Cooperative inhibition of SNARE-mediated vesicle fusion by α-synuclein monomers and oligomers.

The primary hallmark of Parkinson's disease (PD) is the generation of Lewy bodies of which major component is α-synuclein (α-Syn). Because of increasing evidence of the fundamental roles of α-Syn oligomers in disease progression, α-Syn oligomers have become potential targets for therapeutic interventions for PD. One of the potential toxicities of α-Syn oligomers is their inhibition of SNARE-mediated vesicle fusion by specifically interacting with vesicle-SNARE protein synaptobrevin-2 (Syb2), which hampers dopamine release. Here, we show that α-Syn monomers and oligomers cooperatively inhibit neuronal SNARE-mediated vesicle fusion. α-Syn monomers at submicromolar concentrations increase the fusion inhibition by α-Syn oligomers. This cooperative pathological effect stems from the synergically enhanced vesicle clustering. Based on this cooperative inhibition mechanism, we reverse the fusion inhibitory effect of α-Syn oligomers using small peptide fragments. The small peptide fragments, derivatives of α-Syn, block the binding of α-Syn oligomers to Syb2 and dramatically reverse the toxicity of α-Syn oligomers in vesicle fusion. Our findings demonstrate a new strategy for therapeutic intervention in PD and related diseases based on this specific interaction of α-Syn.

Screening and Reverse-Engineering of Estrogen Receptor Ligands as Potent Pan-Filovirus Inhibitors.

Filoviridae, including Ebola (EBOV) and Marburg (MARV) viruses, are emerging pathogens that pose a serious threat to public health. No agents have been approved to treat filovirus infections, representing a major unmet medical need. The selective estrogen receptor modulator (SERM) toremifene was previously identified from a screen of FDA-approved drugs as a potent EBOV viral entry inhibitor, via binding to EBOV glycoprotein (GP). A focused screen of ER ligands identified ridaifen-B as a potent dual inhibitor of EBOV and MARV. Optimization and reverse-engineering to remove ER activity led to a novel compound 30 (XL-147) showing potent inhibition against infectious EBOV Zaire (0.09 µM) and MARV (0.64 µM). Mutagenesis studies confirmed that inhibition of EBOV viral entry is mediated by the direct interaction with GP. Importantly, compound 30 displayed a broad-spectrum antifilovirus activity against Bundibugyo, Tai Forest, Reston, and Menglà viruses and is the first submicromolar antiviral agent reported for some of these strains, therefore warranting further development as a pan-filovirus inhibitor.

The Polyphenolic Compound Curcumin Conjugation with an Alkyne Moiety in the Process of Autophagy.

Curcumin is a hydrophobic polyphenol derived from turmeric: the rhizome of the herb Curcumalonga. Autophagy is an evolutionarily conserved process, in which cellular proteins and organelles are engulfed in autophagosome and then fuses with lysosome for degradation. Our previous study showed that Curcumin activates lysosome and induce autophagy through inhibition of AKT (protein kinase K, PKB)-mammalian target of rapamycin (mTOR) pathway. But whether Curucmin affects the fusion of autophagosome-lysosome is still not clear. Here, we used Curcumin-probe conjugation with an alkyne moiety to label mouse embryonic fibroblasts (MEFs) and found that Curcumin targets autophagy-related proteins, enhances autophagic flux and activates lysosome in cells. Moreover, Curcumin treatment promotes the fusion of autophasosome-lysosome in MEFs. Second, the enhanced fusion of autophagosome-lysosome is attributed to mTOR suppression. Third, blockage of the autophagosome-lysosome fusion leads to cell growth inhibition by Curcumin. Taken together, data from our study indicates the importance of the fusion of autophagosome-lysosome in Curcumin-induced autophagy, which may facilitate the development of Curcumin as a potential therapeutic agent for oxidative stress-related diseases.

A novel autophagy inhibitor berbamine blocks SNARE-mediated autophagosome-lysosome fusion through upregulation of BNIP3.

Increasing evidences reveal that autophagy inhibitor could enhance the effect of chemotherapy to cancer. However, few autophagy inhibitors are currently approved for clinical application in humans. Berbamine (BBM) is a natural compound extracted from traditional Chinese medicine that is widely used for treatment of a variety of diseases without any obvious side effects. Here we found that BBM is a novel auophagy inhibitor, which potently induced the accumulation of autophagosomes by inhibiting autophagosome-lysosome fusion in human breast cancer cells. Mechanistically, we found that BBM blocked autophagosome-lysosome fusion by inhibiting the interaction of SNAP29 and VAMP8. Furthermore, BBM induced upregulation of BNIP3 and the interaction between SNAP29 and BNIP3. BNIP3 depletion or SNAP29 overexpression abrogated BBM-mediated blockade of autophagosome-lysosome fusion through the interaction between SNAP29 and VAMP8, whereas BNIP3 overexpression blocked autophagosome-lysosome fusion through inhibition of the interaction between SNAP29 and VAMP8. These findings suggest that upregulation of BNIP3 and interaction between BNIP3 and SNAP29 could be involved in BBM-mediated blockade of autophagosome-lysosome fusion through inhibition of the interaction between SNAP29 and VAMP8. Our findings identify the critical role of BNIP3 in blockade of autophagosome-lysosome fusion mediated by BBM, and suggest that BBM could potentially be further developed as a novel autophagy inhibitor, which could enhance the effect of chemotherapy to cancer.

A new in vitro hemagglutinin inhibitor screening system based on a single-vesicle fusion assay.

Hemagglutinin (HA) from the influenza virus plays a pivotal role in the infection of host mammalian cells and is, therefore, a druggable target, similar to neuraminidase. However, research involving the influenza virus must be conducted in facilities certified at or above Biosafety Level 2 because of the potential threat of the contagiousness of this virus. To develop a new HA inhibitor screening system without intact influenza virus, we conceived a single-vesicle fusion assay using full-length recombinant HA. In this study, we first showed that full-length recombinant HA can mediate membrane fusion in ensemble and single-vesicle fusion assays. The fluorescence resonance energy transfer (FRET) frequency pattern of single-vesicle complexes completely differed when the inhibitors targeted the HA1 or HA2 domain of HA. This result indicates that analysing the FRET patterns in this assay can provide information regarding the domains of HA inhibited by compounds and compounds' inhibitory activities. Therefore, our results suggest that the assay developed here is a promising tool for the discovery of anti-influenza virus drug candidates as a new in vitro inhibitor screening system against HA from the influenza virus.

Lipid Regulation of Acrosome Exocytosis.

Lipids are critical regulators of mammalian sperm function, first helping prevent premature acrosome exocytosis, then enabling sperm to become competent to fertilize at the right place/time through the process of capacitation, and ultimately triggering acrosome exocytosis. Yet because they do not fit neatly into the "DNA--RNA-protein" synthetic pathway, they are understudied and poorly understood. Here, we focus on three lipids or lipid classes-cholesterol, phospholipids, and the ganglioside G(M1)--in context of the modern paradigm of acrosome exocytosis. We describe how these various- species are precisely segregated into membrane macrodomains and microdomains, simultaneously preventing premature exocytosis while acting as foci for organizing regulatory and effector molecules that will enable exocytosis. Although the mechanisms responsible for these domains are poorly defined, there is substantial evidence for their composition and functions. We present diverse ways that lipids and lipid modifications regulate capacitation and acrosome exocytosis, describing in more detail how removal of cholesterol plays a master regulatory role in enabling exocytosis through at least two complementary pathways. First, cholesterol efflux leads to proteolytic activation of phospholipase B, which cleaves both phospholipid tails. The resultant changes in membrane curvature provide a mechanism for the point fusions now known to occur far before a sperm physically interacts with the zona pellucida. Cholesterol efflux also enables G(M1) to regulate the voltage-dependent cation channel, Ca(V)2.3, triggering focal calcium transients required for acrosome exocytosis in response to subsequent whole-cell calcium rises. We close with a model integrating functions for lipids in regulating acrosome exocytosis.

A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance.

Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus.

The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

Role of D-Limonene in autophagy induced by bergamot essential oil in SH-SY5Y neuroblastoma cells.

Bergamot (Citrus bergamia, Risso et Poiteau) essential oil (BEO) is a well characterized, widely used plant extract. BEO exerts anxiolytic, analgesic and neuroprotective activities in rodents through mechanisms that are only partly known and need to be further investigated. To gain more insight into the biological effects of this essential oil, we tested the ability of BEO (0.005-0.03%) to modulate autophagic pathways in human SH-SY5Y neuroblastoma cells. BEO-treated cells show increased LC3II levels and appearance of dot-like formations of endogenous LC3 protein that colocalize with the lysosome marker LAMP-1. Autophagic flux assay using bafilomycin A1 and degradation of the specific autophagy substrate p62 confirmed that the observed increase of LC3II levels in BEO-exposed cells is due to autophagy induction rather than to a decreased autophagosomal turnover. Induction of autophagy is an early and not cell-line specific response to BEO. Beside basal autophagy, BEO also enhanced autophagy triggered by serum starvation and rapamycin indicating that the underlying mechanism is mTOR independent. Accordingly, BEO did not affect the phosphorylation of ULK1 (Ser757) and p70(S6K) (Thr389), two downstream targets of mTOR. Furthermore, induction of autophagy by BEO is beclin-1 independent, occurs in a concentration-dependent manner and is unrelated to the ability of BEO to induce cell death. In order to identify the active constituents responsible for these effects, the two most abundant monoterpenes found in the essential oil, d-limonene (125-750 µM) and linalyl acetate (62.5-375 µM), were individually tested at concentrations comparable to those found in 0.005-0.03% BEO. The same features of stimulated autophagy elicited by BEO were reproduced by D-limonene, which rapidly increases LC3II and reduces p62 levels in a concentration-dependent manner. Linalyl acetate was ineffective in replicating BEO effects; however, it greatly enhanced LC3 lipidation triggered by D-limonene.

Inhibitory and combinatorial effect of diphyllin, a v-ATPase blocker, on influenza viruses.

An influenza pandemic poses a serious threat to humans and animals. Conventional treatments against influenza include two classes of pathogen-targeting antivirals: M2 ion channel blockers (such as amantadine) and neuraminidase inhibitors (such as oseltamivir). Examination of the mechanism of influenza viral infection has shown that endosomal acidification plays a major role in facilitating the fusion between viral and endosomal membranes. This pathway has led to investigations on vacuolar ATPase (v-ATPase) activity, whose role as a regulating factor on influenza virus replication has been verified in extensive genome-wide screenings. Blocking v-ATPase activity thus presents the opportunity to interfere with influenza viral infection by preventing the pH-dependent membrane fusion between endosomes and virions. This study aims to apply diphyllin, a natural compound shown to be as a novel v-ATPase inhibitor, as a potential antiviral for various influenza virus strains using cell-based assays. The results show that diphyllin alters cellular susceptibility to influenza viruses through the inhibition of endosomal acidification, thus interfering with downstream virus replication, including that of known drug-resistant strains. In addition, combinatorial treatment of the host-targeting diphyllin with pathogen-targeting therapeutics (oseltamivir and amantadine) demonstrates enhanced antiviral effects and cell protection in vitro.

Aqueous extract from a Chaga medicinal mushroom, Inonotus obliquus (higher Basidiomycetes), prevents herpes simplex virus entry through inhibition of viral-induced membrane fusion.

Chaga medicinal mushroom, Inonotus obliquus, a popular prescription in traditional medicine in Europe and Asia, was used to reduce inflammation in the nasopharynx and to facilitate breathing. The aqueous extract from I. obliquus (AEIO) exhibited marked decrease in herpes simplex virus (HSV) infection (the 50% inhibitory concentration was 3.82 µg/mL in the plaque reduction assay and 12.29 µg/mL in the HSV-1/blue assay) as well as safety in Vero cells (the 50% cellular cytotoxicity was > 1 mg/mL, and selection index was > 80). Using a time course assay, effective stage analysis, and fusion inhibition assay, the mechanism of anti-HSV activity was found against the early stage of viral infection through inhibition of viral-induced membrane fusion. Therefore, AEIO could effectively prevent HSV-1 entry by acting on viral glycoproteins, leading to the prevention of membrane fusion, which is different from nucleoside analog antiherpetics.

A botulinum neurotoxin-like function of Potentilla chinensis extract that inhibits neuronal SNARE complex formation, membrane fusion, neuroexocytosis, and muscle contraction.

CONTEXT: Botulinum neurotoxins (BoNTs) are popularly used to treat various diseases and for cosmetic purposes. They act by blocking neurotransmission through specific cleavage of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Recently, several polyphenols were shown to interfere with SNARE complex formation by wedging into the hydrophobic core interface, thereby leading to reduced neuroexocytosis. OBJECTIVE: In order to find industrially-viable plant extract that functions like BoNT, 71 methanol extracts of flowers were screened and BoNT-like activity of selected extract was evaluated. MATERIALS AND METHODS: After evaluating the inhibitory effect of 71 flower methanol extracts on SNARE complex formation, seven candidates were selected and they were subjected to SNARE-driven membrane fusion assay. Neurotransmitter release from neuronal PC12 cells and SNARE complex formation inside the cell was also evaluated. Finally, the effect of one selected extract on muscle contraction and digit abduction score was determined. RESULTS: The extract of Potentilla chinensis Ser. (Rosaceae)(Chinese cinquefoil) flower inhibited neurotransmitter release from neuronal PC12 cells by approximately 90% at a concentration of 10 µg/mL. The extract inhibited neuroexocytosis by interfering with SNARE complex formation inside cells. It reduced muscle contraction of phrenic nerve-hemidiaphragm by approximately 70% in 60 min, which is comparable to the action of the Ca²âº-channel blocker verapamil and BoNT type A. DISCUSSION AND CONCLUSION: While BoNT blocks neuroexocytosis by cleaving SNARE proteins, the Potentilla chinensis extract exhibited the same activity by inhibiting SNARE complex formation. The extract paralyzed muscle as efficiently as BoNT, suggesting the potential versatility in cosmetics and therapeutics.

A natural theaflavins preparation inhibits HIV-1 infection by targeting the entry step: potential applications for preventing HIV-1 infection.

Theaflavins are the major components of tea polyphenols in brewed black tea. We previously reported that theaflavin derivatives, such as TF3, inhibited HIV-1 entry by targeting gp41. However, it is difficult to purify the individual theaflavins and the purified compounds are highly unstable. To develop theaflavins as affordable anti-HIV-1 microbide for preventing HIV sexual transmission, we intended to use an economic natural preparation containing 90% of theaflavins (TFmix). Its antiviral activity against HIV-1 strains was evaluated in vitro using p24 production and luciferase assays. The mechanism by which TFmix inhibits HIV-1 infection was investigated using time-of-addition, cell-cell fusion and biophysical assays. The data suggested TFmix exhibited potent anti-HIV-1 activity on lab-adapted and primary HIV-1 strains with IC(50) less than 1.20 µM. It also effectively inhibited infection by T-20 resistant HIV-1 strains. The mechanism studies suggest that TFmix mainly inhibit the HIV-1 entry by targeting gp41 since it is effective in inhibiting gp41 six-helix bundle (6-HB) formation and HIV-1 envelope protein-mediated cell-cell fusion. TFmix could also inhibit HIV-1 reverse transcriptase (RT) activity, but the IC(50) is about 8-fold higher than that for inhibiting gp41 6-HB formation, suggesting RT is not a major target for TFmix. In conclusion, TFmix is an economic natural product preparation containing high content of theaflavins with potent anti-HIV-1 activity by targeting the viral entry step through the disruption of gp41 6-HB core structure. It has a potential to be developed as a safe and affordable topical microbicide for preventing sexual transmission of HIV.

Dysfunction of the fusion of pre-synaptic plasma membranes and synaptic vesicles caused by oxidative stress, and its prevention by vitamin E.

To define whether hyperoxia induces the dysfunction of membrane fusion between synaptic vesicles with pre-synaptic plasma membranes in the nerve terminals, and whether vitamin E prevents this abnormal event, we investigated the influence of hyperoxia on the fusion ability of isolated synaptic vesicles and the inside-out type pre-synaptic plasma membrane vesicles from rat brain using the fluorescence tracing method. The membrane fusion ability of both membranes from rats subjected to hyperoxia was markedly decreased compared with the membranes from a normal rat. Rats subjected to hyperoxia in the form of oxidative stress showed significant increases in the levels of thiobarbituric acid reactive substances (TBARS), conjugated dienes, and protein carbonyl moieties in both synaptic vesicles and pre-synaptic plasma membranes. When rats were supplemented with vitamin E, these abnormalities were inhibited even when rats were subjected to hyperoxia.

Two novel fusion inhibitors of human respiratory syncytial virus.

To search for novel drugs against human respiratory syncytial virus (RSV), we have screened a diversity collection of 16,671 compounds for anti-RSV activity in cultures of HEp-2 cells. Two of the hit compounds, i.e., the N-(2-hydroxyethyl)-4-methoxy-N-methyl-3-(6-methyl[1,2,4]triazolo[3,4-a]phthalazin-3-yl)benzenesulfonamide (designated as P13) and the 1,4-bis(3-methyl-4-pyridinyl)-1,4-diazepane (designated as C15), reduced the virus infectivity with IC50 values of 0.11 and 0.13µM respectively. The concentration of P13 and C15 that reduced the viability of HEp-2 cells by 50% was 310 and 75µM respectively. Both P13 and C15 exhibited no direct virucidal activity or inhibitory effects on the virus attachment to cells. However, to inhibit formation of RSV-induced syncytial plaques P13 and C15 had to be present during the virus entry into the cells and the cell-to-cell transmission of the virus. The RSV multiplication in HEp-2 cells in the presence of P13 or C15 resulted in rapid selection of viral variants that were ∼1000 times less sensitive to these drugs than original virus. Sequencing of resistant viruses revealed presence of amino acid substitutions in the F protein of RSV, i.e., the D489G for C15-selected, and the T400I and N197T (some clones) for the P13-selected virus variants. In conclusion, we have identified two novel fusion inhibitors of RSV, and the detailed understanding of their mode of antiviral activity including selection for the drug resistant viral variants may help to develop selective and efficient anti-RSV drugs.

Effects of low-molecular-weight heparin on adhesion and vesiculation of phospholipid membranes: a possible mechanism for the treatment of hypercoagulability in antiphospholipid syndrome.

Heparins represent an efficient treatment of acute thrombosis and obstetric complications in antiphospholipid syndrome (APS). Enhanced microvesiculation of cell membranes, as detected by reduced membrane adhesion, can contribute to hypercoagulability in APS. Healthy donor IgG antibodies significantly increased beta2-glycoprotein I (beta2-GPI)-induced membrane adhesion, indicating that IgG antibodies might supplement the role of beta2-GPI in the regulation of membrane microvesiculation in healthy individuals. Anti-beta2-GPI IgG antibodies significantly reduced beta2-GPI-induced membrane adhesion, suggesting a direct role of anti-beta2-GPI antibodies in enhancing membrane microvesiculation in APS. Therapeutic concentration of nadroparin completely restored beta2-GPI-induced membrane adhesion in the presence of anti-beta2-GPI IgG antibodies. A novel anticoagulant mechanism of nadroparin in APS is suggested that supplements its direct effect on the coagulation cascade. Restoration of adhesion between negatively charged membranes in the presence of nadroparin might decrease shedding of microvesicles into the surrounding solution and could thus contribute to the efficacy of heparin treatment in APS.

Synergistic efficacy of combination of enfuvirtide and sifuvirtide, the first- and next-generation HIV-fusion inhibitors.

Enfuvirtide and sifuvirtide, the first- and next-generation HIV-fusion inhibitors, contain different functional domains and have distinct target sites. Here, we found that a combination of enfuvirtide and sifuvirtide resulted in potent synergism in inhibiting HIV-1-mediated cell-cell fusion and infection by X4 and R5 as well as enfuvirtide-resistant HIV-1 strains. These findings suggest that application of enfuvirtide and sifuvirtide in combination may improve their efficacy and resistant profile, leading to a reduction of the dosage and frequency of drug use.

Novel screening systems for HIV-1 fusion mediated by two extra-virion heptad repeats of gp41.

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by its envelope protein gp41 through membrane fusion. Interaction of two extra-virion heptad repeats (HRs) in the gp41 plays a pivotal role in the fusion, and its inhibitor, enfuvirtide (T-20), blocks HIV-1 entry. To identify agents that block HIV-1 fusion, two screening methods based on detection and quantification by the enzyme-linked immunosorbent assay (ELISA) principle have been established. One method uses an alkaline phosphatase (ALP)-conjugated antibody (Ab-ELISA) and the other uses an ALP-fused HR (F-ELISA) to detect and quantify the interaction of the two HRs. The F-ELISA was more simple and rapid, since no ALP-conjugated antibody reaction was required. Both ELISAs detected all the fusion inhibitors tested except for T-20. Interaction of the two HRs was observed in both ELISAs, even in the presence of 10% dimethyl sulfoxide. Ab-ELISA performed best in a pH ranging from 6 to 8, while F-ELISA performed best at a pH ranging from 7 to 8. These results indicate that both established ELISAs are suitable for the identification of HIV-1 fusion inhibitors.

Functional involvement of Annexin-2 in cAMP induced AQP2 trafficking.

Annexin-2 is required for the apical transport in epithelial cells. In this study, we investigated the involvement of annexin-2 in cAMP-induced aquaporin-2 (AQP2) translocation to the apical membrane in renal cells. We found that the cAMP-elevating agent forskolin increased annexin-2 abundance in the plasma membrane enriched fraction with a parallel decrease in the soluble fraction. Interestingly, forskolin stimulation resulted in annexin-2 enrichment in lipid rafts, suggesting that hormonal stimulation might be responsible for a new configuration of membrane interacting proteins involved in the fusion of AQP2 vesicles to the apical plasma membrane. To investigate the functional involvement of annexin-2 in AQP2 exocytosis, the fusion process between purified AQP2 membrane vesicles and plasma membranes was reconstructed in vitro and monitored by a fluorescence assay. An N-terminal peptide that comprises 14 residues of annexin-2 and that includes the binding site for the calcium binding protein p11 strongly inhibited the fusion process. Preincubation of cells with this annexin-2 peptide also failed to increase the osmotic water permeability in the presence of forskolin in intact cells. Altogether, these data demonstrate that annexin-2 is required for cAMP-induced AQP2 exocytosis in renal cells.

[Mechanisms of resistance and failure of treatment with maraviroc]. / Mecanismos de resistencia y fracaso al tratamiento con maraviroc.

Maraviroc (MVC) is a new antagonist of the CCR5 coreceptor and is the first antiviral compound available that has a cell factor essential for HIV entry as a target. The information available from clinical studies with MVC suggests that the main cause of therapeutic failure is, more than the tropism change, the rapid selection of pre-existing strains with an affinity for CXCR4, not detected by the reference test. A recently developed tropism test with an improved sensitivity will help to detect the minority, but clinically significant, presence of strains that use CXCR4. Evidence of resistance to MVC has been shown in vivo in some patients. The mechanism of this resistance appears to be related to changes in gp120 and mainly in the V3 region which enables the virus to recognise the CCR5-co-receptor bound to the MVC molecule. From a practical point of view, standardised tests are currently unavailable to assess susceptibility to MVC, although in dose-response phenotype tests a maximum percentage inhibition (MPI) < 95% would be indicative of resistance to the compound. Similarly, although some mutations associated with resistance in V3, and other zones of gp120, have been described, this preliminary information suggests different resistance patterns and at the moment, we do not know the canonical mutations to be able to establish genotyping algorithms.