Identification of a Conserved B-Cell Epitope on Duck Hepatitis A Type 1 Virus VP1 Protein

BackgroundThe VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.

Methods and Results

To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope ¹⁷³LPAPTS¹⁷⁸ is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.

Conclusions and Significance

We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.     

Investigation of substrate specificity of wildtype and mutant BphK (a glutathione S-transferase) from Burkholderia LB400

The bphK gene located in the bph operon of Burkholderia LB400 encodes a protein, BphK^LB400, with significant sequence similarity to glutathione-S-transferases (GST), a group of enzymes involved in the detoxification of many endobiotic and xenobiotic substances. Comparison of the amino acid sequence of BphK^LB400 with GST from other polychlorinated biphenyl (PCB)-degrading bacteria identified a number of highly conserved amino acids in the C-terminal region of the protein that may be associated with substrate specificity. In this study, two of these conserved amino acids in BphK^LB400 (amino acids 152 and 180) were selected for mutation, using site-directed mutagenesis, and substrate specificity assays. BphK^LB400 (wildtype and mutant) was over-expressed in Escherichia coli where the bphK gene (wildtype and mutant) is under the expression of a lac promoter and is induced by isopropyl thiogalactoside, and bacterial cell extracts were prepared for GST activity assays. Mutations at amino acids 152 and 180 were shown to affect GST activity of BphK^LB400 using 1-chloro-2,4-dinitrobenzene, the model substrate for GST activity assays; 4-chlorobenzoate and 3-chlorobenzoate, intermediates in the polychlorinated biphenyl (PCB) degradation pathway, and 2,4-dichlorophenoxyacetate and atrazine, commonly used herbicides; as substrates. A BphK^LB400 mutant (Ala180Pro) is identified in this study as having increased activity towards all substrates tested. This mutant may have potential in bioremediation.     

Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution

Antibodies raised against the synthetic peptide corresponding to the carboxy-terminal 24 amino acids (305-328) of the heavy chain of the hemagglutinin molecule of influenza virus A/X-31 (H3) bind this peptide at three antigenic sites. These sites were identified by assaying binding of polyclonal BALB/c mouse antipeptide sera to the complete set of all possible di-, tri, tetra-, penta-, hexa-, hepta-, and octapeptides homologous with the 24-residue sequence. Individual epitopes were defined and essential residues identified by testing the binding of monoclonal antibodies to sets of peptide analogues in which every one of the homologous residues was replaced in turn by each of the 19 alternative genetically coded amino acids. The immunodominant epitope was shown to be a linear sequence of five amino acids, 314LKLAT318. Replacement of any one of these residues with any other amino acid resulted in loss of antibody binding, indicating that all five are essential to the interaction and that they are probably contact residues. Another antigenic site contains at least two overlapping epitopes: polyclonal sera recognize predominantly an epitope or epitopes encompassed by the linear sequence 320MRNVPEKQT328, whereas the epitope defined by a particular monoclonal antibody comprises the seven amino acids 322NVPEKQT328, of which N322, E325, and Q327 were implicated as contact residues.     

Exquisite specificity and peptide epitope recognition promiscuity, properties shared by antibodies from sharks to humans

This review considers definitions of the specificity of antibodies including the development of recent concepts of recognition polyspecificity and epitope promiscuity. Using sets of homologous and unrelated peptides derived from the sequences of immunoglobulin and T cell receptor chains we offer operational definitions of cross-reactivity by investigating correlations of either identities in amino acid sequence, or in hydrophobicity/hydrophilicity profiles with degree of binding in enzyme-linked immunosorbent assays. Polyreactivity, or polyspecificity, are terms used to denote binding of a monoclonal antibody or purified antibody preparation to large complex molecules that are structurally unrelated, such as thyroglobulin and DNA. As a first approximation, there is a linear correlation between degree of sequence identity or hydrophobicity/hydrophilicity and antigenic cross-binding. However, catastrophic interchanges of amino acids can occur where changing of one amino acid out of 16 in a synthetic peptide essentially eliminates binding to certain antibodies. An operational definition of epitope promiscuity for peptides is the case where two peptides show little or no identity in amino acid sequence but bind strongly to the same antibody as shown by either direct binding or competitive inhibition. Analysis of antibodies of humans and sharks, the two most divergent species in evolution to express antibodies and the combinatorial immune response, indicates that the capacity for both exquisite specificity and epitope recognition promiscuity are essential conserved features of individual vertebrate antibodies.     

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues ⁵AIDITRK¹¹, ⁷²RDELNVL⁷⁸, ²⁵¹KSKHNRREGY²⁶⁰, ²⁶⁹DENGIVLD²⁷⁶, and ³⁴¹DETTLVRS³⁴⁸. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.     

Characterization of a Discontinuous Neutralizing Epitope on Glycoprotein B of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and newborn infants infected in utero. The viral envelope glycoprotein B (gB) is an attractive molecule for active vaccination and passive immunoprophylaxis and therapy. Using human monoclonal antibodies (MAbs), we have recently identified antigenic region 4 (AD-4) on gB as an important target for neutralizing antibodies. AD-4 is formed by a discontinuous sequence comprising amino acids 121 to 132 and 344 to 438 of gB of HCMV strain AD169. To map epitopes for human antibodies on this protein domain, we used a three-dimensional (3D) model of HCMV gB to identify surface-exposed amino acids on AD-4 and selected juxtaposed residues for alanine scans. A tyrosine (Y) at position 364 and a lysine (K) at position 379 (the YK epitope), which are immediate neighbors on the AD-4 surface, were found to be essential for binding of the human MAbs. Recognition of AD-4 by sera from HCMV-infected individuals also was largely dependent on these two residues, indicating a general importance for the antibody response against AD-4. A panel of AD-4 recombinant viruses harboring mutations at the crucial antibody binding sites was generated. The viruses showed significantly reduced susceptibility to neutralization by AD-4-specific MAbs or polyclonal AD-4-specific antibodies, indicating that the YK epitope is dominant for the AD-4-specific neutralizing antibody response during infection. To our knowledge, this is the first molecular identification of a functional discontinuous epitope on HCMV gB. Induction of antibodies specific for this epitope may be a desirable goal following vaccination with gB.     

The impact of the antibody 2F5 biotinylation on the selection of the peptides from combinatorial phage library

The impact of monoclonal antibodies (mAb) biotinylation on the output and the repertoire of selected peptides in the biopanning procedure were tested. A comparative analysis of the peptides selected from phage library using the biotinylated and non-biotinylated mAb 2F5 was performed. It was shown that the output of peptides homologous to the native epitope was 1.7-fold higher for biotinylated antibodies, whereas the binding capacity of the selected phages with mAb 2F5 in ELISA was higher in the case of using non-biotinylated antibodies. It should be noted that the phages exposing peptides, which have 4-5 amino acid sequence similarity with the native epitope, demonstrate the highest binding affinity. The phages that expose peptides with 3 amino acid sequence similarity demonstrate different binding affinity: from the smallest to the largest. Based on the obtained data, it is safe to suggest that the rational biopanning may proceed in accordance with the task.     

Investigation of substrate specificity of wildtype and mutant BphKLB400 (a glutathione S-transferase) from Burkholderia LB400

The bphK gene located in the bph operon of Burkholderia LB400 encodes a protein, BphK^LB400, with significant sequence similarity to glutathione-S-transferases (GST), a group of enzymes involved in the detoxification of many endobiotic and xenobiotic substances. Comparison of the amino acid sequence of BphK^LB400 with GST from other polychlorinated biphenyl (PCB)-degrading bacteria identified a number of highly conserved amino acids in the C-terminal region of the protein that may be associated with substrate specificity. In this study, two of these conserved amino acids in BphK^LB400 (amino acids 152 and 180) were selected for mutation, using site-directed mutagenesis, and substrate specificity assays. BphK^LB400 (wildtype and mutant) was over-expressed in Escherichia coli where the bphK gene (wildtype and mutant) is under the expression of a lac promoter and is induced by isopropyl thiogalactoside, and bacterial cell extracts were prepared for GST activity assays. Mutations at amino acids 152 and 180 were shown to affect GST activity of BphK^LB400 using 1-chloro-2,4-dinitrobenzene, the model substrate for GST activity assays; 4-chlorobenzoate and 3-chlorobenzoate, intermediates in the polychlorinated biphenyl (PCB) degradation pathway, and 2,4-dichlorophenoxyacetate and atrazine, commonly used herbicides; as substrates. A BphK^LB400 mutant (Ala180Pro) is identified in this study as having increased activity towards all substrates tested. This mutant may have potential in bioremediation.     

Molecular cloning and characterization of a copper chaperone for copper/zinc superoxide dismutase from the rat

Copper chaperone is an essential cytosolic factor that maintains copper homeostasis in living cells. Cytosolic metallochaperones have been recently identified in plant, yeast, rodents, and human cells. During our investigation, we found a new member of the copper chaperone family for copper/zinc superoxide dismutase, which was cloned from rats. The new copper chaperone was named rCCS (rat Copper Chaperone for Superoxide dismutase). The cDNA of rCCS was found to have a length of 1094 bp, and the protein analyzed from the cDNA was deduced to contain 274 amino acids. The amino acid sequence of rCCS consists of three domains: A metal binding domain, which has a MXCXXC motif in domain I, a homolog of the Cu/Zn SOD in domain II, and a CXC motif in domain III. The binding of rCCS to Cu/Zn SOD was analyzed by GST column binding assay, and the domain II of rCCS was found to be essential for binding to Cu/Zn SOD, which in turn activates Cu/Zn SOD.     

Identification of the beta-dystroglycan binding epitope within the C-terminal region of alpha-dystroglycan.

Dystroglycan is a receptor for extracellular matrix proteins that plays a crucial role during embryogenesis in addition to adult tissue stabilization. A precursor product of a single gene is post-translationally cleaved to form two different subunits, alpha and beta. The extracellular alpha-dystroglycan is a membrane-associated, highly glycosylated protein that binds to various extracellular matrix molecules, whereas the transmembrane beta-dystroglycan binds, via its cytosolic domain, to dystrophin and many other proteins. alpha- and beta-Dystroglycan interact tightly but noncovalently. We have previously shown that the N-terminal region of beta-dystroglycan, beta-DG(654-750), binds to the C-terminal region of murine alpha-dystroglycan independently from glycosylation. Preparing a series of deleted recombinant fragments and using solid-phase binding assays, the C-terminal sequence of alpha-dystroglycan containing the binding epitope for beta-dystroglycan has been defined more precisely. We found that a region of 36 amino acids, from position 550-585, is required for binding the extracellular region, amino acids 654-750 of beta-dystroglycan. Recently, a dystroglycan-like gene was identified in Drosophila that showed a moderate degree of conservation with vertebrate dystroglycan (31% identity, 48% similarity). Surprisingly, the Drosophila sequence contains a region showing a higher degree of identity and conservation (45% and 66%) that coincides with the 550-585 sequence of vertebrate alpha-dystroglycan. We have expressed this Drosophila dystroglycan fragment and measured its binding to the extracellular region of vertebrate (murine) beta-dystroglycan (Kd = 6 +/- 1 microM). These data confirm the proper identification of the beta-dystroglycan binding epitope and stress the importance of this region during evolution. This finding might help the rational design of dystroglycan-specific binding drugs, that could have important biomedical applications.     

Epitope structures recognised by antibodies against the major coat protein (g8p) of filamentous bacteriophage fd (Inoviridae).

To map the accessible surface of filamentous bacteriophage fd particles, the epitope structures of polyclonal rabbit serum and three mouse monoclonal antibodies raised against complete phage were analysed. Western blot analysis confirmed the major coat protein, gene VIII product (g8p or pVIII), to be the antigen. Overlapping peptides were synthesised by spot synthesis on cellulose membranes, covering the whole sequence of g8p. Each of the three tested monoclonal antibodies, B62-FE2, B62-GF3/G12 and B62-EA11, reacted with a core epitope covering ten amino acid residues at or near the amino terminus of g8p. The epitope recognised by B62-FE2 consists of the ten N-terminal amino acid residues of g8p. Extension of the amino terminus by various sequences did not inhibit binding, indicating that a terminal amino group is not essential for the interaction. Both B62-GF3/G12 and B62-EA11 recognise internal epitopes covering amino acid residues 3 to 12 of g8p. The epitopes of the polyclonal rabbit serum were also confined to the 12 N-terminal amino acid residues. The contribution of individual amino acid residues to the binding was analysed by a set of peptides containing individual amino acids exchanged by glycine. Accessible residues were Glu2, Asp4, Asp5, Pro6, Lys8, Phe11 and Asp12. The positions of the essential amino acid residues within the epitope are in accordance with a helical conformation of the amino-terminal region of g8p. Further, the results suggest new designs of phage display screening vectors to improve their performance in analysing non-linear epitopes.     

An immunologically cryptic epitope of Plasmodium falciparum circumsporozoite protein facilitates liver cell recognition and induces protective antibodies that block liver cell invasion

Circumsporozoite, a predominant surface protein, is involved in invasion of liver cells by Plasmodium sporozoites, which leads to malaria. We have previously reported that the amino terminus region (amino acids 27-117) of P. falciparum circumsporozoite protein plays a critical role in the invasion of liver cells by the parasite. Here we show that invasion-blocking antibodies are induced by a polypeptide encoding these 91 amino acids, only when it is presented in the absence of the rest of the protein. This suggests that when present in the whole protein, the amino terminus remains immunologically cryptic. A single reactive epitope was identified and mapped to a stretch of 21 amino acids from position 93 to 113. The epitope is configurational in nature, since its recognition was affected by deleting as little as 3 amino acids from either end of the 21-residue peptide. Lysine 104, the only known polymorphic position in the epitope, affected its recognition by the antibodies, and its conversion to leucine in the protein led to a substantial loss of binding activity of the protein to the hepatocytes. This indicated that in the protein, the epitope serves as a binding ligand and facilitates the interaction between sporozoite and hepatic cells. When considered along with the observation that in its native state this motif is immunologically unresponsive, we suggest that hiding functional moieties of the protein from the immune system is an evasion strategy to preserve liver cell binding function and may be of importance in designing anti-sporozoite vaccines.     

Mapping and Characterization of a Sequential Epitope on the Rabies Virus Glycoprotein Which Is Recognized by a Neutralizing Monoclonal Antibody,RG719

We have established a murine hybridoma cell line RG719 which produces a rabies virus-neutralizing IgM-type monoclonal antibody (referred to as MAb RG719). Immunoblot analysis indicated that the antibody recognized a sequential epitope of G protein. Among four rabies virus strains tested, the antigenicity to MAb RG719 was absent from the Nishigahara strain, while the other three strains (HEP, ERA and CVS) reacted to the MAb. Studies with deletion mutants of the G protein indicated that the epitope was located in a middle region of the primary structure of G protein, ranging from position 242 to 300. By comparing the estimated amino acid sequence of the four strains, we found in this region two amino acids (at positions 263 and 291) which are common to three of those strains but are not shared by the Nishigahara strain. The site-directed point mutagenesis revealed that replacement of phenylalanine-263 by leucine destroyed the epitope of the HEP G protein, while the epitope was generated on the Nishigahara G protein whose leucine-263 was replaced by phenylalanine. These observations suggest that phenylalanine-263 is essential for constructing the epitope for MAb RG719. The synthetic 20-mer peptide produced by mimicking the amino acid sequence (ranging from amino acid positions 249 to 268) of the presumed epitope region was shown to bind specifically to MAb RG719 and also to raise the virus-neutralizing antibodies in rabbits. Vaccination with the HEP vaccine produced in Japan induced in humans and rabbits production of significant amounts of the antibodies which reacted with the 20-mer peptide.     

Selective Pressure to Increase Charge in Immunodominant Epitopes of the H3 Hemagglutinin Influenza Protein

The evolutionary speed and the consequent immune escape of H3N2 influenza A virus make it an interesting evolutionary system. Charged amino acid residues are often significant contributors to the free energy of binding for protein–protein interactions, including antibody–antigen binding and ligand–receptor binding. We used Markov chain theory and maximum likelihood estimation to model the evolution of the number of charged amino acids on the dominant epitope in the hemagglutinin protein of circulating H3N2 virus strains. The number of charged amino acids increased in the dominant epitope B of the H3N2 virus since introduction in humans in 1968. When epitope A became dominant in 1989, the number of charged amino acids increased in epitope A and decreased in epitope B. Interestingly, the number of charged residues in the dominant epitope of the dominant circulating strain is never fewer than that in the vaccine strain. We propose these results indicate selective pressure for charged amino acids that increase the affinity of the virus epitope for water and decrease the affinity for host antibodies. The standard PAM model of generic protein evolution is unable to capture these trends. The reduced alphabet Markov model (RAMM) model we introduce captures the increased selective pressure for charged amino acids in the dominant epitope of hemagglutinin of H3N2 influenza (R ² > 0.98 between 1968 and 1988). The RAMM model calibrated to historical H3N2 influenza virus evolution in humans fit well to the H3N2/Wyoming virus evolution data from Guinea pig animal model studies.     

Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modeling

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing the PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive with MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.     

Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library

Background

The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported.

Methods and Results

This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched ²¹³SVQYHPL²¹⁹ of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that ²¹³SVQYHPL²¹⁹ was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group.

Conclusions and Significance

We identified ²¹³SVQYHPL²¹⁹ as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.     

Epitope analysis of human monoclonal antibody specific for rice allergenic protein generated by in vitro immunization

We previously established an in vitro immunization protocol for generating antigen specific human monoclonal antibodies (mAbs). In vitro immunization was performed against the soluble protein of rice allergenic protein (RA), resulting in the generation of three B cell clones, AC7-1/F9, CB7-1/E2 and CB7-8/F5, all of which produce a RA-specific human monoclonal IgM antibody. We attempted to map the epitope regions recognized by thesem Abs to characterize their specificities. We performed two rounds of epitope mapping, rough mapping using 10-mer peptides covering the full-length RA with 5 amino acids overlapping, and fine mapping using 8-mer peptides covering the putative epitope regions from the rough mapping with 1amino acid overlapping. As a result of the fine mapping,we identified the epitope regions of these three mAbs as⁴⁵QVWQDCCRQ⁵⁴L, ⁵⁶AVDDGWCRCGA⁶⁷L and⁹¹FPGCRRG⁹⁸D on the RA molecule and found to be identical. Furthermore, we determined the putative core epitope regions, which are critical for mAb binding to each region, ⁴⁷WQDCC⁵²R and ⁶⁰GWC⁶³R. The information about the epitope region on the RA molecule,which might trigger the allergenic response, would be useful to establish a specific immunotherapy against rice allergy. This revised version was published online in July 2006 with corrections to the Cover Date.     

The dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific.

Bacterial fusion proteins, constructed from overlapping fragments of the open reading frame coding for gp86 of human cytomegalovirus (HCMV) strain AD169, were used to localize antigenic regions recognized by antibodies from human convalescent sera. A major domain for binding of conformation-independent antibodies was localized on fusion protein AP86, containing amino acids 15 to 142 of gp86. Human antibodies, affinity purified on AP86, neutralized infectious virus in tissue culture. In addition, a mouse monoclonal antibody (AP86-SA4), raised against AP86, also neutralized HCMV. AP86-SA4 was reactive with viral gp86 in immunoblot assays and showed a plasma membrane staining on intact HCMV-infected fibroblasts late in infection. After exonuclease III deletions of the viral gene, the binding site of neutralizing human as well as mouse antibodies was localized between amino acid residues 34 and 43. The domain has sequence variation between laboratory strains AD169 and Towne, and binding of the antibodies was strain specific. To our knowledge, this is the first characterization of a strain-specific neutralizing epitope on HCMV.     

Cloning and characterization of the mouse Interleukin Enhancer Binding Factor 3 (Ilf3) homolog in a screen for RNA binding proteins

In a screen for RNA-binding proteins expressed during murine spermatogenesis, we have identified a cDNA that encodes a protein of 911 amino acids that contains two copies of the double-stranded RNA-binding motif and has 80% identity with human Interleukin Enhancer Binding Factor 3 (ILF3). Linkage and cytogenetic analyses localized the Ilf3 cDNA to a portion of mouse Chr 9, which shows conserved synteny with a region of human Chr 19 where the human ILF3 gene had been previously localized, supporting that we had cloned the murine homolog of ILF3. Northern analysis indicated the Ilf3 gene is ubiquitously expressed in mouse adult tissues with high levels of expression in the brain, thymus, testis, and ovary. Polyclonal antibodies detected multiple protein species in a subset of the tissues expressing Ilf3 RNA. Immunoreactive species are present at high levels in the thymus, testis, ovary, and the spleen to a lesser extent. The high degree of sequence similarity between the mouse ILF3 protein and other dsRNA binding motif-containing proteins suggests a role in RNA metabolism, while the differential expression indicates the mouse ILF3 protein predominantly functions in tissues containing developing lymphocyte and germ cells. Received: 21 October 1998 / Accepted: 15 January 1999