Resistance to neuraminidase inhibitors conferred by an R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population.

UNLABELLED: We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was completely inhibited at 250 µM and 31.25 µM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2'-(4-methylumbelliferryl)-α-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay. IMPORTANCE: The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants. We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir. Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection.

Neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir.

The influenza virus neuraminidase (NA) inhibitors zanamivir and oseltamivir were introduced into clinical practice in various parts of the world between 1999 and 2002. In order to monitor the potential development of resistance, the Neuraminidase Inhibitor Susceptibility Network was established to coordinate testing of clinical isolates collected through the World Health Organization influenza surveillance network from different regions of the world (M. Zambon and F. G. Hayden, Antivir. Res. 49:147-156, 2001). The present study establishes the baseline susceptibilities prior to and shortly after the introduction of the NA inhibitors. Over 1000 clinical influenza isolates recovered from 1996 to 1999 were tested. Susceptibilities were determined by enzyme inhibition assays with chemiluminescent or fluorescent substrates with known NA inhibitor-resistant viruses as controls. The 50% inhibitory concentrations (IC(50)s) depended upon the assay method, the drug tested, and the influenza virus subtype. By both assays, the mean zanamivir IC(50)s were 0.76, 1.82, and 2.28 nM for the subtype H1N1 (N1), H3N2 (N2), and B NAs, respectively, and the oseltamivir IC(50)s were 1.2, 0.5, and 8.8 nM for the N1, N2, and B NAs, respectively. The drug susceptibilities of known zanamivir- and oseltamivir-resistant viruses with the NA mutations E119V, R292K, H274Y, and R152K fell well outside the 95% confidence limits of the IC(50)s for all natural isolates. Sequence analysis of the NAs of viruses for which the IC(50)s were above the 95% confidence limits and several control isolates for which the IC(50)s were in the normal range revealed variations in some previously conserved residues, including D151, A203, T225, and E375 (N2 numbering). Known resistance mutations are both influenza virus subtype and drug specific, but there was no evidence of naturally occurring resistance to either drug in any of the isolates.

Evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network.

The increasing use of influenza virus neuraminidase (NA) inhibitors (NIs) necessitates the development of reliable methods for assessing the NI susceptibility of clinical isolates. We evaluated three NA inhibition assays against a panel of five clinical isolates each of influenza virus A/H1N1, A/H3N2, and B strains and four viruses with a defined resistance genotype (R292K, H274Y, R152K, and E119V). For fluorometric enzyme assay (FA) 1 (FA-1), 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUNANA) at 100 microM was used as the substrate, with pretitration of the virus input. For FA-2, MUNANA at 200 microM was used as the substrate, with a fixed 1:10 dilution of input virus. For the chemiluminescence (CL) assay, the 1,2-dioxetane derivative of sialic acid at 100 microM was used as the substrate, with pretitration of the virus. Four different operators repeated the assays several times in a blinded fashion with both zanamivir and oseltamivir carboxylate (GS4071) to determine intra- and interassay variations. Mean 50% inhibitory concentration (IC(50)) values were lower and generally less variable with the CL assay. FA-1 displayed greater variation than the CL assay or FA-2 and the highest IC(50) values with zanamivir; FA-2 showed the highest values with oseltamivir, particularly for influenza virus B, and was more variable with zanamivir than was the CL assay. All three assays detected 40-fold or greater changes in IC(50) values for the resistant viruses with at least one drug. Mixing experiments, whereby increasing fractions (0, 20, 40, 60, 80, and 100%) of NA from a known NI-resistant virus were mixed with the corresponding NI-sensitive parental NA, indicated that the resolution of IC(50) values was clearer with the CL assay than with FA-2 for two of the resistant variants (R152K and E119V). The FA and CL methods were reliable for the detection of NI resistance, but all assays have certain limitations. Based on reproducibility, ease of automation, time required for the assay, and greater sensitivity, the CL assay was selected for future susceptibility testing of influenza virus isolates circulating globally.

Antiviral activity and structural characteristics of the nonglycosylated central subdomain of human respiratory syncytial virus attachment (G) glycoprotein.

Segments of the cystine noose-containing nonglycosylated central subdomain, residues 149-197, of the attachment (G) glycoprotein of human respiratory syncytial virus (HRSV) have been assessed for impact on the cytopathic effect (CPE) of respiratory syncytial virus (RSV). Nalpha-acetyl residues 149-197-amide (G149-197), G149-189, and G149-177 of the A2 strain of HRSV protected 50% of human epithelial HEp-2 cells from the CPE of the A2 strain at concentrations (IC(50)) between 5 and 80 microm. Cystine noose-containing peptides G171-197 and G173-197 did not inhibit the CPE even at concentrations above 150 microm. Systematic C- and N-terminal truncations from G149-189 and G149-177 and alanine substitutions within G154-177 demonstrated that residues 166-170 (EVFNF), within a sequence that is conserved in HRSV strains, were critical for inhibition. Concordantly, G154-177 of bovine RSV and of an antibody escape mutant of HRSV with residues 166-170 of QTLPY and EVSNP, respectively, were not inhibitory. Surprisingly, a variant of G154-177 with an E166A substitution had an IC(50) of 750 nm. NMR analysis demonstrated that G149-177 adopted a well-defined conformation in solution, clustered around F168 and F170. G154-170, particularly EVFNF, may be important in binding of RSV to host cells. These findings constitute a promising platform for the development of antiviral agents for RSV.

The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion.

BACKGROUND: Membrane fusion within the Paramyxoviridae family of viruses is mediated by a surface glycoprotein termed the "F", or fusion, protein. Membrane fusion is assumed to involve a series of structural transitions of F from a metastable (prefusion) state to a highly stable (postfusion) state. No detail is available at the atomic level regarding the metastable form of these proteins or regarding the transitions accompanying fusion. RESULTS: The three-dimensional structure of the fusion protein of Newcastle disease virus (NDV-F) has been determined. The trimeric NDV-F molecule is organized into head, neck, and stalk regions. The head is comprised of a highly twisted beta domain and an additional immunoglobulin-like beta domain. The neck is formed by the C-terminal extension of the heptad repeat region HR-A, capped by a four-helical bundle. The C terminus of HR-A is encased by a further helix HR-C and a 4-stranded beta sheet. The stalk is formed by the remaining visible portion of HR-A and by polypeptide immediately N-terminal to the C-terminal heptad repeat region HR-B. An axial channel extends through the head and neck and is fenestrated by three large radial channels located approximately at the head-neck interface. CONCLUSION: We propose that prior to fusion activation, the hydrophobic fusion peptides in NDV-F are sequestered within the radial channels within the head, with the central HR-A coiled coil being only partly formed. Fusion activation then involves, inter alia, the assembly of a complete HR-A coiled coil, with the fusion peptides and transmembrane anchors being brought into close proximity. The structure of NDV-F is fundamentally different than that of influenza virus hemagglutinin, in that the central coiled coil is in the opposite orientation with respect to the viral membrane.

VP-14637 ViroPharma.

VP-14637 is the lead compound in a series of low molecular weight viral replication inhibitors which are under preclinical investigation by ViroPharma for the potential treatment of RSV infection [322651]. Phase I trials designed to evaluate the safety and pharmacokinetic prpfile of VR-14637 in healthy human volunteers have begun [385870]. VP-14637 is most active against pneumoviruses and the available data suggest that it is an inhibitor of RSV viral fusion activity [363716], [361097].

Biochemical Methods for the Characterization of Influenza Viruses with Reduced Sensitivity to 4-Guanidino-Neu5Ac2en.

Viruses that are less sensitive to the influenza neuraminidase (NA)-specific inhibitor 4-guanidino-Neu5Ac2en (zanamavir) (1) can be isolated after several passages in MDCK cells in the presence of the inhibitor. Although there are three reports of a mutation in the NA gene at the same conserved site, glu119 (2-4), most of the variants have mutations in the hemagglutinin (HA) gene (5). Many of these mutations appear to lower the affinity of the HA for the cellular receptor, so there is less requirement for significant NA activity for the newly synthesized progeny virus to elute. In this chapter we describe noncell culture-based methods for characterization of both HA and NA variants.

Virological Methods for the Generation and Characterization of Influenza Viruses with Reduced Sensitivity to 4-Guanidino-Neu5Ac2en.

The compound 4-guanidino-Neu5Ac2en (zanamivir) has been described as a selective inhibitor of the influenza virus neuraminidase (NA) (1). Viruses that are less sensitive to this inhibitor can be isolated after several passages in MDCK cells in the presence of the inhibitor. Variants isolated so far have had mutations predominantly in the hemagglutinin (HA) gene (2). Many of these mutations appear to lower the affinity of the HA for the cellular receptor, so that there is less requirement for significant NA activity for the newly synthesized progeny virus to elute. There are three reports of a mutation in the NA gene, all at the same conserved site, glu 119 (3-5). In this chapter, the authors describe methods for the isolation of the mutants, and for their characterization in cell culture based assays.

Substrate, inhibitor, or antibody stabilizes the Glu 119 Gly mutant influenza virus neuraminidase.

We have previously reported the isolation and characterization of an influenza virus variant with decreased sensitivity to the neuraminidase-specific inhibitor zanamivir. This variant, which has a mutation in the active site, Glu 119 Gly (E119G), has the same specific activity as the wild-type neuraminidase (NA), but is inherently unstable, as measured by loss of both enzyme activity and NC10 monoclonal antibody reactivity. However, despite the instability of the NA, replication of the virus in liquid culture is not adversely affected. We demonstrate here that in addition to enhanced temperature sensitivity the mutant NA was significantly more sensitive to formaldehyde and to specimen preparation for electron microscopy. Substrate, inhibitor, or monoclonal antibodies stabilized the NA against all methods of denaturation. These results suggest that the instability of the variant is primarily at the level of polypeptide chain folding rather than at the level of association of monomers into tetramers. Furthermore the presence of high levels of substrate, either cell or virus associated, may be sufficient to stabilize the NA during virus replication.

The interaction of neuraminidase and hemagglutinin mutations in influenza virus in resistance to 4-guanidino-Neu5Ac2en.

We have previously described a 4-guanidino-Neu5Ac2en (zanamivir)-resistant neuraminidase (NA) variant G70C4-G, with an active site mutation Glu 119 to Gly. This variant has been found to also harbor a hemagglutinin (HA) mutation in the receptor binding site, Ser 186 to Phe. Examination of early passages of the G70C4-G virus revealed that this HA mutation had arisen by the first passage. From a subsequent passage two transient variants were isolated which had each acquired a different second HA mutation, Ser 165 to Asn and Lys 222 to Thr. Both were highly drug resistant and drug dependent and their ability to adsorb to and penetrate cells was decreased. Comparison of drug sensitivities between the variant, with the additional HA mutation at Ser 165, and viruses with either mutation alone revealed that these two HA mutations acted synergistically to increase resistance. To determine the contribution to resistance of each of the NA and HA mutations in G70C4-G, the NA mutation was separated from the HA mutation by reassorting. The NA mutation and the HA mutation each conferred low-level resistance to zanamivir, while the two mutations interacted synergistically in the double mutant to give higher resistance in vitro. Infectivity was not adversely affected in the double mutant and while there was a small decrease in sensitivity to zanamivir in the mouse model, there was no detectable resistance to zanamivir in the ferret model.

Drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase.

BACKGROUND: Inhibitors of the influenza virus neuraminidase have been shown to be effective antiviral agents in humans. Several studies have reported the selection of novel influenza strains when the virus is cultured with neuraminidase inhibitors in vitro. These resistant viruses have mutations either in the neuraminidase or in the viral haemagglutinin. Inhibitors in which the glycerol sidechain at position 6 of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) has been replaced by carboxamide-linked hydrophobic substituents have recently been reported and shown to select neuraminidase variants. This study seeks to clarify the structural and functional consequences of replacing the glycerol sidechain of the inhibitor with other chemical constituents. RESULTS: The neuraminidase variant Arg292-->Lys is modified in one of three arginine residues that encircle the carboxylate group of the substrate. The structure of this variant in complex with the carboxamide inhibitor used for its selection, and with other Neu5Ac2en analogues, is reported here at high resolution. The structural consequences of the mutation correlate with altered inhibitory activity of the compounds compared with wild-type neuraminidase. CONCLUSIONS: The Arg292-->Lys variant of influenza neuraminidase affects the binding of substrate by modification of the interaction with the substrate carboxylate. This may be one of the structural correlates of the reduced enzyme activity of the variant. Inhibitors that have replacements for the glycerol at position 6 are further affected in the Arg292-->Lys variant because of structural changes in the binding site that apparently raise the energy barrier for the conformational change in the enzyme required to accommodate such inhibitors. These results provide evidence that a general strategy for drug design when the target has a high mutation frequency is to design the inhibitor to be as closely related as possible to the natural ligands of the target.

Mutations in a conserved residue in the influenza virus neuraminidase active site decreases sensitivity to Neu5Ac2en-derived inhibitors.

The influenza virus neuraminidase (NA)-specific inhibitor zanamivir (4-guanidino-Neu5Ac2en) is effective in humans when administered topically within the respiratory tract. The search for compounds with altered pharmacological properties has led to the identification of a novel series of influenza virus NA inhibitors in which the triol group of zanamivir has been replaced by a hydrophobic group linked by a carboxamide at the 6 position (6-carboxamide). NWS/G70C variants generated in vitro, with decreased sensitivity to 6-carboxamide, contained hemagglutinin (HA) and/or NA mutations. HA mutants bound with a decreased efficiency to the cellular receptor and were cross-resistant to all the NA inhibitors tested. The NA mutation, an Arg-to-Lys mutation, was in a previously conserved site, Arg292, which forms part of a triarginyl cluster in the catalytic site. In enzyme assays, the NA was equally resistant to zanamivir and 4-amino-Neu5Ac2en but showed greater resistance to 6-carboxamide and was most resistant to a new carbocyclic NA inhibitor, GS4071, which also has a hydrophobic side chain at the 6 position. Consistent with enzyme assays, the lowest resistance in cell culture was seen to zanamivir, more resistance was seen to 6-carboxamide, and the greatest resistance was seen to GS4071. Substrate binding and enzyme activity were also decreased in the mutant, and consequently, virus replication in both plaque assays and liquid culture was compromised. Altered binding of the hydrophobic side chain at the 6 position or the triol group could account for the decreased binding of both the NA inhibitors and substrate.

Use of oligonucleotide probes for selecting potential high-yielding influenza reassortants.

A new method for rapid screening of high-yielding reassortants of influenza virus, as candidates for vaccine production, is described. Oligonucleotide probes specific for all the parent genes of A/PR/8/34 (PR8), except the HA and the NA were designed based on database information available for different influenza strains. Digoxigenin labelled probes were tested by slot-blot hybridizations to purified RNA from a panel of A/PR/8/34 wild type and A/PR/8/34 reassortant viruses. The results show that the vast majority of reassortants selected for their high growth yield had acquired the non-structural (NS), matrix (M) and RNA polymerase 2 (PB2) genes from the PR8 parent. It is proposed that probes for these genes provide the potential for a simple and rapid procedure for selection of candidate high-yield reassortants for vaccine production.

Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases.

The x-ray structure of a complex of sialic acid (Neu5Ac) with neuraminidase N9 subtype from A/tern/Australia/G70C/75 influenza virus at 4 degrees C has revealed the location of a second Neu5Ac binding site on the surface of the enzyme. At 18 degrees C, only the enzyme active site contains bound Neu5Ac. Neu5Ac binds in the second site in the chair conformation in a similar way to which it binds to hemagglutinin. The residues that interact with Neu5Ac at this second site are mostly conserved in avian strains, but not in human and swine strains, indicating that it has some as-yet-unknown biological function in birds.

Mutation in the influenza virus neuraminidase gene resulting in decreased sensitivity to the neuraminidase inhibitor 4-guanidino-Neu5Ac2en leads to instability of the enzyme.

We previously isolated a variant of the influenza virus NWS/G70C, with a decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en in vitro, which has a mutation in one of the conserved residues of the neuraminidase Glu 119 to Gly. Despite the mutation, purified neuraminidase demonstrated the same specific activity as the parent neuraminidase. In contrast, characterization of a similar mutant by another group revealed a low specific activity of the enzyme. We confirm here that the specific activity of our variant is the same as that of the parent, but report that this mutation makes the enzyme inherently unstable, at high and low temperatures, either on the virion or as purified neuraminidase. Thus, for a valid determination of specific activity the concentration of native NA needs to be determined at the time of enzyme assay. Structurally, the instability may be partially explained by the introduction of a side chain (Gly), which carries a greater entropy penalty in condensation of the structure from the unfolded to the folded state and this, together with the loss of stabilizing interaction between Glu 119 and its neighbors in the active site, is not compensated for by the water molecule occupying the position of the carboxylate group (6).

Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en.

The compounds 4-amino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-amino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid) and 4-guanidino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-guanidino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid), which selectively inhibit the influenza virus neuraminidase, have been tested in vitro for their ability to generate drug-resistant variants. NWS/G70C virus (H1N9) was cultured in each drug by limiting-dilution passaging. After five or six passages in either compound, there emerged viruses which had a reduced sensitivity to the inhibitors in cell culture. Variant viruses were up to 1,000-fold less sensitive in plaque assays, liquid culture, and a hemagglutination-elution assay. In addition, cross-resistance to both compounds was seen in all three assays. Some isolates demonstrated drug dependence with an increase in both size and number of plaques in a plaque assay and an increase in virus yield in liquid culture in the presence of inhibitors. No significant difference in neuraminidase enzyme activity was detected in vitro, and no sequence changes in the conserved sites of the neuraminidase were found. However, changes in conserved amino acids in the hemagglutinin were detected. These amino acids were associated with either the hemagglutinin receptor binding site, Thr-155, or the left edge of the receptor binding pocket, Val-223 and Arg-229. Hence, mutations at these sites could be expected to affect the affinity or specificity of the hemagglutinin binding. Compensating mutations resulting in a weakly binding hemagglutinin thus seem to be circumventing the inhibition of the neuraminidase by allowing the virus to be released from cells with less dependence on the neuraminidase.

Generation and characterization of an influenza virus neuraminidase variant with decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en.

A variant of the influenza virus NWS/G70C has been generated which has decreased sensitivity in vitro to the neuraminidase-specific inhibitor, 4-guanidino-Neu5Ac2en. The virus is 1000-fold less sensitive to the 4-guanidino-Neu5Ac2en in a plaque assay, but only 10-fold less sensitive to 4-amino-Neu5Ac2en. In an enzyme inhibition assay 250-fold more drug was needed to achieve inhibition comparable to that observed with the parent virus. In contrast to the plaque assay, the virus was fully sensitive to 4-amino-Neu5Ac2en in the enzyme inhibition assay. Kinetic analysis of 4-guanidino-Neu5Ac2en binding demonstrated that the variant no longer exhibited the slow binding characteristic seen with the parent and other influenza viruses and inhibition by Neu5Ac2en was also decreased. However, binding to 4-amino-Neu5Ac2en remained the same as the parent. Sequence analysis of this virus revealed a mutation at a previously conserved site in the enzyme active site of the neuraminidase, Glu 119 to Gly. Crystallographic analysis of the mutant neuraminidase with and without bound inhibitor confirmed this mutation and suggested that the reduced affinity for the 4-guanidino-Neu5Ac2en derives partly from the loss of a stabilizing interaction between the guanidino moiety and the carboxylate at residue 119, and partly from alterations to the solvent structure of the active site.

Changes in the NS gene of neurovirulent strains of influenza affect splicing.

The nonstructural (NS) gene has been identified as an accessory factor in determining neurovirulence for influenza A virus. The nucleotide sequence of the NS gene of the neurovirulent variant A/NWS/33 was determined and compared with its parent, A/WS/33. Alterations in the mRNA structure of the gene were observed that serve to mask the 3' splice site. Changes in this region were shown to correlate with reduced splicing of the NS gene in neurovirulent strains.

The structure of a complex between the NC10 antibody and influenza virus neuraminidase and comparison with the overlapping binding site of the NC41 antibody.

BACKGROUND: While it is well known that different antibodies can be produced against a particular antigen, and even against a particular site on an antigen, up until now there have been no structural studies of cross-reacting antibodies of this type. One antibody-antigen complex whose structure is known is that of the influenza virus antigen, neuraminidase, in complex with the NC41 antibody. Another anti-neuraminidase antibody, NC10, binds to an overlapping site on the antigen. The structure of the complex formed by this antibody with neuraminidase is described here and compared with the NC41-containing complex. RESULTS: The crystal structure of the NC10 Fab-neuraminidase complex has been refined to a nominal resolution of 2.5A. Approximately 80% of the binding site of the NC10 antibody on neuraminidase overlaps with that of the NC41 antibody. The epitope residues of neuraminidase are often engaged in quite different interactions with the two antibodies. Although the NC10 and NC41 antibodies have identical amino acid sequences within the first complementarity determining region of their heavy chains, this is not the basis of the cross-reaction. CONCLUSIONS: The capacity of two different proteins to bind to the same target structure on a third protein need not be based on the existence of identical or homologous amino acid sequences within those proteins. As we have demonstrated, amino acid residues on the common target structure may be in quite different chemical environments, and may also adopt different conformations within two protein-protein complexes.