targets. higher than the concentration required to inhibit the repli-

Therefore an HIV-1 strain was selected in the presence of cation of wild-type HIV-1(NL4.3) by 50% (IC50) SPL2923 (NL4.3/SPL2923). Several mutations were found (0.3 µg/ml). The strains NL4.3/DS and NL4.3/T20 have in gp120 of NL4.3/ SPL2923, whereas no mutations were been previously selected in our laboratory by serial passage found in RT or integrase (Witvrouw et al., 2000). of HIV1(NL4.3) in the presence of increasing concentra- Here, we further analysed the (cross) resistance profile of tions of DS as described in Esté et al. (1997), or T20, as the NL4.3/SPL2923 strain. In addition to the mutations in described in Fikkert et al. (2002). gp120, we identified mutations in gp41 of NL4.3/ SPL2923. Phenotypically, this showed reduced susceptibility to the PCR amplification and sequencing of the inhibitory effect of SPL2923 and cross resistance to the entry different env regions inhibitors DS and T20. We used env chimeric virus tech- PCR Amplification of the gp120, gp41 and gp160 nology (CVT) (Fikkert et al., 2002) to determine to what encoding sequences and the subsequent sequencing of extent the mutations are accountable for the phenotypic resis- these genes have been previously described in Fikkert et al. tance of NL4.3/SPL2923. Unexpectedly, the reduced sensi- (2002). Primer positions correspond to the HIV1(LAV-1) tivity to SPL2923 was not reproduced by the recombination recombinant clone pNL4.3 (GenBank accession no. of gp120, gp41 or gp160 of NL4.3/SPL2923 in wild-type M19921). background, whereas the cross resistance towards DS and T20 was reproduced by gp160-recombination. Apparently, Env chimeric virus technology regions outside env play a role in the phenotypic resistance of The construction of the gp120-, gp41- and gp160-deleted NL4.3/SPL2923 towards SPL2923. HIV-1(NL4.3) clones and the env-recombination assays using specific primer sets for PCR amplification of the Materials and methods different env-genes and the different linerarized gp120-, gp41- or gp160-deleted HIV-1(NL4.3) clones are Compounds described in detail in Fikkert et al. (2002). SPL2923 was synthesized as described in Witvrouw et al. (Witvrouw et al., 2000). DS (average molecular weight, PCR amplification and sequencing of the p17 5,000) was purchased from Sigma (Bornem, Belgium). and p24 gene AMD3100 was provided by G Bridger, AnorMED (Langley, BC, Canada) and was synthesized as described PCR Amplification of p17 and p24 encoding (Bridger et al., 1995). 3′-Azido-3′-deoxythymidine (AZT) sequences was synthesized according to the method described by DNA extraction of proviral DNA from MT-4 cells infected Horwitz et al. (1964). PNU-90152T was obtained from B with the different selected strains was performed using the Bruce (Pharmacia and Upjohn, Kalamazoo, MI, USA). QIAamp blood kit (Qiagen, Hilden, Germany). A 1423 Saquinavir (Ro31-8959) was a gift from N Roberts (Roche nucleotide base pair fragment of gag (corresponding to Products Limited, Welwyn Garden City, UK). position 681–2104) encoding p17 and p24 was PCR amplified on a Biometra Trioblock (Westburg, Leusden, Cells The Netherlands) using the primers AV14 (5′ CTC TCT MT-4 cells (Miyoshi et al., 1982) were grown in a humidi- CGA CGC AGG ACT CGG CTT GCT GAA 3′ corre- ′ fied atmosphere with 5% CO2 at 37°C and maintained in sponding to position 681–710) and BVR 2 (5 GCC ARA RPMI 1640 medium supplemented with 10% heat-inacti- TYT TCC CTA AAA AAT TAG CC 3′ corresponding to vated fetal calf serum, 2 mM L-glutamine, 0.1% sodium position 2079–2104). Primer positions correspond to the bicarbonate and 20 µg/ml of gentamycin. HIV-1(LAV-1) recombinant clone pNL4.3 (GenBank accession no. M19921). The cycling conditions were as Virus strains follows: a first denaturation step of 2 min at 95°C followed The plasmid pNL4.3 (Adachi et al., 1986) is a molecular by 40 cycles of 30 sec 95°C, 30 sec 55°C, 90 sec 72°C. A clone obtained from the National Institutes of Health final extension was performed at 72°C for 10 min.

Antiviral Chemistry & Chemotherapy 16.4 255 A Hantson et al.

Sequencing of the p17 and p24 encoding signal), a tail sequence comprising both restriction sites. regions The cycling conditions were as follows: 40 cycles consisting PCR products were purified using the PCR purification kit of 15 sec 95°C, 30 sec 55°C and 30 sec 68°C. A final exten- (Qiagen). To carry out the sequencing reaction, the ABI sion was performed at 72°C for 10 min. The resulting PCR PRISM™ Dye terminator cycle sequencing core kit (Perkin fragment was digested with the restriction endonucleases Elmer, Brussels, Belgium) was used. The primers used to BssHII and SpeI and subsequently purified by phenol/chlo- sequence the amplified region of gag (AV14-BVR2) were: roform extraction. This linker sequence was ligated into the AV58 (5′ GGG TGC GAG AGC GTC 3′ corresponding vector to recirculize the plasmid. This clone was further to position 792–806), AV13 (5′ CTG CGA ATC GTT referred to as the p17-deleted clone. CTA GCT CCC TGC TTG CCC 3′ corresponding to position 886–925), AV103 (5′ GCC ATA TCA CCT Construction of an HIV-1(NL4.3) clone deleted AGA ACT TT 3′ corresponding to position 1225–1244), for p17 and gp160 AV26 (5′ GCT ATG TCA CTT CCC CTT GGT TCT The p17/gp160-deleted clone was generated analogously C 3′ corresponding to position 1475–1499), AV159 (5′ with the previously described clones. The gp160-deleted GGG ATT AAA TAA AAT AGT AAG 3′ corresponding clone described in Fikkert et al. (2002) was digested with to position 1593–1613), BVR3 (5′ TTT CCA ACA GCC the restriction enzymes BssHII and SpeI (Figure 1) and the CTT TTT CCT AG 3′ corresponding to position linker sequence as described above, was ligated into the 2012–2034). Primer positions correspond to the HIV- vector in order to recirculize the p17/gp160-deleted 1(LAV-1) recombinant clone pNL4.3 (GenBank accession plasmid. no. M19921). The samples were loaded on the ABI PRISM 310 Genetic Analyzer (Perkin Elmer, Brussels, p17- and p17/gp160-recombination assay Belgium). The sequences were analysed using the software MT-4 cells were subcultured at a density of program Vector NTI Suite 7 (InforMax Inc, Oxford, UK). 500 000 cells/ml the day before transfection. Cells were Mutations present in more than 25% of the global virus pelleted and resuspended in culture medium at a concen- population could be detected as a mixture with the wild- tration of 6.25×106 cells/ml. For each transfection type amino acid by means of population sequencing. 5×106 cells (0.8 ml) were used. Transfections were performed by electroporation using EASYJECT Plus Construction of an HIV-1(NL4.3) clone deleted (Immunosource, Halle-Zoersel, Belgium) and electropora- for p17 tion cuvettes (Immunosource). For use in p17-recombina- The proviral molecular clone pNL4.3 (Adachi et al., 1986), tion experiments plasmid DNA preparations of the consisting of the plasmid pUC18, wherein the complete p17-deleted clone were linearized by MluI digestion. HIV-1 genome flanked with chromosomal DNA is MT-4 cells were co-transfected with 10 µg of the inserted, was digested with the restriction enzymes BssHII linearized p17-deleted clone and 2 µg purified and concen- and SpeI to generate the clone deleted for p17 and the 5′ trated AV14-BVR2 PCR product (PCR Purification Kit, part of the region coding for p24. Since part of the pack- Qiagen). aging signal was subsequently deleted by this digest, a For p17/gp160-recombination experiments, the cells linker sequence, designed to contain the packaging signal were co-transfected with 7 µg of MluI linearized and the novel MluI restriction site besides both BssHII and p17/gp160-deleted clone, and 7 µg of SalI linearized SpeI restriction sites, was created by means of PCR ampli- p17/gp160-deleted clone, 2 µg AV14-BVR2 PCR product fication. The theory behind this reasoning is explained in and 2 µg AV310-AV319 PCR product (Figure 2A) or more detail in the discussion. To construct the linker 15 µg of MluI and SalI digested p17/gp160-deleted clone, sequence, a 126-nucleotide base pair fragment of pNL4.3 2 µg AV14-BVR2 PCR product and 2 µg AV310-AV319 was amplified with Expand™ High Fidelity PCR system PCR product (Figure 2B). Electroporation was performed (Roche, Mannheim, Germany), which is composed of an at 250 V and 1500 µF. The transfected cells were enzyme mix containing thermostable Taq DNA and Pwo suspended in 5 ml of culture medium and incubated at ′ ′ DNA polymerase with 3 5 exonuclease proofreading 37°C in a humidified atmosphere with 5% CO2. The next capacity, using the primers AV14 and PC-VAB (5′ ACT day, the cells were centrifuged and resuspended in 10 ml AGT AGT TCC TGC TAT GTC ACG CGT CTC culture medium. When full cytopathic effect (CPE) was TCT CCT TCT AGC C 3′). PC-VAB was specifically observed in the culture, the recombinant virus was designed to insert the MluI and SpeI restriction sites in harvested by centrifugation and stored in 1 ml aliquots at the PCR amplicon. Therefore this primer contains, as 80°C for subsequent infectivity and drug susceptibility well as the sequence necessary to anneal and amplify the determinations (50% inhibitory concentration or IC50) in fragment upstream the start codon of p17 (the packaging the MT4/MTT assay.

Figure 1. Construction of the p17/gp160-deleted pNL4.3 clone LTR

3‘ end of nef pHIV ∆ gp160 (gp160 deleted SalI proviral plasmid)

vif vpr LTR pol

gag packaging signal

BssHII SpeI

Position of NL4.3 718 1514

BssHII MluI SpeI

5‘ GAAG CGCG C . . . packaging signal A CGCG TGACATAGCAGGAACTA CTAG TACC 3’

3‘ CTTC GCGC G . . . . . T GCGC ACTGTATCGTCCTTGAT GATC ATGG 5’ LTR 3‘ end of nef

pHIV ∆ p17 ∆ gp160 Sal 1

vif vpr

LTR pol

gag packaging signal 3‘ end of p24 Mlu 1 p2 p7 p1 p6

To generate the p17/gp160-deleted clone, the gp160-deleted pNL4.3 clone (described in Fikkert et al., 2002) was digested with BssHI and SpeI. A linker sequence containing the MluI restriction site and the packaging signal was designed and subsequently ligated into the vector. The p17-deleted clone was constructed analogously using the full-length pNL4.3 clone.

Antiviral Chemistry & Chemotherapy 16.4 257 A Hantson et al.

Figure 2. Generation of p17/gp160-recombined HIV strains

A PCR p17 (AV14-BVR2) PCR gp160 (AV310-AV319)

vif packaging signal 3‘ end of p24 vpr 3‘ end of nef

MluI digestion SalI digestion at p17 at gp160

MluI 3‘ end of p24 SalI vif packaging signal p2 p7 vpr 3‘ end of nef p1 p6 pol LTR pol LTR

vif pHIV ∆ p17 / ∆ gp160 vpr pHIV ∆ p17 / ∆ gp160 SalI 3‘ end of p24 p2 p7 3‘ end p1 LTR of nef MluI LTR p6

packaging signal

(A) Approach 1: For p17/gp160-recombination experiments, MT-4 cells were co-transfected with the p17/gp160-deleted clone linearized by MluI digest, the p17/gp160-deleted clone linearized by SalI digest and both PCR products amplified by primer sets AV14-BVR2 and AV310- AV319. (B) Approach 2: Co-transfection of MT-4 cells with the p17/gp160-deleted clone digested in both MluI and SalI restriction sites and both PCR products amplified by primer sets AV14-BVR2 and AV310-AV319.

Figure 2 continued.

PCR p17 PCR gp160 (AV14-BVR2) (AV310-AV319)

3‘ end vif of p24 vpr

packaging signal pHIV - 1(NL4.3) 3‘ end of nef ∆p17 / ∆gp160

MluI digestion at p17 SalI digestion at gp160

3‘ end of p24 p2 p7 MluI p1 packaging signal p6

pol LTR

vif pHIV ∆ p17 / ∆ gp160 vpr SalI

3‘ end of nef LTR

Antiviral Chemistry & Chemotherapy 16.4 259 A Hantson et al.

Drug susceptibility assay The inhibitory effect of antiviral drugs on the HIV- induced CPE in human lymphocyte MT-4 cell culture was determined by the MT-4/MTT-assay (Pauwels et al., 1988). This assay is based on the reduction of the yellow coloured 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetra- zolium bromide (MTT) by mitochondrial dehydrogenase of metabolically active cells to a blue formazan derivative, which can be measured spectrophotometrically. The 50%

cell culture infective dose (CCID50) of the different HIV strains was determined by titration of the virus stock using MT-4 cells. For the drug-susceptibility assays MT-4 cells

FNSTW R F K were infected with 100–300 CCID50 of the virus stock in the presence of fivefold serial dilutions of the antiviral drugs. The concentration of various compounds achieving 50% protection against the CPE of the different HIV

strains, which is defined as the IC50, was determined. g/ml).

µ Results 0 ition in the rest of population, as analysed by population Genotypic analysis of the gp120 genes of the T– drug-resistant strains Multiple mutations were detected in the gp120 gene of NL4.3/SPL2923 in comparison with the wild-type HIV 1(NL4.3) strain. As a result the gp120 protein of (2002). NL4.3/SPL2923 contained less basic and more acidic (1997). et al.

QY–EV amino acids residues than wild-type gp120. Some of these

et al. mutations have already been reported to be associated with resistance towards inhibitors of the HIV entry process (Table 1 and (Witvrouw et al., 2000). The gp120 mutations found in NL4.3/DS and NL4.3/T20 have been previously described in Esté et al. (1997) and Fikkert et al. (2002), respectively.

Genotypic analysis of the gp41 genes of the drug-resistant strains Sequencing analysis of the NL4.3/SPL2923 strain revealed § the mutations A22V, L33S, P216L and A308T in gp41, while the mutations V2V/M, N126K L210L/I and

genes of various HIV-1(NL4.3) strains selected in the presence of viral entry inhibitors relative to wild-type genes of various HIV-1(NL4.3) P213P/L were found in gp41 of NL4.3/DS (Table 2). The mutations L33S and N43K present in gp41 of NL4.3/T20

gp120 have been previously described in Fikkert et al. (2002).

Genotypic analysis of the gag genes of the drug-resistant strains The p17-gene of the strain selected in the presence of NN I E H D K– T N SSFVFNQKNAFN SPL2923 contained the following substitutions; E11K, K25R, V34I and G61R. In addition, the mixture A/E was found at position A92 of the p24-encoding region of gag in the NL4.3/SPL2923 strain. The mutations E11K and ‡

Genotypic analysis of the V34I were present in the p17 gene of the complete virus † population or part of the virus population of NL4.3/DS or NL4.3/T20, respectively (Table 3). HIV-1(NL4.3) strain selected in MT-4 cells in the presence of T20, as previously described Fikkert strain selected in MT-4 HIV-1(NL4.3) HIV-1(NL4.3) strain selected in MT-4 cells in the presence of DS, as previously described Esté strain selected in MT-4 HIV-1(NL4.3) Part of the virus population (at least 25%) contains this mutation, while wild-type amino acid is still present at pos NL4.3/T20 Table 1. Table strain HIV-1(NL4.3) Amino acid ofgp120 NL4.3 115 136 147 154 cells during 30 passages in the presence of increasing concentrations SPL2923 (up to 2 strain selected in MT-4 *HIV-1(NL4.3) † ‡ 247§ sequencing. DS, dextran sulfate; T20, enfuvirtide. 271 280 292 295 299 323 325 366–370 389 393 460 NL4.3/SPL2923*NL4.3/DS L V/E

Table 2. Genotypic analysis of the gp41 genes of various HIV-1(NL4.3) strains selected in the presence of viral entry inhibitors relative to the wild-type HIV-1(NL4.3) strain

Amino acid of 2 22 33 43 126 210 213 216 308 gp41 NL4.3 V A L N N L P P A

NL4.3/SPL2923* V S L T NL4.3/DS† V/M§ K L/I P/L NL4.3/T20‡ SK

*HIV-1(NL4.3) strain selected in MT-4 cells during 30 passages in the presence of increasing concentrations of SPL2923 (up to 20 µg/ml). †HIV-1(NL4.3) strain selected in MT-4 cells in the presence of DS, as previously described in Esté et al. (1997). ‡HIV-1(NL4.3) strain selected in MT-4 cells in the presence of T20, as previously described in Fikkert et al. (2002). §Part of the virus population (at least 25%) contains this mutation, while the wild-type amino acid is still present at this position in the rest of the population, as analysed by population sequencing. DS, dextran sulfate; T20, enfuvirtide.

Evaluation of phenotypic (cross) resistance of wild-type strain (1.6-, 2.0-, 1.8- and 1.0-fold increase in

NL4.3/SPL2923 IC50, respectively) (Table 4 and data not shown). We determined the antiviral activity of SPL2923 against the virus strains HIV-1(NL4.3) and NL4.3/SPL2923 in MT-4 env chimeric virus technology cell culture. In parallel, the sensitivity of both strains for the env recombination assays for gp120, gp41 or gp160 were HIV entry inhibitors DS, AMD3100 and T20 were deter- performed for the virus strains HIV-1(NL4.3) and mined. The inhibitory effects of the nucleoside RT inhib- NL4.3/SPL2923. gp120-recombined strains were referred itor zidovudine (AZT), the non-nucleoside RT inhibitor to as Rgp120/NL4.3 and Rgp120/SPL2923; whereas gp41 delavirdine (PNU-90152T) and the PRO inhibitor or gp160 recombined strains were referred to as saquinavir (Ro31-8959) were evaluated as well (Table 2). Rgp41/NL4.3 and Rgp41/SPL2923 or Rgp160/NL4.3 NL4.3/SPL2923 displayed a reduced susceptibility and Rgp160/SPL2923, respectively. compared to the HIV1(NL4.3) strain to SPL2923 (7.1-fold increase in IC50), while cross resistance was observed for this p17 and p17/gp160 chimeric virus technology strain towards the entry inhibitors DS and T20 (163.6- and The recombination of the PCR amplified gag-region (p17 ′ 39.5-fold increase in IC50, respectively) (Table 4). The sensi- and the 5 part of p24) derived from the HIV-1(NL4.3) tivity of NL4.3/SPL2923 to AMD3100, AZT, delavirdine and NL4.3/SPL2923 strains in the clone deleted for this and saquinavir was comparable to the sensitivity of the region resulted in the p17-recombined strains Rp17/NL4.3 and Rp17/SPL2923. A first approach for p17/gp160- recombination experiments consisted of the co-transfection of MT-4 cells with the p17/gp160-deleted clone linearized Table 3. Genotypic analysis of the gag genes of various HIV-1(NL4.3) strains selected in the presence of at the position of the p17 deletion, the p17/gp160-deleted viral entry inhibitors relative to the wild-type HIV- clone linearized at the position of the gp160 deletion and 1(NL4.3) strain both p17 and gp160 PCR products. In a second approach, Gene p17 p24 the p17/gp160-deleted clone was digested at both the position of the p17 and the gp160 deletion preceding co-trans- Position of amino acid 11 25 34 61 92 fection with the PCR-derived p17 and gp160 sequences. We obtained proof-of-principle that both approaches HIV-1(NL4.3) E K V G A resulted in productive virus infection, though the second NL4.3/SPL2923* K R I R A/E§ approach resulted in a productive virus infection more NL4.3/DS† KI NL4.3/T20‡ E/K V/I quickly than the first approach. The strains resulting from p17/gp160-recombination of the HIV-1(NL4.3) and *HIV-1(NL4.3) strain selected in MT-4 cells during 30 passages in the NL4.3/SPL2923 strains were referred to as Rp17gp160/ presence of increasing concentrations of SPL2923 (up to 20 µg/ml). †HIV-1(NL4.3) strain selected in MT-4 cells in the presence of DS, as NL4.3 and Rp17gp160/SPL2923, respectively. previously described in Esté et al. (1997). ‡HIV-1(NL4.3) strain selected in MT-4 cells in the presence of T20, as previously described in Fikkert et al. (2002). Evaluation of the different recombined HIV-1 §Part of the virus population (at least 25%) contains this mutation, strains while the wild-type amino acid is still present at this position in the rest of the population, as analysed by population sequencing. The env- and gag-sequences after recombination were veri- DS, dextran sulfate; T20, enfuvirtide. fied to be identical to those of the respective wild-type or

Antiviral Chemistry & Chemotherapy 16.4 261 A Hantson et al. 0.0021 0.0011 0.0002 0.0005 0.0019 0.0008 0.0008 0.0011 0.0028 0.0002 ± ± ± ± ± ± ± ± ± ± l compounds † ) 50 0.0040.005 0.0030 0.0022 0.006 0.0010 0.047 0.0011 ± ± ± ± 0.810.210.12 0.0022 0.012 0.0025 0.0025 0.34 0.0016 0.0033 0.24 0.0005 ) (2.0) )) (1.9) (1.5) ) (0.5) ± ± ± ± ± ± g/ml), (fold increase in IC µ in MT-4 cells. in MT-4 of the compound against the parental HIV-1(NL4.3) or recombined of the compound against parental HIV-1(NL4.3) * ( g/ml). 50 50 µ 0.0250.0050.001 2.17 0.002 0.32 0.005 0.19 0.004 0.020 0.003 0.58 0.028 0.62 0.007 0.026 0.014 0.055 0.011 0.72 IC ± ± ± ± ± ± ± ± ± ± 15.20 0.020 HIV entry inhibitors HIV RT inhibitor HIV entry inhibitors RT ) (1.6) (39.5 ± 22.50.302.93 0.025 0.08 0.013 0.007 0.2623.9 0.004 0.012 0.023 0.34 0.014 0.24 0.015 selected strain in the presence of SPL2923. )) (0.5)) (2.0) (0.6) (1.9)) (29.0 (1.0) (22.1 (1.4) (27.5 ± ± ± ± ± ± ± ± 0.09 0.008 ± in vitro selected strain in the presence of SPL2923. selected strain in the presence of SPL2923. selected strain in the presence of SPL2923. selected strain in the presence of SPL2923. in vitro in vitro in vitro in vitro genes of HIV-1(NL4.3). genes of the gp160 gp160 gene of HIV-1(NL4.3). gene of the selected or recombined NL4.3/SPL2923 strain compared to the IC 1.420.170.27 45.8 0.090.17 0.29 3.82 0.200.16 0.14 1.6 0.230.31 0.29 26.4 0.0060.69 1.54 2.11 0.40 29.45 0.028 0.029 0.22 0.17 0.0013 0.0014 0.20 0.28 gene of the gene of HIV-1(NL4.3). gene of the ± ± ± ± ± ± ± ± ± ± ± ± gene of HIV-1(NL4.3). gene of the and gene of HIV-1(NL4.3). and ) (163.6 ) (73.6 p17 p17 gp160 gp160 p17 gp41 p17 2.20 0.23 0.43 0.20 0.27 0.29 0.73 0.69 0.72 (1.0)0.42 2.14 (1.4) (1.0) (0.8) (1.0) (7.1 (1.9)(1.4) (13.2 (2.5) (11.4 (91.0 (5.1 gp120 gp120 gp41 in vitro selected during 30 passages in the presence of increasing concentrations SPL2923 (up to 20 ⊕ of the compound against in vitro 50 ¶ ♠ ‡ § $ Susceptibility of the HIV-1 strain selected in the presence of SPL2923 and its different recombined strains to various antivira strain selected in the presence of SPL2923 and its different Susceptibility of the HIV-1 HIV-1(NL4.3) wild-type strain recombined with the HIV-1(NL4.3) wild-type strain recombined with the HIV-1(NL4.3) HIV-1(NL4.3) wild-type strain recombined with the HIV-1(NL4.3) HIV-1(NL4.3) wild-type strain recombined with the HIV-1(NL4.3) wild-type strain recombined with the HIV-1(NL4.3) HIV-1(NL4.3) wild-type strain recombined with the HIV-1(NL4.3) HIV-1(NL4.3) wild-type strain recombined with the HIV-1(NL4.3) HIV-1(NL4.3) wild-type strain recombined with the HIV-1(NL4.3) Fold increase in IC strain HIV-1(NL4.3) wild-type strain recombined with the HIV-1(NL4.3) wild-type strain recombined with the HIV-1(NL4.3) Rp17gp160/NL4.3 Underlined text indicates reduced susceptibility. AZT, zidovudine; DS, dextran sulfate; T20, enfuvirtide AZT, Underlined text indicates reduced susceptibility. Rgp41/NL4.3 HIV-1(NL4.3) strain, respectively. HIV-1(NL4.3) Table 4. Table compared to HIV-1(NL4.3) StrainsNL4.3 0.31 SPL2923 DS HIV strains by 50% (CPE) of different *50% inhibitory concentration or required to inhibit the cytopathic effect † AMD3100‡ § ¶ $ ♠ T20 ⊕ AZT Rgp120/NL4.3 Rgp160/NL4.3 Rp17/NL4.3 Rp17/SPL2923 Rgp41/SPL2923 Rgp160/SPL2923 Rp17gp160/SPL2923 NL4.3/SPL2923 Rgp120/SPL2923

in vitro selected NL4.3/SPL2923 strains (data not shown). al., 2002). The different env-recombined NL4.3/SPL2923 Antiviral susceptibility of the different recombined strains strains were analysed phenotypically (Table 4). The cross for the entry inhibitors SPL2923, DS, AMD3100 and T20 resistance of NL4.3/SPL2923 towards DS (249-fold) is and the RT inhibitor AZT was determined by the standard partly reproduced by the recombination of either gp120 or MT4/MTT assay (Table 4). gp120-recombination of gp41 of this strain (13.2- and 11.4-fold, respectively), and NL4.3/SPL2923 in wild-type background resulted in a fully reproduced by gp160-recombination (91.0-fold). strain with sensitivity comparable to HIV-1(NL4.3) for all Conversely, the cross resistance towards T20 (39.5 fold) compounds evaluated, with the exception of DS (13.2-fold was completely reproduced by gp41-recombination (29- reduced sensitivity). The gp41-recombination resulted in a fold). Surprisingly, the gp120-, gp41- and even the gp160- strain with a 11.4-fold and 29-fold reduced susceptibility recombined NL4.3/SPL2923 strains displayed no for DS and T20, respectively. The gp160-recombined significant reduction in susceptibility (up to 2.5-fold) for NL4.3/SPL2923 strain displayed a comparable loss in the compound SPL2923 (Table 4). So it appeared that susceptibility for the compounds DS (91.0-fold) and T20 resistance against SPL2923 is at least partially attributed to (22.1-fold) as compared to the originally selected strain; mutations in genes distinct from gp160. however, only a 2.5-fold reduced sensitivity was observed Genetic studies of HIV-1 gag and env mutants imply for SPL2923. Introduction of the p17 mutations of interactions between the cytoplasmic domain of gp41 and NL4.3/SPL2923 in wild-type background did not render the MA protein in order to mediate Env glycoprotein the resulting virus less sensitive than the wild-type strain. incorporation into virions. The mutations L12E and L30E The loss in sensitivity of the p17/gp160-recombined in MA (Freed & Martin, 1996; Ono et al., 1997) or small NL4.3/SPL2923 strain with respect to the wild-type deletions in α-helix 2 (between amino acid residues 277- p17/gp160-recombined strain mirrored the decreased 296) of the cytoplasmic domain of gp41 (Murakami & sensitivity of the corresponding parental selected strain as Freed, 2000) were found to impair virus replication as a compared to HIV-1(NL4.3) for all antiviral compounds result of a defect in Env incorporation. The substitution evaluated, that is 5.1-, 73.6-, 1.4-, 27.5-, and 0.5-fold V34I in MA was able to reverse these replication defects increase in IC50 for SPL2923, DS, AMD3100, T20 and imposed by some of the gp41 deletions and the MA muta- AZT, respectively (Table 4). tion L12E, through an increase in Env incorporation. In contrast, the MA mutation V34E by itself was associated Discussion with a reduction in gp120 content of the virions. These observations indicate an important role of the amino acids Primary studies regarding the mode of action of polyan- at positions 12 and 34 in MA and α-helix 2 of the cyto- ionic dendrimers pointed to the interaction with gp120 as plasmic domain of gp41 (Freed & Martin, 1996; the basis of the anti-HIV activity of the negatively charged Murakami & Freed, 2000). Based on these grounds, the dendrimers. However, it appeared that the RT or integrase MA protein represented the most likely candidate to affect steps could not be excluded as antiviral targets of interac- resistance against SPL2923. Indeed, the sequencing tion of SPL2923. To identify the target in cell culture, an analysis of gag of NL4.3/SPL2923 revealed several muta- HIV1 strain was selected in the presence of SPL2923. tions in the MA protein, that is, E11K, K25R, V34I and Several mutations were found in gp120 and gp41 of G61R; and the additional substitution A92A/E in the NL4.3/SPL2923, whereas no mutations were found in RT capsid protein (CA, p24) (Table 3). The mutations in MA or integrase (Table 1, Table 2 and (Witvrouw et al,. 2000)). result in an overall change towards more basic amino acids. We further analysed the (cross) resistance profile of The amino acids at position 11 and 25 of MA are both NL4.3/SPL2923. The strain was found to display cross located in the basic domain of p17 which is known to play resistance against DS and the fusion inhibitor T20 (Table an important role in membrane binding by interacting with 4). gp41 of NL4.3/DS (Esté et al., 1997) was genotypically acidic phospholipids on the inner face of the lipid bilayer analysed and four mutations, V2V/M, N126K, L210L/I (Ono et al., 2000). Genetic analysis of the MA of and P213P/L were identified (Table 2). We previously NL4.3/DS and NL4.3/T20 also revealed mutations. Since reported the mutations L33S and N43K in gp41 of gp120- or gp41-recombination of NL4.3/DS or NL4.3/T20 (Fikkert et al., 2002). The observed cross resis- NL4.3/T20 reproduces the resistant phenotypes of both tance can probably be attributed to the substitution L33S, strains, mutations outside these regions as such do not seem present in both NL4.3/T20 and NL4.3/SPL2923. To find to affect the phenotypic resistance of both strains to DS or out to what extent the mutations present in the distinct T20 [unpublished observations and Fikkert et al. (2002)]. envelope genes are responsible for the elicited phenotypic The CA proteins binds cyclophilin A (CyPA), a cellular (cross) resistance of NL4.3/SPL2923, chimeric virus tech- enzyme involved in folding of host proteins, through a nology for the gp120, gp41 and gp160 was used (Fikkert et proline-rich region between residues 85–93 resulting in the

Antiviral Chemistry & Chemotherapy 16.4 263 A Hantson et al.

incorporation of CyPA into the virion. Blocking CyPA both approaches resulted in productive virus infection. The incorporation results in a decrease in virus infectivity second approach though, that is recombination at the (Franke et al., 1994). It has been suggested that CyPA cellular level, has a higher chance to result in infective virus promotes disassembly of the viral core during uncoating by production compared to the first approach, being recombi- weakening the association of the CA proteins (Gamble et nation at the viral level. al., 1996). In addition, after incorporation CyPA may relo- Phenotypical analysis of the NL4.3/SPL2923 p17- cate to the viral surface, where the exposed CyPA can recombined strain revealed that the observed mutations in mediate viral entry by attachment to heparans (Saphire et MA and CA on their own do not have an effect on the al., 2000) or CD147 (Pushkarsky et al., 2001). (cross) resistance against SPL2923, DS and T20. On the Cyclosporine A prevents CyPA incorporation as a result of contrary, p17/gp160 recombination reproduced the resis- its high affinity binding to it. Resistance selection of HIV- tance of the NL4.3/SPL2923 strain to SPL2923 (5.1- 1 in the presence of cyclosporine A results in the CA muta- fold), implying that the mutations in gag (p17 and p24) tions A92E or G94D. Both drug-resistant mutants do not have to be present in combination with the env-mutations require virion-associated CyPA to initiate infection to reproduce the resistant phenotype. However, these gag (Braaten et al., 1996). The phenotypic role of the A92A/E mutations did not seem to contribute to the cross resistance mutation in CA concerning HIV resistance to SPL2923 or of NL4.3/SPL2923 to DS and T20 (Table 4). CyPA incorporation needs additional examination. Mutations in gag have never before been reported to be To investigate the impact of the mutations revealed in associated with resistance towards entry inhibitors. However, gag on the phenotypic resistance of the NL4.3/SPL2923 additional gag mutations have been observed in strains resis- strain towards SPL2923, recombination assays were devel- tant to protease inhibitors (Carillo et al., 1998; Robinson et oped, wherein the HIV1(NL4.3) wild-type or mutant al., 2002). Duplication of a proline-rich p6 (PTAP) motif NL4.3/SPL2923 p17 gene and the 5′ part of p24 by itself has been identified to be associated with resistance to a and in combination with the HIV-1(NL4.3) wild-type or nucleoside RT inhibitor by improving assembly and pack- mutant NL4.3/SPL2923 gp160-gene were placed in the aging at membrane locations and increasing infectivity of the wild-type backbone of HIV-1(NL4.3). However, the low mutant virion (Peters et al., 2001). percentage of homologous recombination after transfec- This is the first reported phenotypical recombination tion, reduces the likelihood of recombination of two assay that is able to assess the impact of mutations in discontinuous regions. For this reason, two approaches for discontinuous genes. This opens perspectives for the the recombination assay were evaluated. In a first approach, further establishment of different resistance assays, wherein the cells were co-transfected with the p17/gp160-deleted the phenotypical effect of separated HIV genes coding for clone linearized at the p17-deletion, the p17/gp160-deleted two distinct target proteins for antiretroviral compounds, clone linearized at gp160 deletion and the PCR-derived can both be evaluated in the context of recombination into p17- and gp160-sequences (Figure 2A). In this way, the a wild-type genotype. p17- and gp160-sequences each recombine in their homol- The more we learn about HIV resistance development ogous regions on different plasmids (unless linearisation is against different classes of anti-HIV compounds, the more not that essential for homologous recombination) and it becomes clear that the mechanisms responsible for HIV viable virus particles will only be generated by an additional resistance development are multifaceted and additional recombination step during virus replication. This approach genes may be involved besides the genes encoding the requires the packaging into the HIV virion of one RNA protein directly targeted by the antiretroviral agent. strand containing the p17 recombined HIV sequence, still lacking gp160, and the other RNA strand containing the Acknowledgements gp160 recombined HIV sequence, still lacking p17.In order to achieve the packaging of the HIV genome that These investigations were supported by the Belgian lacks p17, the p17/gp160-deleted vector has been Geconcerteerde Onderzoeksacties (GOA 00/12, Vlaamse completed for the packaging signal via the reintroduction Gemeenschap), by grants from the Fonds voor of a linker sequence. In a second approach, the p17/gp160- Wetenschappelijk Onderzoek, FWO-Vlaanderen (G. deleted plasmid was digested at the position of the p17 0104.98), by the Stimulating Flemish Participation in EU deletion as well as the gp160 deletion (Figure 2B). This Research Programs (Verkennende Internationale approach required two recombination events to occur in the Samenwerking-VISA6895, Vlaamse Gemeenschap) and same p17/gp160-deleted plasmid. In this case however, the by the European TRIoH grant (contract number LSHB- disruption of the packaging signal did not affect recombi- CT-2003-503480). A Hantson is funded by a grant from nation, since no additional recombination step during viral the Flemish Institute supporting Science-Technological replication is required. We obtained proof-of-principle that Research in Industry (IWT).

References with the emergence of specific mutations in the envelope gp120 glycoprotein. Molecular Pharmacology 52:98–104. Adachi A, Gendelman HE, Koenig E, Folks T, Willey R, Rabson A Fikkert V, Cherepanov P, Van Laethem K, Hantson A, Van & Martin MA (1986) Production of acquired immunodeficiency Remoortel B, Pannecouque C, De Clercq E, Debyser Z, syndrome-associated retrovirus in human and nonhuman cells Vandamme AM & Witvrouw M (2002) Chimeric virus technolo- transfected with an infectious molecular clone. Journal of Virology gy (CVT) for HIV env-genes to evaluate the resistance/suscepti- 59:284–291. bility profile towards HIV entry inhibitors. Antimicrobial Agents & Allaway GP, Davis-Bruno KL, Beaudry GA, Garcia EB, Wong EL, Chemotherapy 46:3954–3962. Ryder AM, Hasel KW, Gauduin MC, Koup RA, McDougal JS & Franke EK, Yuan HE & Luban J (1994) Specific incorporation of Maddon PJ (1995) Expression and characterization of CD4-IgG2, cyclophilin A into HIV-1 virions. Nature 372:359–362. a novel hetrotetramer, which neutralizes primary HIV type 1, iso- Freed EO & Martin MA (1996) Domains of the human immunode- lates. AIDS Research & Human Retroviruses 11:533–539. ficiency virus type 1 matrix and gp41 cytoplasmic tail required for Arakaki R, Tamamura H, Premanathan M, Kanbara K, Ramanan S, envelope incorporation into virions. Journal of Virology 70:341–351. Mochizuki K, Baba M, Fujii N & Nakashima H (1999) T134, a Gamble TR, Vajdos FF, Yoo S, Worthylake DK, Houseweart M, small molecule CXCR4 inhibitor, has no cross-drug resistance Sundquist WI & Hill CP (1996) Crystal structure of human with AMD3100, a CXCR4 antagonist with a different structure. cyclophilin A bound to the amino-terminal domain of HIV-1 cap- Journal of Virology 73:1719–1723. sid. Cell 87:1285–1294. Baba M, Pauwels R, Balzarini J, Arnout J, Desmyter J & De Clercq Guo Q, Ho HT, Dicker I, Fan L, Zhou N, Friborg J, Wang T, E (1988) Mechanism of inhibitory effect of dextran sulfate and McAuliffe BV, Wang HG, Rose RE, Fang H, Scarnati HT, heparin on replication of human immunodeficiency virus in vitro. Langley DR, Meanwell NA, Abraham R, Colonno RJ & Lin PF Proceedings of the National Academy of Sciences, USA 85:6132–6136. (2003) Biochemical and genetic characterizations of a novel human Braaten D, Aberham C, Franke EK, Yin L, Phares W & Luban J immunodeficiency virus type 1 inhibitor that blocks gp120-CD4 (1996) Cyclosporine A-resistant human immunodeficiency virus interactions. Journal of Virology 77:10528–10536. type 1 mutants demonstrate that Gag encodes the functional target Horwitz JP, Chua J & Noel M (1964) Nucleosides. V. The monome- of cyclophilin. American Journal of Virology 70:5170–5176. sylates of 1-(2′-deoxy-β-D-lyxofuranosyl)thymidine. Journal of Bridger GJ, Skerlj RT, Padmanabhan S, Martellucci SA, Henson Organic Chemistry 29:2076–2078. GW, Struyf S, Witvrouw M, Schols D & De Clercq E (1995) Jacobson JM, Lowy I, Fletcher CV, O’Neill TJ, Tran DN, Ketas TJ, Synthesis and structure-activity relationship of phenylenebis(meth- Trkola A, Klotman ME, Maddon PJ, Olson WC & Israel RJ ylene)-linked bis-tetraazamacrocycles that inhibit HIV replication. (2000) Single-dose safety, pharmacology, and antiviral activity of Effect of heteroaromatic linkers on the activity of bicyclams. the human immunodeficiency virus (HIV) type 1 entry inhibitor Journal of Medical Chemistry 38:366–378. PRO 542 in HIV-infected adults. Journal of Infectious Diseases Carrillo A, Stewart KD, Sham HL, Norbeck D, Kohlbrenner WE, 182:326–329. Leonard JM, Kempf DJ & Molla A (1998) In vitro selection and Lalezari JP, Henry K, O’Hearn M, Montaner JS, Piliero PJ, Trottier characterization of human immunodeficiency virus type 1 variants B, Walmsley S, Cohen C, Kuritzkes DR, Eron JJ Jr, Chung J, with increased resistance to ABT-378, a novel protease inhibitor. DeMasi R, Donatacci L, Drobnes C, Delehanty J, Salgo M & Journal of Virology 72:7532–7541. TORO 1 Study Group (2003) Enfuvirtide, an HIV-1 fusion Chan DC & Kim PS (1998) HIV entry and its inhibition. Cell inhibitor, for drug-resistant HIV infection in North and South 93:681–684. America. New England Journal of Medicine 348:2175–2185. Daelemans D, Schols D, Witvrouw M, Pannecouque C, Hatse S, Lazzarin A, Clotet B, Cooper D, Reynes J, Arasteh K, Nelson M, Vandooren S, Hamy F, Klimkait T, De Clercq E & Vandamme A- Katlama C, Stellbrink HJ, Delfraissy JF, Lange J, Huson L, M (2000) A second target for the peptoid Tat/TAR inhibitor DeMasi R, Wat C, Delehanty J, Drobnes C, Salgo M & TORO 2 CGP64222: inhibition of HIV replication by blocking CXCR4- Study Group (2003) Efficacy of enfuvirtide in patients infected mediated virus entry. Molecular Pharmacology 57:116–124. with drug-resistant HIV-1 in Europe and Australia. New England Journal of Medicine 348:2186–2195. De Clercq E, Yamamoto N, Pauwels R, Baba M, Schols D, Nakashima H, Balzarini J, Debyser Z, Murrer BA, Schwartz D, Lin PF, Blair W, Wang T, Spicer T, Guo Q, Zhou N, Gong YF, Thornton D, Bridger G, Fricker S, Henson G, Abrams M & Wang HG, Rose R, Yamanaka G, Robinson B, Li CB, Fridell R, Picker D (1992) Potent and selective inhibition of human immun- Deminie C, Demers G, Yang Z, Zadjura L, Meanwell N & odeficiency virus (HIV)-1 and HIV-2 replication by a class of Colonno R (2003) A small molecule HIV-1 inhibitor that targets bicyclams interacting with a viral uncoating event. Proceedings of the HIV1 envelope and inhibits CD4 receptor binding. Proceedings the National Academy of Sciences, USA 89:5286–5290. of the National Academy of Sciences, USA 100:11013–11018. De Vreese K, Kofler-Mongold V, Leutgeb C, Weber V, Vermeire K, Miyoshi I, Taguchi H, Kobonishi I, Yoshimoto S, Ohtsuki Y & Schacht S, Anne J, De Clercq E, Datema R & Werner G (1996) Shiraishi Y (1982) Type C virus-producing cell lines derived from The molecular target of bicyclams potent inhibitors of human adult T cell leukemia. Gann Monograph on Cancer Research immunodeficiency virus replication. Journal of Virology 70:689–696. 28:219–228. Doranz BJ, Grovit-Ferbas K, Sharron MP, Mao SH, Goetz MB, Murakami T, Nakajima T, Koyanagi Y, Tachibana K, Fujii N, Daar ES, Doms RW & O’Brien WA (1997) A small molecule Tamamura H, Yoshida N, Waki M, Matsumoto A, Yoshie O, inhibitor directed against the chemokine receptor CXCR4 pre- Kishimoto T, Yamamoto N & Nagasawa T (1997) A small mole- vents its use as an HIV-1 coreceptor. Journal of Experimental cule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 infec- Medicine 186:1395–1400. tion. Journal of Experimental Medicine 186:1389–1393. Eckert DM & Kim PS (2001) Design of potent inhibitors of HIV-1 Murakami T & Freed EO (2000) Genetic evidence for an interac- entry from the gp41 N-peptide region. Proceedings of the National tion between human immunodeficiency virus type 1 matrix and Academy of Sciences, USA 98:11187–11192. alpha-helix 2 of the gp41 cytoplasmic tail. Journal of Virology 74:3548–3554. Esté JA, Schols D, De Vreese K, Van Laethem K, Vandamme AM, Desmyter J & De Clercq E (1997) Development of resistance of Ono A, Huang M & Freed EO (1997) Characterization of human human immunodeficiency virus type 1 to dextran sulfate associated immunodeficiency virus type 1 matrix revertants: effects on virus

Antiviral Chemistry & Chemotherapy 16.4 265 A Hantson et al.

assembly, Gag processing, and Env incorporation into virions. in vivo. Proceedings of the National Academy of Sciences, USA Journal of Virology 71:4409–4418. 98:12718–12723. Ono A, Orenstein JM & Freed EO (2000) Role of the Gag matrix Tamamura H, Arakaki A, Funakoshi H, Imai M, Otaka A, Ibuka T, domain in targeting human immunodeficiency virus type 1 assem- Nakashima H, Murakami T, Waki M, Matsumoto A, Yamamoto bly. Journal of Virology 74:2855–2866. N & Fujii N (1998a) Effective lowly cytotoxic analogs of an HIV- Pauwels R, Balzarini J, Baba M, Snoeck R, Schols D, Herdewijn P, cell fusion inhibitor, T22 (Tyr5,12, Lys7-polyphemusin II). Desmyter J & De Clercq E (1988) Rapid and automated tetrazoli- Bioorganic & Medicinal Chemistry 6:231–238. um-based colorimetric assay for the detection of anti-HIV com- Tamamura H, Xu Y, Hattori T, Zhang X, Arakaki R, Kanbara K, pounds. Journal of Virological Methods 20:309–321. Omagari A, Otaka A, Ibuka T, Yamamoto N, Nakashima H & Peters S, Munoz M, Yerly S, Sanchez-Merino V, Lopez-Galindez C, Fujii N (1998b) A low-molecular-weight inhibitor against the Perrin L, Larder B, Cmarko D, Fakan S, Meylan P & Telenti A chemokine receptor CXCR4: a strong anti-HIV peptide T140. (2001) Resistance to nucleoside analog reverse transcriptase Biochemical & Biophysical Research Communications 253:877–882. inhibitors mediated by human immunodeficiency virus type 1 p6 Tashima KT & Carpenter CC (2003) Fusion inhibition – a major protein. Journal of Virology 75:9644–9653. but costly step forward in the treatment of HIV-1. New England Pushkarsky T, Zybarth G, Dubrovsky L, Yurchenko V, Tang H, Guo Journal of Medicine 348:2249–2250. H, Toole B, Sherry B & Bukrinsky M (2001) CD147 facilitates Vandamme AM, Van Laethem K & De Clercq E (1999) Managing HIV-1 infection by interacting with virus-associated cyclophilin A. Resistance to anti-HIV drugs. An important consideration for Proceedings of the National Academy of Sciences, USA 98:6360–6365. effective disease management. Drugs 57:337–361. Richman DD (2001) HIV chemotherapy. Nature 410:995–1001. Wang T, Zhang Z, Wallace O-B, Deshpande M, Fang H, Yang Z, Robinson LH, Gale CV & Kleim JP (2002) Inclusion of full length Zadjura LM, Tweedie DL, Huang S, Zhao F, Ranadive S, human immunodeficiency virus type 1 (HIV-1) gag sequences in Robinson BS, Gong YF, Ricarrdi K, Spicer TP, Deminie C, Rose viral recombinants applied to drug susceptibility phenotyping. R, Wang HG, Blair WS, Shi PY, Lin PF, Colonno RJ & Journal of Virological Methods 104:147–160. Meanwell NA (2003) Discovery of 4-benzoyl-1-[(4-methoxy-1H- Saphire AC, Bobardt MD & Gallay PA (2000) Human immunode- pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2- (R)-methylpiperazine ficiency virus type 1 hijacks host cyclophilin A for its attachment (BMS-378806): a novel HIV-1 attachment inhibitor that inter- to target cells. Immunologic Research 21:211–217. feres with CD4-gp120 interactions. Journal of Medicinal Chemistry 46:4236–4239. Schols D, Struyf S, Van Damme J, Esté JA, Henson G & De Clercq E (1997) Inhibition of T tropic HIV strains by selective antagoni- Witvrouw M & De Clercq E (1997) Sulfated polysaccharides zation of the chemokine receptor CXCR4. Journal of Experimental extracted from sea algae as potential antiviral drugs. General Medicine 186:1393–1388. Pharmacology 29:497–511. Strizki JM, Xu S, Wagner NE, Wojcik L, Liu J, Hou Y, Endres M, Witvrouw M, Fikkert V, Pluymers W, Matthews B, Mardel K, Palani A, Shapiro S, Clader JW, Greenlee WJ, Tagat JR, Schols D, Raff J, Debyser Z, De Clercq E, Holan G & McCombie S, Cox K, Fawzi AB, Chou CC, Pugliese-Sivo C, Pannecouque C (2000) Polyanionic (i.e. polysulfonate) dendrimers Davies L, Moreno M-E, Ho DD, Trkola A, Stoddart CA, Moore can inhibit the replication of human immunodeficiency virus by JP, Reyes GR & Baroudy BM (2001) SCH-C (SCH 351125), an interfering with both virus adsorption and later steps (reverse tran- orally bioavailable, small molecule antagonist of the chemokine scriptase/integrase) in the virus replicative cycle. Molecular receptor CCR5, is a potent inhibitor of HIV-infection in vitro and Pharmacology 58:1100–1108.

Received 23 December 2004, accepted 18 April 2005