Further studies on the mechanism of rabies virus neutralization by a viral glycoprotein-specific monoclonal antibody, #1-46-12

We previously reported that a conformational epitope-specific monoclonal antibody (mAb; #1-46-12) neutralized the rabies virus by binding only a small number (less than 20) of the antibody molecules per virion, while a linear epitope-specific mAb (#7-1-9) required more than 250 IgG molecules for the neutralization. We also isolated both the epitope-negative (R-31) and-positive (R-61) escape mutants that resisted mAb #1-46-12. Co-infection studies with wild type (wt) and R-61 mutant have shown that although the infectivity of R-61 mutant was not affected by the binding of about 300 IgG molecules per virion, incorporation of a small number of wt G protein into the R-61 virion resulted in dramatic loss of the resistance. In this study, we further investigated properties of the mutant G proteins. The R-61 G protein lost reactivity to the mAb when solubilized, even keeping a trimer form, suggesting that membrane-anchorage is essential for the maintenance of its epitope-positive conformation. On the other hand, incorporation of wt G proteins into the R-31 virions did not affect their resistance to the mAb very much. Although we have not so far found the presumed conformational changes induced by the mAb-binding, we think that these results are not inconsistent with our previously proposed novel model (referred to as a domino effect model) for the virus neutralization by mAb #1-46-12 other than a classical spike-blocking model, which implicates successive spreading of the postulated antibody-induced conformational changes of G protein to the neighboring spikes until abolishing the host cell-binding ability of the virion.     

Direct and indirect interactions in the recognition between a cross‐neutralizing antibody and the four serotypes of dengue virus

Dengue fever is the most important vector‐borne viral disease. Four serotypes of dengue virus, DENV1 to DENV4, coexist. Secondary infection by a different serotype is a risk factor for severe dengue. Monoclonal antibody mAb4E11 neutralizes the four serotypes of DENV with varying efficacies by recognizing an epitope located within domain‐III (ED3) of the viral envelope (E) protein. To better understand the cross‐reactivities between mAb4E11 and the four serotypes of DENV, we constructed mutations in both Fab4E11 fragment and ED3, and we searched for indirect interactions in the crystal structures of the four complexes. According to the serotype, 7 to 12 interactions are mediated by one water molecule, 1 to 10 by two water molecules, and several of these interactions are conserved between serotypes. Most interfacial water molecules make hydrogen bonds with both antibody and antigen. Some residues or atomic groups are engaged in both direct and water‐mediated interactions. The doubly‐indirect interactions are more numerous in the complex of lowest affinity. The third complementarity determining region of the light chain (L‐CDR3) of mAb4E11 does not contact ED3. The structures and double‐mutant thermodynamic cycles showed that the effects of (hyper)‐mutations in L‐CDR3 on affinity were caused by conformational changes and indirect interactions with ED3 through other CDRs. Exchanges of residues between ED3 serotypes showed that their effects on affinity were context dependent. Thus, conformational changes, structural context, and indirect interactions should be included when studying cross‐reactivity between antibodies and different serotypes of viral antigens for a better design of diagnostics, vaccine, and therapeutic tools against DENV and other Flaviviruses. Copyright © 2014 John Wiley & Sons, Ltd.     

From resistance to stimulation: the evolution of a virus in the presence of a dominant lethal inhibitory scaffolding protein

By acquiring resistance to an inhibitor, viruses can become dependent on that inhibitor for optimal fitness. However, inhibitors rarely, if ever, stimulate resistant strain fitness to values that equal or exceed the uninhibited wild-type level. This would require an adaptive mechanism that converts the inhibitor into a beneficial replication factor. Using a plasmid-encoded inhibitory external scaffolding protein that blocks φX174 assembly, we previously demonstrated that such mechanisms are possible. The resistant strain, referred to as the evolved strain, contains four mutations contributing to the resistance phenotype. Three mutations confer substitutions in the coat protein, whereas the fourth mutation alters the virus-encoded external scaffolding protein. To determine whether stimulation by the inhibitory protein coevolved with resistance or whether it was acquired after resistance was firmly established, the strain temporally preceding the previously characterized mutant, referred to as the intermediary strain, was isolated and characterized. The results of the analysis indicated that the mutation in the virus-encoded external scaffolding protein was primarily responsible for stimulating strain fitness. When the mutation was placed in a wild-type background, it did not confer resistance. The mutation was also placed in cis with the plasmid-encoded dominant lethal mutation. In this configuration, the stimulating mutation exhibited no activity, regardless of the genotype (wild type, evolved, or intermediary) of the infecting virus. Thus, along with the coat protein mutations, stimulation required two external scaffolding protein genes: the once inhibitory gene and the mutant gene acquired during evolution.     

Concordant modulation of neutralization resistance and high infectivity of the primary human immunodeficiency virus type 1 MN strain and definition of a potential gp41 binding site in gp120

Efforts to develop a vaccine against human immunodeficiency virus type 1 (HIV-1) are complicated by resistance of virus to neutralization. The neutralization resistance phenotype of HIV-1 has been linked to high infectivity. We studied the mechanisms determining this phenotype using clones of the T-cell-line-adapted (TCLA) MN strain (MN-TCLA) and the neutralization-resistant, primary MN strain (MN-P). Mutations in the amino- and carboxy-terminal halves of gp120 and the carboxy terminus of gp41 contributed to the neutralization resistance, high-infectivity phenotype but depended upon sequences in the leucine zipper (LZ) domain of gp41. Among 23 clones constructed to map the contributing mutations, there was a very strong correlation between infectivity and neutralization resistance (R(2) = 0.81; P < 0.0001). Mutations that distinguished the gp120s of MN-P and MN-TCLA clones were clustered in or near the CD4 and coreceptor binding sites and in regions distant from those binding sites. To test the hypothesis that some of these distant mutations may interact with gp41, we determined which of them contributed to high infectivity and whether those mutations modulated gp120-gp41 association in the context of MN-P LZ sequences. In one clone, six mutations in the amino terminus of gp120, at least four of which clustered closely on the inner domain, modulated infectivity. This clone had a gp120-gp41 association phenotype like MN-P: in comparison to MN-TCLA, spontaneous dissociation was low, and dissociation induced by soluble CD4 binding was high. These results identify a region of the gp120 inner domain that may be a binding site for gp41. Our studies clarify mechanisms of primary virus neutralization resistance.     

Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors

Background

HIV-1 typically develops resistance to any single antiretroviral agent. Combined anti-retroviral therapy to reduce drug-resistance development is necessary to control HIV-1 infection. Here, to assess the utility of a combination of antibody and fusion inhibitor treatments, we investigated the potency of monoclonal antibodies at neutralizing HIV-1 variants that are resistant to fusion inhibitors.

Results

Mutations that confer resistance to four fusion inhibitors, enfuvirtide, C34, SC34, and SC34EK, were introduced into the envelope of HIV-1_JR-FL, a CCR5-tropic tier 2 strain. Pseudoviruses with these mutations were prepared and used for the assessment of neutralization sensitivity to an array of antibodies. The resulting neutralization data indicate that the potencies of some antibodies, especially of those against the CD4 binding site, V3 loop, and membrane-proximal external region epitopes, were increased by the mutations in gp41 that conferred resistance to the fusion inhibitors. C34-, SC34-, and SC34EK-resistant mutants showed more sensitivity to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the “conformational unmasking” of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant mutants in vivo was shown by its high rate of neutralization by IgG from HIV patient samples.

Conclusions

Mutations in gp41 that confer fusion inhibitor resistance exert enhanced sensitivity to broad neutralizing antibodies (e.g., VRC01 and 10E8) and other conventional antibodies developed in HIV-1 infected patients. Therefore, next-generation fusion inhibitors and monoclonal antibodies could be a potential combination for future regimens of combined antiretroviral therapy.

     

Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization

Mutations were made at 64 positions on the external surface of the adeno-associated virus type 2 (AAV-2) capsid in regions expected to bind antibodies. The 127 mutations included 57 single alanine substitutions, 41 single nonalanine substitutions, 27 multiple mutations, and 2 insertions. Mutants were assayed for capsid synthesis, heparin binding, in vitro transduction, and binding and neutralization by murine monoclonal and human polyclonal antibodies. All mutants made capsid proteins within a level about 20-fold of that made by the wild type. All but seven mutants bound heparin as well as the wild type. Forty-two mutants transduced human cells at least as well as the wild type, and 10 mutants increased transducing activity up to ninefold more than the wild type. Eighteen adjacent alanine substitutions diminished transduction from 10- to 100,000-fold but had no effect on heparin binding and define an area (dead zone) required for transduction that is distinct from the previously characterized heparin receptor binding site. Mutations that reduced binding and neutralization by a murine monoclonal antibody (A20) were localized, while mutations that reduced neutralization by individual human sera or by pooled human, intravenous immunoglobulin G (IVIG) were dispersed over a larger area. Mutations that reduced binding by A20 also reduced neutralization. However, a mutation that reduced the binding of IVIG by 90% did not reduce neutralization, and mutations that reduced neutralization by IVIG did not reduce its binding. Combinations of mutations did not significantly increase transduction or resistance to neutralization by IVIG. These mutations define areas on the surface of the AAV-2 capsid that are important determinants of transduction and antibody neutralization.     

Sequences in Glycoprotein gp41, the CD4 Binding Site,and the V2 Domain Regulate Sensitivity and Resistance of HIV-1 to Broadly Neutralizing Antibodies

The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals.     

Mutations in ftsZ that confer resistance to SulA affect the interaction of FtsZ with GTP.

Mutations in the essential cell division gene ftsZ confer resistance to SulA, a cell division inhibitor that is induced as part of the SOS response. In this study we have purified and characterized the gene products of six of these mutant ftsZ alleles, ftsZ1, ftsZ2, ftsZ3, ftsZ9, ftsZ100, and ftsZ114, and compared their properties to those of the wild-type gene product. The binding of GTP was differentially affected by these mutations. FtsZ3 exhibited no detectable GTP binding, and FtsZ9 and FtsZ100 exhibited markedly reduced GTP binding. In contrast, FtsZ1 and FtsZ2 bound GTP almost as well as the wild type, and FtsZ114 displayed increased GTP binding. Furthermore, we observed that all mutant FtsZ proteins exhibited markedly reduced intrinsic GTPase activity. It is likely that mutations in ftsZ that confer sulA resistance alter the conformation of the protein such that it assumes the active form.     

Resistance of human immunodeficiency virus type 1 to neutralization by natural antisera occurs through single amino acid substitutions that cause changes in antibody binding at multiple sites.

The ability of human immunodeficiency virus type 1 (HIV-1) to replicate in the presence of strong immune responses to the virus may be due to its high mutation rate, which provides envelope gene variability for selection of neutralization-resistant variants. Understanding neutralization escape mechanisms is therefore important for the design of HIV-1 vaccines and our understanding of the disease process. In this report, we analyze mutations at amino acid positions 281 and 582 in the HIV-1 envelope, where substitutions confer resistance to broadly reactive neutralizing antisera from seropositive individuals. Neither of these mutations lies within an antibody-binding site, and therefore the mechanism of immune escape in both cases is by alteration of the shape of the envelope proteins. The conformation of the CD4-binding site is shown to be critical with regard to presentation of other discontinuous epitopes. From our analysis of the neutralization of these variants, we conclude that escape from polyclonal sera occurs through alterations at several different epitopes, generally resulting from single amino acid substitutions which influence envelope conformation. Experiments on a double mutant showed that the combination of both mutations is not additive, suggesting that these variants utilized alternate pathways to elicit similar alterations of the HIV-1 envelope structure.     

The molecular mechanism behind resistance of the kinase FLT3 to the inhibitor quizartinib

Fms‐like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is a drug target for leukemias. Several potent inhibitors of FLT3 exist, and bind to the inactive form of the enzyme. Unfortunately, resistance due to mutations in the kinase domain of FLT3 limits the therapeutic effects of these inhibitors. As in many other cases, it is not straightforward to explain why certain mutations lead to drug resistance. Extensive fully atomistic molecular dynamics (MD) simulations of FLT3 were carried out with an inhibited form (FLT‐quizartinib complex), a free (apo) form, and an active conformation. In all cases, both the wild type (wt) proteins and two resistant mutants (D835F and Y842H) were studied. Analysis of the simulations revealed that impairment of protein‐drug interactions cannot explain the resistance mutations in question. Rather, it appears that the active state of the mutant forms is perturbed by the mutations. It is therefore likely that perturbation of deactivation of the protein (which is necessary for drug binding) is responsible for the reduced affinity of the drug to the mutants. Importantly, this study suggests that it is possible to explain the source of resistance by mutations in FLT3 by an analysis of unbiased MD simulations.     

Selection for Neutralization Resistance of the Simian/Human Immunodeficiency Virus SHIVSF33A Variant In Vivo by Virtue of Sequence Changes in the Extracellular Envelope Glycoprotein That Modify N-Linked Glycosylation

We previously reported on the in vivo adaptation of an infectious molecular simian/human immunodeficiency virus (SHIV) clone, SHIVSF33, into a pathogenic biologic viral variant, designated SHIVSF33A. In the present study, we show that SHIVSF33A is resistant to neutralization by human immunodeficiency virus (HIV) and SHIV antisera. Multiple amino acid substitutions accumulated over time throughout the env gene of SHIVSF33A; some of them coincided with the acquisition of the neutralization resistance of the virus. Of interest are changes that resulted in the removal, repositioning, and addition of potential glycosylation sites within the V1, V2, and V3 regions of envelope gp120. To determine whether potential glycosylation changes within these principal neutralization domains of HIV type 1 formed the basis for the resistance to serum neutralization of SHIVSF33A, mutant viruses were generated on the backbone of parental SHIVSF33 and tested for their neutralization sensitivity. The mutations generated did not alter the in vitro replication kinetics or cytopathicity of the mutant viruses in T-cell lines. However, the removal of a potential glycosylation site in the V1 domain or the creation of such a site in the V3 domain did allow the virus to escape serum neutralization antibodies that recognized parental SHIVSF33. The combination of the V1 and V3 mutations conferred an additive effect on neutralization resistance over that of the single mutations. Taken together, these data suggest that (i) SHIV variants with changes in the Env SU can be selected in vivo primarily by virtue of their ability to escape neutralizing antibody recognition and (ii) carbohydrates play an important role in conferring neutralization escape, possibly by altering the structure of envelope gp120 or by shielding principal neutralization sites.     

Porin-mediated antibiotic resistance in Neisseria gonorrhoeae: ion, solute, and antibiotic permeation through PIB proteins with penB mutations

Neisseria gonorrhoeae has two porins, PIA and PIB, whose genes (porA and porB, respectively) are alleles of a single por locus. We recently demonstrated that penB mutations at positions 120 and 121 in PIB, which are presumed to reside in loop 3 that forms the pore constriction zone, confer intermediate-level resistance to penicillin and tetracycline (M. Olesky, M. Hobbs, and R. A. Nicholas, Antimicrob. Agents Chemother. 46:2811-2820, 2002). In the present study, we investigated the electrophysiological properties as well as solute and antibiotic permeation rates of recombinant PIB proteins containing penB mutations (G120K, G120D/A121D, G120P/A121P, and G120R/A121H). In planar lipid bilayers, the predominant conducting state of each porin variant was 30 to 40% of the wild type, even though the anion selectivity and maximum channel conductance of each PIB variant was similar to that of the wild type. Liposome-swelling experiments revealed no significant differences in the permeation of sugars or beta-lactam antibiotics through the wild type or PIB variants. Although these results are seemingly contradictory with the ability of these variants to increase antibiotic resistance, they are consistent with MIC data showing that these porin mutations confer resistance only in strains containing an mtrR mutation, which increases expression of the MtrC-MtrD-MtrE efflux pump. Moreover, both the mtrR and penB mutations were required to decrease in vivo permeation rates below those observed in the parental strain containing either mtrR or porin mutations alone. Thus, these data demonstrate a novel mechanism of porin-mediated resistance in which mutations in PIB have no affect on antibiotic permeation alone but instead act synergistically with the MtrC-MtrD-MtrE efflux pump in the development of antibiotic resistance in gonococci.     

Role of the kdr and super-kdr sodium channel mutations in pyrethroid resistance: correlation of allelic frequency to resistance level in wild and laboratory populations of horn flies (Haematobia irritans)

The kdr and super-kdr point mutations found in the insect sodium channel gene are postulated to confer knockdown resistance (kdr) to pyrethroids. Using an allele-specific PCR assay to detect these mutations in individual horn flies, Haematobia irritans (L.), we determined the allelic frequency of the kdr and super-kdr mutations in several wild and laboratory populations. Wild populations with very similar allelic frequencies had resistance levels that ranged widely from 3- to 18-fold relative to a susceptible population. Conversely, the kdr allele frequency in a lab population with 17-fold resistance was nearly double that found in a heavily pressured wild population with 18-fold resistance. We conclude that, although the kdr mutation confers significant levels of pyrethroid resistance, a substantial component of resistance in insecticidally pressured populations is conferred by mechanisms that are PBO-suppressible. High super-kdr allele frequencies were detected in two resistant lab populations, but in wild populations with equivalent resistance the super-kdr allele frequency was very low. Interestingly, in over 1200 individuals assayed, the super-kdr mutation was never detected in the absence of the kdr mutation.     

Mutations conferring resistance to neutralization with monoclonal antibodies in type 1 poliovirus can be located outside or inside the antibody-binding site.

Antigenic variants resistant to eight neutralizing monoclonal antibodies were selected from wild (Mahoney) and attenuated (Sabin) type 1 infectious poliovirions. Cross-immunoprecipitation revealed interrelationships between epitopes which were not detected by cross-neutralization. Operational analysis of antigenic variants showed that seven of eight neutralization epitopes studied were interrelated. Only one neutralization epitope, named Kc, varied independently from all the others. This latter, recognized by C3 neutralizing monoclonal antibody, was present not only on infectious virions but also on heat-denatured (C-antigenic) particles and on isolated capsid protein VP1. Loss of the neutralization function of an epitope did not necessary result from the loss of its antibody-binding capacity. Such potential, but not functional, neutralization epitopes exist naturally on Mahoney and Sabin 1 viruses. Their antibody-binding property could be disrupted by isolating antigenic variants in the presence of the nonneutralizing monoclonal antibody and anti-mouse immunoglobulin antibodies. Single-point mutations responsible for the acquisition of resistance to neutralization in the antigenic variants were located by sequence analyses of their genomes. Mutants selected in the presence of C3 neutralizing monoclonal antibody always had the mutation located inside the antibody-binding site (residues 93 through 103 of VP1) at the amino acid position 100 of VP1. On the contrary, antigenic variants selected in the presence of neutralizing monoclonal antibodies reacting only with D-antigenic particles had mutations situated in VP3, outside the antibody-binding site (residues 93 through 103 of VP1). The complete conversion of the Mahoney to the Sabin 1 epitope map resulted from a threonine-to-lysine substitution at position 60 of VP3.     

Computational design of an integrin I domain stabilized in the open high affinity conformation

We have taken a computational approach to design mutations that stabilize a large protein domain of approximately 200 residues in two alternative conformations. Mutations in the hydrophobic core of the alphaMbeta2 integrin I domain were designed to stabilize the crystallographically defined open or closed conformers. When expressed on the cell surface as part of the intact heterodimeric receptor, binding of the designed open and closed I domains to the ligand iC3b, a form of the complement component C3, was either increased or decreased, respectively, compared to wild type. Moreover, when expressed in isolation from other integrin domains using an artificial transmembrane domain, designed open I domains were active in ligand binding, whereas designed closed and wild type I domains were inactive. Comparison to a human expert designed open mutant showed that the computationally designed mutants are far more active. Thus, computational design can be used to stabilize a molecule in a desired conformation, and conformational change in the I domain is physiologically relevant to regulation of ligand binding.     

Coordinate changes in drug resistance and drug-induced conformational transitions in altered-function mutants of the multidrug transporter P-glycoprotein

The MDR1 P-glycoprotein (Pgp), responsible for a clinically important form of multidrug resistance in cancer, is an ATPase efflux pump for multiple lipophilic drugs. The G185V mutation near transmembrane domain 3 of human Pgp increases its relative ability to transport several drugs, including etoposide, but decreases the transport of other substrates. MDR1 cDNA with the G185V substitution was used in a function-based selection to identify mutations that would further increase Pgp-mediated resistance to etoposide. This selection yielded the I186N substitution, adjacent to G185V. Pgps with G185V, I186N, or both mutations were compared to the wild-type Pgp for their ability to confer resistance to different drugs in NIH 3T3 cells. In contrast to the differential effects of G185V, I186N mutation increased resistance to all the tested drugs and augmented the effect of G185V on etoposide resistance. The effects of the mutations on conformational transitions of Pgp induced by different drugs were investigated using a conformation-sensitive antibody UIC2. Ligand-binding analysis of the drug-induced increase in UIC2 reactivity was used to determine the K(m) value that reflects the apparent affinity of drugs for Pgp, and the Hill number reflecting the apparent number of drug-binding sites. Both mutations altered the magnitude of drug-induced increases in UIC2 immunoreactivity, the K(m) values, and the Hill numbers for individual drugs. Mutation-induced changes in the magnitude of UIC2 reactivity shift did not correlate with the effects of the mutations on resistance to the corresponding drugs. In contrast, an increase or a decrease in drug resistance relative to that of the wild type was accompanied by a corresponding increase or decrease in the K(m) or in both the K(m) and the Hill number. These results suggest that mutations that alter the ability of Pgp to transport individual drugs change the apparent affinity and the apparent number of drug-binding sites in Pgp.     

Structural and biological identification of residues on the surface of NS3 helicase required for optimal replication of the hepatitis C virus

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains. A crystal structure of the NS3 helicase domain (NS3h) was generated in the presence of a single-stranded oligonucleotide long enough to accommodate binding of two molecules of enzyme. Several amino acid residues at the interface of the two NS3h molecules were identified that appear to mediate a protein-protein interaction between domains 2 and 3 of adjacent molecules. Mutations were introduced into domain 3 to disrupt the putative interface and subsequently examined using an HCV subgenomic replicon, resulting in significant reduction in replication capacity. The mutations in domain 3 were then examined using recombinant NS3h in biochemical assays. The mutant enzyme showed RNA binding and RNA-stimulated ATPase activity that mirrored wild type NS3h. In DNA unwinding assays under single turnover conditions, the mutant NS3h exhibited a similar unwinding rate and only approximately 2-fold lower processivity than wild type NS3h. Overall biochemical activities of the mutant NS3h were similar to the wild type enzyme, which was not reflective of the large reduction in HCV replicative capacity observed in the biological experiment. Hence, the biological results suggest that the known biochemical properties associated with the helicase activity of NS3h do not reveal all of the likely biological roles of NS3 during HCV replication. Domain 3 of NS3 is implicated in protein-protein interactions that are necessary for HCV replication.     

Enfuvirtide resistance mutations: impact on human immunodeficiency virus envelope function, entry inhibitor sensitivity, and virus neutralization

Enfuvirtide (ENF/T-20/Fuzeon), the first human immunodeficiency virus (HIV) entry inhibitor to be licensed, targets a structural intermediate of the entry process. ENF binds the HR1 domain in gp41 after Env has bound CD4, preventing conformational changes needed for membrane fusion. Mutations in HR1 that confer ENF resistance can arise following ENF therapy. ENF resistance mutations were introduced into an R5- and X4-tropic Env to examine their impact on fusion, infection, and sensitivity to different classes of entry inhibitors and neutralizing antibodies. HR1 mutations could reduce infection and fusion efficiency and also delay fusion kinetics, likely accounting for their negative impact on viral fitness. HR1 mutations had minimal effect on virus sensitivity to other classes of entry inhibitors, including those targeting CD4 binding (BMS-806 and a CD4-specific monoclonal antibody [MAb]), coreceptor binding (CXCR4 inhibitor AMD3100 and CCR5 inhibitor TAK-779), or fusion (T-1249), indicating that ENF-resistant viruses can remain sensitive to other entry inhibitors in vivo. Some HR1 mutations conferred increased sensitivity to a subset of neutralizing MAbs that likely target fusion intermediates or with epitopes preferentially exposed following receptor interactions (17b, 48D, 2F5, 4E10, and IgGb12), as well as sera from some HIV-positive individuals. Mechanistically, enhanced neutralization correlated with reduced fusion kinetics, indicating that, in addition to steric constraints, kinetics may also limit virus neutralization by some antibodies. Therefore, escape from ENF comes at a cost to viral fitness and may confer enhanced sensitivity to humoral immunity due to prolonged exposure of epitopes that are not readily accessible in the native Env trimer. Resistance to other entry inhibitors was not observed.