Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence.

Twelve monoclonal antibodies neutralizing the CVS strain of rabies virus were used to characterize antigenic site II of the viral glycoprotein. Nineteen antigenic mutants resistant to neutralization by some of these antibodies were selected; some continued to normally or partially bind the antibody, whereas others did not. Mutations conferring resistance to neutralization by site II-specific monoclonal antibodies were localized into two clusters, the first between amino acids 34 and 42 (seven groups of mutants) and the second at amino acids 198 and 200 (three groups of mutants). Two intermediate mutations were identified at positions 147 and 184. Four mutations resulted in reduced pathogenicity after intramuscular inoculation of the virus in adult mice. One of the mutants, M23, was 300 times and the others were 10 to 30 times less pathogenic than CVS. In three cases the attenuated phenotype was related to an important modification of antigenic site II, whereas the other known antigenic sites were unchanged.     

An Avirulent Mutant of Rabies Virus Is Unable To Infect Motoneurons In Vivo and In Vitro

An antigenic double mutant of rabies virus (challenge virus standard [CVS] strain) was selected by successive use of two neutralizing antiglycoprotein monoclonal antibodies, both specific for antigenic site III. This mutant differed from the original virus strain by two amino acid substitutions in the ectodomain of the glycoprotein. The lysine in position 330 and the arginine in position 333 were replaced by asparagine and methionine, respectively. This double mutant was not pathogenic for adult mice. When injected intramuscularly into the forelimbs of adult mice, this virus could not penetrate the nervous system, either by the motor or by the sensory route, while respective single mutants infected motoneurons in the spinal cord and sensory neurons in the dorsal root ganglia. In vitro experiments showed that the double mutant was able to infect BHK cells, neuroblastoma cells, and freshly prepared embryonic motoneurons, albeit with a lower efficiency than the CVS strain. Upon further incubation at 37°C, the motoneurons became resistant to infection by the mutant while remaining permissive to CVS infection. These results suggest that rabies virus uses different types of receptors: a molecule which is ubiquitously expressed at the surface of continuous cell lines and which is recognized by both CVS and the double mutant and a neuron-specific molecule which is not recognized by the double mutant.     

Extensive Attenuation of Rabies Virus by Simultaneously Modifying the Dynein Light Chain Binding Site in the P Protein and Replacing Arg333 in the G Protein

Rabies virus (RV) is a highly neurotropic virus that migrates from the portal of entry to the central nervous system (CNS). The cytoplasmic dynein light chain (LC8), which is involved in a variety of intracellular motile events, was shown to interact with RV phosphoprotein (P). In order to determine the functional significance of this interaction, P residues 143 to 149 or 139 to 149 encompassing a conserved LC8-interacting motif (K/RXTQT) were deleted from recombinant viruses SAD-L16 and SAD-D29. These viruses are identical except for a replacement of the arginine at position 333 (R333) of the RV glycoprotein by an aspartic acid in SAD-D29. SAD-L16 virus is fully pathogenic for mice, whereas SAD-D29 is nonpathogenic for adult mice but retained pathogenicity for suckling mice. The deletions introduced into the LC8 binding site abolished the P-LC8 interaction and blocked LC8 incorporation into virions. All the mutants propagated in cell culture as efficiently as the parent strains. The pathogenicity of the mutants was then compared with that of the parent viruses by inoculating suckling mice. SAD-L16 derivatives were as pathogenic as their parent virus after intramuscular inoculation, indicating that LC8 is dispensable for the spread of a pathogenic RV from a peripheral site to the CNS. In contrast, SAD-D29-derived deletion mutants were attenuated by as much as 30-fold after intramuscular inoculation but remained as pathogenic as the parent virus when inoculated directly into the brain. This remarkable attenuation after intramuscular but not after intracranial inoculation suggested that abolishing the P-LC8 interaction reduces the efficiency of peripheral spread of the more attenuated SAD-D29 strain. These results demonstrate that elimination of the LC8 ligand and simultaneous substitution of R333 considerably attenuate RV pathogenicity and may be helpful in designing and developing highly safe live-RV-based vaccines.     

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

Mapping of the low pH-sensitive conformational epitope of rabies virus glycoprotein recognized by a monoclonal antibody #1-30-44

Monoclonal antibody (mAb) #1-30-44 recognized an acid-sensitive conformational epitope of rabies virus glycoprotein (G). The antigenicity of G protein exposed on the cell surface was lost when the infected cells were exposed to pH 5.8. By comparing the deduced amino acid sequence of G protein between the HEP-Flury strain and the epitope-negative CVS strain as well as the mAb-resistant escape mutants, two distant sites that contained Lys-202 and Asn-336 were shown to be involved in the epitope formation. Lys-202 is located in the so-called neurotoxin-like sequence, while Asn-336 is included in antigenic site III and is very near the amino acid at position 333, which is known to affect greatly the neuropathogenicity of rabies virus when changed. Consistent with this finding, antigenicity of a neurovirulent revertant of the HEP-Flury strain, in which Gln-333 of G protein was replaced by Arg, was also affected as shown by its greatly decreased reactivity with mAb #1-30-44 compared to that of the original avirulent HEP virus. Based on these results, we hypothesize that the neurotoxin-like domain and some amino acids in antigenic site III come into contact with each other to form a conformational epitope for mAb #1-30-44, and such a configuration would be lost when exposed to acidic conditions to perform a certain low pH-dependent function of G protein.     

Neutralization epitopes on rotavirus SA11 4fM outer capsid proteins.

The VP7 and VP4 genes of seven antigenic mutants of simian rotavirus SA11 4fM (serotype 3) selected after 39 passages in the presence of SA11 4fM hyperimmune antiserum, were sequenced. Nucleotide sequence analysis indicated the following. (i) Twice as many amino acid substitutions occurred in the VP7 protein than in VP4, which has a molecular weight twice that of VP7. (ii) Most amino acid changes that occurred clustered in six variable regions of VP7 and in two variable regions of VP4; these variable regions may represent immunodominant epitopes. (iii) Most amino acid substitutions that occurred in VP7 and VP4 of these mutants were also observed in antigenic mutants selected with neutralizing monoclonal antibodies (NMAbs); however, some amino acid substitutions occurred that were not selected for NMAbs. (iv) On VP7, some of the neutralization epitopes appeared to be interrelated because amino acid substitution in one site affected binding of specific NMAbs to other sites, while other neutralization epitopes on VP7 appeared to be independent, in that amino acid substitution in one site did not affect the binding of NMAbs to another distant site.     

A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity

Several rabies virus (RV) vaccine strains containing an aspartic acid (Asp) or glutamic acid (Glu) instead of an arginine (Arg) at position 333 of the RV glycoprotein (G) are apathogenic for immunocompetent mice even after intracranial inoculation. However, we previously showed that the nonpathogenic phenotype of the highly attenuated RV strain SPBNGA, which contains a Glu at position 333 of G, is unstable when this virus is passaged in newborn mice. While the Glu(333) remained unchanged after five mouse passages, an Asn(194)-->Lys(194) mutation occurred in RV G. This mutation was associated with increased pathogenicity for adult mice. Using site-directed mutagenesis to exchange Asn(194) with Lys(194) in the G protein of SPBNGA, resulting in SPBNGA-K, we show here that this mutation is solely responsible for the increase in pathogenicity and that the Asn(194)-->Lys(194) mutation does not arise when Asn(194) is exchanged with Ser(194) (SPBNGA-S). Our data presented indicate that the increased pathogenicity of SPBNGA-K is due to increased viral spread in vivo and in vitro, faster internalization of the pathogenic virus into cells, and a shift in the pH threshold for membrane fusion. These results are consistent with the notion that the RV G protein is a major contributor to RV pathogenesis and that the more pathogenic RVs escape the host responses by a faster spread than that of less pathogenic RVs.     

Dominance of a nonpathogenic glycoprotein gene over a pathogenic glycoprotein gene in rabies virus

The nonpathogenic phenotype of the live rabies virus (RV) vaccine SPBNGAN is determined by an Arg-->Glu exchange at position 333 in the glycoprotein, designated GAN. We recently showed that after several passages of SPBNGAN in mice, an Asn-->Lys mutation arose at position 194 of GAN, resulting in GAK, which was associated with a reversion to the pathogenic phenotype. Because an RV vaccine candidate containing two GAN genes (SPBNGAN-GAN) exhibits increased immunogenicity in vivo compared to the single-GAN construct, we tested whether the presence of two GAN genes might also enhance the probability of reversion to pathogenicity. Comparison of SPBNGAN-GAN with RVs constructed to contain either both GAN and GAK genes (SPBNGAN-GAK and SPBNGAK-GAN) or two GAK genes (SPBNGAK-GAK) showed that while SPBNGAK-GAK was pathogenic, SPBNGAN-GAN and SPBNGAN-GAK were completely nonpathogenic and SPBNGAK-GAN showed strongly reduced pathogenicity. Analysis of genomic RV RNA in mouse brain tissue revealed significantly lower virus loads in SPBNGAN-GAK- and SPBNGAK-GAN-infected brains than those detected in SPBNGAK-GAK-infected brains, indicating the dominance of the nonpathogenic phenotype determined by GAN over the GAK-associated pathogenic phenotype. Virus production and viral RNA synthesis were markedly higher in SPBNGAN-, SPBNGAK-GAN-, and SPBNGAN-GAK-infected neuroblastoma cells than in the SPBNGAK- and SPBNGAK-GAK-infected counterparts, suggesting control of GAN dominance at the level of viral RNA synthesis. These data point to the lower risk of reversion to pathogenicity of a recombinant RV carrying two identical GAN genes compared to that of an RV carrying only a single GAN gene.     

Epitope mapping of the hemagglutinin molecule of a highly pathogenic H5N1 influenza virus by using monoclonal antibodies

We mapped the hemagglutinin (HA) antigenic epitopes of a highly pathogenic H5N1 influenza virus on the three-dimensional HA structure by characterizing escape mutants of a recombinant virus containing A/Vietnam/1203/04 (H5N1) ΔHA and neuraminidase genes in the genetic background of A/Puerto Rico/8/34 (H1N1) virus. The mutants were selected with a panel of eight anti-HA monoclonal antibodies (MAbs), seven to A/Vietnam/1203/04 (H5N1) virus and one to A/Chicken/Pennsylvania/8125/83 (H5N2) virus, and the mutants’ HA genes were sequenced. The amino acid changes suggested three MAb groups: four MAbs reacted with the complex epitope comprising parts of the antigenic site B of H3 HA and site Sa of H1 HA, two MAbs reacted with the epitope corresponding to the antigenic site A in H3 HA, and two MAbs displayed unusual behavior: each recognized amino acid changes at two widely separate antigenic sites. Five changes were detected in amino acid residues not previously reported as changed in H5 escape mutants, and four others had substitutions not previously described. The HA antigenic structure differs substantially between A/Vietnam/1203/04 (H5N1) virus and the low-pathogenic A/Mallard/Pennsylvania/10218/84 (H5N2) virus we previously characterized (N. V. Kaverin et al., J. Gen. Virol. 83:2497-2505, 2002). The hemagglutination inhibition reactions of the MAbs with recent highly pathogenic H5N1 viruses were consistent with the antigenic-site amino acid changes but not with clades and subclades based on H5 phylogenetic analysis. These results provide information on the recognition sites of the MAbs widely used to study H5N1 viruses and demonstrate the involvement of the HA antigenic sites in the evolution of highly pathogenic H5N1 viruses, findings that can be critical for characterizing pathogenesis and vaccine design.     

Cross-reactive and serotype-specific neutralization epitopes on VP7 of human rotavirus: nucleotide sequence analysis of antigenic mutants selected with monoclonal antibodies.

The neutralization epitopes of human and simian rotavirus protein VP7 were studied by producing six neutralizing monoclonal antibodies (N-MAbs) and using these N-MAbs to select antigenic mutants that resisted neutralization by the N-MAbs used for their selection. Cross-neutralization tests between the N-MAbs and the antibody-selected antigenic mutants identified one cross-reactive and five distinct serotype-specific neutralization epitopes which operationally overlapped one another and constituted a single antigenic site. In addition, the amino acid substitutions in human rotavirus VP7 that are responsible for the antigenic alterations in the mutants selected with anti-VP7 cross-reactive or serotype-specific N-MAbs were identified. All the amino acid substitutions in the antigenic mutants occurred in one of two variable regions: amino acids 87 to 101 and 208 to 221.     

Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity

The genetic diversity of representative members of the Lyssavirus genus (rabies and rabies-related viruses) was evaluated using the gene encoding the transmembrane glycoprotein involved in the virus-host interaction, immunogenicity, and pathogenicity. Phylogenetic analysis distinguished seven genotypes, which could be divided into two major phylogroups having the highest bootstrap values. Phylogroup I comprises the worldwide genotype 1 (classic Rabies virus), the European bat lyssavirus (EBL) genotypes 5 (EBL1) and 6 (EBL2), the African genotype 4 (Duvenhage virus), and the Australian bat lyssavirus genotype 7. Phylogroup II comprises the divergent African genotypes 2 (Lagos bat virus) and 3 (Mokola virus). We studied immunogenic and pathogenic properties to investigate the biological significance of this phylogenetic grouping. Viruses from phylogroup I (Rabies virus and EBL1) were found to be pathogenic for mice when injected by the intracerebral or the intramuscular route, whereas viruses from phylogroup II (Mokola and Lagos bat viruses) were only pathogenic by the intracerebral route. We showed that the glycoprotein R333 residue essential for virulence was naturally replaced by a D333 in the phylogroup II viruses, likely resulting in their attenuated pathogenicity. Moreover, cross-neutralization distinguished the same phylogroups. Within each phylogroup, the amino acid sequence of the glycoprotein ectodomain was at least 74% identical, and antiglycoprotein virus-neutralizing antibodies displayed cross-neutralization. Between phylogroups, the identity was less than 64.5% and the cross-neutralization was absent, explaining why the classical rabies vaccines (phylogroup I) cannot protect against lyssaviruses from phylogroup II. Our tree-axial analysis divided lyssaviruses into two phylogroups that more closely reflect their biological characteristics than previous serotypes and genotypes.     

Identification of mar mutations in herpes simplex virus type 1 glycoprotein B which alter antigenic structure and function in virus penetration.

Analysis of six monoclonal antibody-resistant (mar) mutants in herpes simplex virus type 1 glycoprotein B identified two type-common (II and III) and two type-specific (I and IV) antigenic sites on this molecule. To derive additional information on the location of these sites, mar mutations were mapped and nucleotide alterations were identified by DNA sequencing. Each mutant carried a single amino acid substitution resulting from a G-to-A base transition. Alterations affecting antibody neutralization were identified at residues 473, 594, 305, and 85 for mutants in sites I through IV, respectively. Two clonally distinct site II antibodies each selected mar mutants (Gly to Arg at residue 594) that exhibited a reduction in the rate of entry (roe) into host cells. A site II mar revertant that regained sensitivity to neutralization by site II antibodies also showed normal entry kinetics. DNA sequencing of this virus identified a single base reversion of the site II mar mutation, resulting in restoration of the wild-type sequence (Arg to Gly). This finding demonstrated that the mar and roe phenotypes were the result of a single mutation. To further define structures that contributed to antibody recognition, monoclonal antibodies specific for all four sites were tested for their ability to immune precipitate a panel of linker-insertion mutant glycoprotein B molecules. Individual polypeptides that contained single insertions of 2 to 28 amino acids throughout the external domain were not recognized or were recognized poorly by antibodies specific for sites II and III, whereas no insertion affected antibody recognition of sites I and IV. mar mutations affecting either site II or III were previously shown to cause temperature-sensitive defects in glycoprotein B glycosylation, and variants altered in both these sites were temperature sensitive for virus production. Taken together, the data indicate that antigenic sites II and III are composed of higher-order structures whose integrity is linked with the ability of glycoprotein B to function in virus infectivity.     

Sindbis virus mutations which coordinately affect glycoprotein processing, penetration, and virulence in mice

Rapid penetration of baby hamster kidney cells was used as a selective pressure for the isolation of pathogenesis mutants of the S.A.AR86 strain of Sindbis virus. Unlike most Sindbis virus strains, S.A.AR86 is virulent in adult as well as neonatal mice. Two classes of mutants were defined. One class was attenuated in adult mice inoculated intracerebrally as well as in neonatal mice inoculated either intracerebrally or subcutaneously. Sequence analysis of the glycoprotein genes of the parent virus and three such mutant strains revealed a single point mutation which resulted in an amino acid change at position 1 in the E2 glycoprotein. The change from a serine in S.A.AR86 to an asparagine in the mutants created a new site for N-linked glycosylation which appeared to be utilized. This mutation did not retard release of infectious particles; however, mutant virions contained the E2 precursor protein (PE2) rather than the E2 glycoprotein itself. The mutants also lost the ability to bind two E2-specific monoclonal antibodies, R6 and R13. A second class of mutants was attenuated in neonatal mice upon subcutaneous inoculation but remained virulent in adults and in neonates when inoculated intracerebrally. Sequence analysis of three such strains revealed the substitution of an arginine residue for a serine at position 114 in the E2 glycoprotein. Reactivity with monoclonal antibodies R6 and R13 was reduced, yet members of this mutant class were more susceptible than S.A.AR86 to neutralization by these antibodies.     

Comparison of epitope structures of H3HAs through protein modeling of influenza A virus hemagglutinin: mechanism for selection of antigenic variants in the presence of a monoclonal antibody

Starting with nine plaques of influenza A/Kamata/14/91(H3N2) virus, we selected mutants in the presence of monoclonal antibody 203 (mAb203). In total, amino acid substitutions were found at nine positions (77, 80, 131, 135, 141, 142, 143, 144 and 146), which localized in the antigenic site A of the hemagglutinin (HA). The escape mutants differed in the extent to which they had lost binding to mAb203. HA protein with substitutions of some amino acid residues created by site-directed mutagenesis in the escape mutants retained the ability to bind to mAb203. Changes in the amino acid character affecting charge or hydrophobicity accounted for the binding capacity to the antibody of the HA with most of the substitutions in the escape mutants and binding-positive mutants. However, the effect of some amino acid substitutions remained unexplained. A three-dimensional model of the 1991 HA was constructed and used to analyze substituted amino acids in these mutants for the accessible surface hydrophobic and hydrophilic characters. One amino acid substitution in an escape mutant and another amino acid substitution in a binding-positive mutant seemed to be explained by the changes noted on this model.     

A single amino acid substitution in envelope protein E of tick-borne encephalitis virus leads to attenuation in the mouse model.

We have determined the virulence characteristics of seven monoclonal antibody escape mutants of tick-borne encephalitis virus in the mouse model. One of the mutants with an amino acid substitution from tyrosine to histidine at residue 384 revealed strongly reduced pathogenicity after peripheral inoculation of adult mice but retained its capacity to replicate in the mice and to induce a high-titered antibody response. Infection with the attenuated mutant resulted in resistance to challenge with virulent virus. Assessment of nonconservative amino acid substitutions in other attenuated flaviviruses suggests that a structural element including residue 384 may represent an important determinant of flavivirus virulence in general.     

Use of monoclonal antibodies to identify four neutralization immunogens on a common cold picornavirus, human rhinovirus 14.

A collection of 35 mouse monoclonal antibodies, raised against human rhinovirus 14 (HRV-14), was used to isolate 62 neutralization-resistant mutants. When cross-tested against the antibodies in a neutralization assay, the mutants fell into four antigenic groups, here called neutralization immunogens: NIm-IA, -IB, -II, and -III. Sequencing the mutant RNA in segments corresponding to serotype-variable regions revealed that the amino acid substitutions segregated into clusters, which correlated exactly with the immunogenic groups (NIm-IA mutants at VP1 amino acid residue 91 or 95; NIm-II mutants at VP2 residue 158, 159, 161, or 162; NIm-III mutants at VP3 residue 72, 75, or 78; and NIm-IB mutants at two sites, either VP1 residue 83 or 85, or residue 138 or 139). Examination of the three-dimensional structure of the virus (M. G. Rossmann, E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H.-J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, Nature [London], 317:145-153, 1985) revealed that each of the substitution clusters formed a protrusion from the virus surface, and the side chains of the substituted amino acids pointed outward. Moreover, four of the amino acid substitutions, which initially appeared to be anomalous because they were encoded well outside the cluster groups, could be traced to surface positions immediately adjacent to the appropriate viral protrusions. We conclude that three of the four antigens, NIm-IB, -II, and -III, are discontinuous. Thus, the amino acid substitutions in all 62 mutants fell within the proposed immunogenic sites; there was no evidence for alteration of any antigenic site by a distal mutation.     

Mutational analysis of the octapeptide sequence motif at the NS1-NS2A cleavage junction of dengue type 4 virus.

We have previously shown that proper processing of dengue type 4 virus NS1 from the NS1-NS2A region of the viral polyprotein requires a hydrophobic N-terminal signal and the downstream NS2A. Results from deletion analysis indicate that a minimum length of eight amino acids at the C terminus of NS1 is required for cleavage at the NS1-NS2A junction. Comparison of this eight-amino-acid sequence with the corresponding sequences of other flaviviruses suggests a consensus cleavage sequence of Met/Leu-Val-Xaa-Ser-Xaa-Val-Xaa-Ala. Site-directed mutagenesis was performed to construct mutants of NS1-NS2A that contained a single amino acid substitution at different positions of the consensus cleavage sequence or at the immediate downstream position. Three to eight different substitutions were made at each position. A total of 50 NS1-NS2A mutants were analyzed for their cleavage efficiency relative to that of the wild-type dengue type 4 virus sequence. As predicted, nearly all substitutions at positions P1, P3, P5, P7, and P8, occupied by conserved amino acids, yielded low levels of cleavage, with the exception that Pro or Ala substituting for Ser (P5) was tolerated. Substitutions of an amino acid at the remaining positions occupied by nonconserved amino acids generally yielded high levels of cleavage. However, some substitutions at nonconserved positions were not tolerated. For example, substitution of Gly or Glu for Gln (P4) and substitution of Val or Glu for Lys (P6) each yielded a low level of cleavage. Overall, these data support the proposed cleavage sequence motif deduced by comparison of sequences among the flaviviruses. This study also showed that in addition to the eight-amino-acid sequence, the amino acid immediately following the NS1-NS2A cleavage site plays a role in cleavage.     

In vitro mutagenesis of a mouse MHC class I gene for the examination of structure-function relationships

Oligonucleotide-directed, site-specific mutagenesis has been employed to elucidate the role of individual amino acids on the expression and function of a MHC class I antigen. Two oligonucleotides were synthesized to introduce single amino acid substitutions in the murine H-2Ld gene. The highly conserved glycosylation site at amino acid position 86 was changed from asparagine to lysine to remove the carbohydrate moiety from the first external domain of the H-2 molecule, and the phenylalanine at position 116 was changed to tyrosine, replacing the Ld residue with the Kb type amino acid analogous to Kb mutants: bm5 and bm16 mutants derived from the Kb antigen have the Ld-type residue at this position. The mutant genes were constructed by annealing the mutagenic oligomers to the single stranded H-2Ld gene, followed by chain elongation reaction. The expected mutations were confirmed by DNA sequence determination. The mutant genes were introduced into mouse L cells by DNA-mediated gene transfer. Both mutant genes expressed the antigens on the cell surface, as detected by antibody binding; these antigens were reactive with the cytotoxic T cells specific for the H-2Ld antigen. Detailed examination with 16 monoclonal anti-H-2Ld antibodies revealed that the binding of some antibodies was significantly reduced in the glycosylation mutant, implying a certain contribution of the carbohydrates to the antigenic activity of some determinants. No detectable changes have been observed in the mutant of the substitution at position 116 by the parameters we tested.     

Mutations conferring resistance to neutralization by a soluble form of the neurotrophin receptor (p75NTR) map outside of the known antigenic sites of the rabies virus glycoprotein

The neurotrophin receptor (p75NTR) serves as a receptor for rabies virus (RV). We expressed and purified a soluble chimera consisting of the p75NTR ectodomain fused to the human immunoglobulin G1 (IgG1) Fc fragment (p75-Fc). Although p75-Fc interacts with RV, the infectivity of RV did not decrease significantly when it was incubated in the presence of the soluble receptor alone. However, when it was subsequently incubated with an antihuman IgG directed against the Fc fragment of p75-Fc, the infectivity of RV was significantly lowered (>90%), whereas incubation with antihuman IgG alone had no effect. We then selected eight independent RV mutants that were not neutralized by p75-Fc and antihuman IgG (srr [soluble receptor resistant] mutants). Each mutant carried a single mutation in the glycoprotein gene leading to one amino acid substitution in the protein. A total of four different substitutions were found. Two of the mutations were located at position 318 (phenylalanine replaced by a serine or a valine residue), and two were located at position 352 (histidine replaced by a tyrosine or an arginine residue). All of the mutations prevented the interaction with p75NTR as either a soluble or a membrane-anchored form. Two mutants (F318S) and (H352R) resulted in the formation of small plaques on BSR cells, probably due to the slower maturation of the glycoprotein. Immunoprecipitation, immunofluorescence, and neutralization assays showed that the four mutated glycoproteins still interacted with representative anti-RV glycoprotein monoclonal antibodies (MAbs), indicating that p75NTR binds outside of the known RV glycoprotein antigenic sites.     

Antigenic structure of human hepatitis A virus defined by analysis of escape mutants selected against murine monoclonal antibodies.

We examined the antigenic structure of human hepatitis A virus (HAV) by characterizing a series of 21 murine monoclonal-antibody-resistant neutralization escape mutants derived from the HM175 virus strain. The escape phenotype of each mutant was associated with reduced antibody binding in radioimmunofocus assays. Neutralization escape mutations were identified at the Asp-70 and Gln-74 residues of the capsid protein VP3, as well as at Ser-102, Val-171, Ala-176, and Lys-221 of VP1. With the exception of the Lys-221 mutants, substantial cross-resistance was evident among escape mutants tested against a panel of 22 neutralizing monoclonal antibodies, suggesting that the involved residues contribute to epitopes composing a single antigenic site. As mutations at one or more of these residues conferred resistance to 20 of 22 murine antibodies, this site appears to be immunodominant in the mouse. However, multiple mutants selected independently against any one monoclonal antibody had mutations at only one or, at the most, two amino acid residues within the capsid proteins, confirming that there are multiple epitopes within this antigenic site and suggesting that single-amino-acid residues contributing to these epitopes may play key roles in the binding of individual antibodies. A second, potentially independent antigenic site was identified by three escape mutants with different substitutions at Lys-221 of VP1. These mutants were resistant only to antibody H7C27, while H7C27 effectively neutralized all other escape mutants. These data support the existence of an immunodominant neutralization site in the antigenic structure of hepatitis A virus which involves residues of VP3 and VP1 and a second, potentially independent site involving residue 221 of VP1.