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PubMed1998

PAH Mutation Analysis Consortium Database: 1997. Prototype for relational locus-specific mutation databases.

PAHdb (http://www.mcgill.ca/pahdb ) is a curated relational database (Fig. 1) of nucleotide variation in the human PAH cDNA (GenBank U49897). Among 328 different mutations by state (Fig. 2) the majority are rare mutations causing hyperphenylalaninemia (HPA) (OMIM 261600), the remainder are polymorphic variants without apparent effect on phenotype. PAHdb modules contain mutations, polymorphic haplotypes, genotype-phenotype correlations, expression analysis, sources of information and the reference sequence; the database also contains pages of clinical information and data on three ENU mouse orthologues of human HPA. Only six different mutations account for 60% of human HPA chromosomes worldwide, mutations stratify by population and geographic region, and the Oriental and Caucasian mutation sets are different (Fig. 3). PAHdb provides curated electronic publication and one third of its incoming reports are direct submissions. Each different mutation receives a systematic (nucleotide) name and a unique identifier (UID). Data are accessed both by a Newsletter and a search engine on the website; integrity of the database is ensured by keeping the curated template offline. There have been >6500 online interrogations of the website.

Animals↗
PubMed1991

Linker-scanning mutational analysis of the transcriptional activity of the human immunodeficiency virus type 1 long terminal repeat.

We have compared the relative importance of transcription regulatory regions in the U3 and R regions of the human immunodeficiency virus type 1 long terminal repeat (LTR) by using linker-scanning mutational analysis. Twenty-six mutant LTR-chloramphenicol acetyltransferase (CAT) transient expression plasmids were prepared in which consecutive 18-bp regions of wild-type LTR were replaced with an NdeI-XhoI-SalI (NXS) polylinker. The mutant LTR-CAT plasmids were transfected into unstimulated Jurkat cells, Jurkat cells stimulated with phytohemagglutinin and tetradecanoylphorbol acetate, and Jurkat cells which constitutively express the human immunodeficiency virus type 1 trans-activator protein, Tat. Transcriptional activity was measured by analysis of CAT activity. The activities of these mutants identified one major and several minor transcription control elements in addition to previously identified elements. In addition, this fine-structure analysis identified differences in utilization of regulatory regions between unstimulated, stimulated, and Tat-expressing Jurkat cells. A significant regulatory region was indicated by linker-scanning mutations between nucleotides -183 and -130 (relative to the transcription start site, +1). These mutations caused marked decreases in activity of the LTR in unstimulated and especially in stimulated Jurkat cells but had no effect in Tat-expressing Jurkat cells. DNA mobility shift studies comparing probes of wild-type and mutant sequences in the -183 to -130 region indicated that alterations in specific DNA binding correspond to the altered transcriptional activity of the mutants. The effects of mutations in several regulatory regions, in addition to the -183 to -130 region described above, differ between Tat-expressing and -nonexpressing Jurkat cells. For example, the NF-kB sites are necessary for transcription in both Tat-expressing and -nonexpressing cells. However, Tat-expressing Jurkat cells primarily require only the 3'-proximal site, while both stimulated and unstimulated Jurkat cells appear to require both sites. Mutants downstream of the TATA element cause a more significant decrease in activity in Tat-expressing Jurkat cells than in the others. Finally, several mutations in the 5' half of the LTR (-453 to -184) show modest increases in transcription (1.5-fold or less) in unstimulated Jurkat cells only, suggesting possible negative regulatory sites. In summary, our studies have identified a control region (-183 to -130) upstream of the NF-kB sites and have more precisely defined significant differences in the utilization of regulatory regions between unstimulated, stimulated, and Tat-expressing Jurkat cells.

Base Sequence↗
PubMed2005

Mutation analysis of the PLOD1 gene: an efficient multistep approach to the molecular diagnosis of the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA).

The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA) is an inheritable connective tissue disorder characterized by a deficiency of lysyl hydroxylase due to mutations in PLOD1. We describe a mutation analysis strategy for the PLOD1 gene using either cDNA or gDNA or a combination thereof, which allows for reliable, time-effective and efficient mutation detection in patients with EDS VIA. We report the results obtained in 9 index patients from 12 unrelated families: three patients were homozygous for three novel mutations (p.Ile454IlefsX2, p.Ala667Thr, and p.His706Arg), four patients were homozygous for the common duplication of exons 10-16, one patient was compound heterozygous for the common duplication and p.Ile454IlefsX2, and one patient was homozygous for p.Arg319X.

Adolescent↗
PubMed1997

Mutation analysis for prenatal diagnosis of hereditary tyrosinaemia type 1.

Hereditary tyrosinaemia type 1 is a rare but serious metabolic disorder with an autosomal recessive mode of inheritance. We describe the prenatal diagnosis of an affected fetus performed by DNA-mutation analysis and a subsequent pregnancy with a healthy child in the same family.

Amino Acid Metabolism, Inborn Errors↗
PubMed2000

Mot protein assembly into the bacterial flagellum: a model based on mutational analysis of the motB gene.

The 308 residue MotB protein anchors the stator complex of the Escherichia coli flagellar motor to the peptidoglycan of the cell wall. Together with MotA, it comprises the transmembrane channel that delivers protons to the motor. At the outset of the mutational analysis of MotB described here, we found that the non-motile phenotype of a DeltamotAB strain was rescued better by a pmotA(+)B(+) plasmid than the non-motile phenotype of a DeltamotB strain was rescued by a pmotB(+) plasmid. Transcription in each case was from the inducible tac promoter but relied on the native ribosome-binding site (RBS). This result confirms that translational coupling to motA is important for normal translation of the motB mRNA, since overproduction of MotA in trans did not improve complementation by pmotB. However, introduction of an optimized RBS into pmotB (to generate pmotB(o)) did. To dissect the function of the periplasmic domain of MotB, site-directed mutagenesis was used to replace Gln, Ser, and Tyr codons scattered throughout motB with amber (UAG) codons. Plasmid-borne motB(am) genes were introduced into sup(o), supE, and supF strains to see what motility defects were imposed by particular amber mutations and whether the defects could be suppressed by amber-suppressor tRNAs inserting the native or heterologous amino acids. Amber mutations at codon 268 or earlier in pmotB, and at codon 261 or earlier in pmotB(o) or pmotAB, eliminated motility. Thus, in agreement with the deletion analysis of motB by another laboratory, we conclude that the portion of MotB carboxyl-terminal to its peptidoglycan-binding motif (residues 161 to 264) is not essential. In strains containing supE or supF alleles, motility defects associated with motB(am) mutations were suppressed weakly, if at all, in pmotB. In contrast, motility defects conferred by most motB(am) mutations in pmotB(o) or pmotAB could be suppressed to a significant extent. However, the S18(am), Q100(am), Q112(am), Q124(am), Y201(am), and Y208(am) mutations were still suppressed extremely poorly. Full-length MotB was present at very low levels in suppressor strains containing the first four mutations, but Y201(am) and Y208(am) were suppressed efficiently at the translational level. We suggest that a translational pause by suppressor tRNAs reading UAG at these two positions may divert the nascent polypeptide into an alternative folding pathway that traps MotB in a non-functional conformation. We further propose that MotA and MotB form a stable pre-assembly complex in the membrane. In this complex, MotB exists in a form that cannot associate with peptidoglycan and blocks the proton-conducting channel. Opening of the channel and attachment to the cell wall may occur when the complex collides with a flagellar basal body and MotA makes specific contacts with the C ring and/or the MS ring.

Amino Acid Sequence↗
PubMed2001

Estimate of the frequency of Wilson's disease in the US Caucasian population: a mutation analysis approach.

The frequency of Wilson's disease in many populations is thought to be about one in 40000 persons, based on case and autopsy studies. Although the Wilson's disease gene has been identified, there is such a large number of mutations already known that it is not currently feasible to determine disease gene frequency by mutation analysis of a population. We have used a novel approach to obtain an estimate of the number of cases of Wilson's disease expected at birth in the US Caucasian population. We used data from four studies to determine that approximately one-third of Wilson's disease mutations in US Caucasian Wilson's disease patients are due to His-->Gln at the 1069 position. We then determined the frequency of this mutation in random DNA samples from 2601 US Caucasian newborns to be 0.285%. Multiplying by three gives an expected Wilson's disease heterozygote frequency of 0.855% and an allele frequency of 0.428%, or 0.00428. These data translate into a Wilson's disease frequency of about one in 55000 births. The 95% confidence interval is rather broad, ranging from about one in 18000 to one in 700000 births, but will be reduced as more data are added.

DNA Primers↗
PubMed1997

Mutation analysis of the HLA-H gene in Italian hemochromatosis patients.

Hemochromatosis (HH) is an inborn error of iron metabolism, frequent among Caucasians, characterized by progressive iron loading that, if untreated, causes high morbidity and death. HLA-H, a putative HH gene, has recently been isolated. The large majority of patients so far studied are homozygous for a single mutation, which results in a cysteine-to-tyrosine substitution at amino acid 282 of the protein. A second, less frequent, variant, His63Asp, has an undefined role in the pathogenesis of the disease. Here we report that the Cys282Tyr change accounts for 69% of HH chromosomes in a series of 75 unrelated Italian patients who fulfilled well-defined criteria for HH diagnosis. Sixty-four percent of patients were Cys282Tyr homozygous, 10% were heterozygous, and 21% carried the normal allele. The same mutation was rare in normal controls. The His63Asp variant was less frequent but had a similar frequency among affected and normal chromosomes. Subjects without two copies of the Cys282Tyr change were both isolated patients and individuals from families with a 6p-linked disease. Mutation analysis of the HLA-H gene, carried out by RNA-SSCP in the latter patients, did not reveal any significant nucleotide abnormality in coding sequences and intron-exon boundaries. The absence of mutations in HLA-H gene was confirmed in three cases by direct sequencing. Major deletions or rearrangements of the gene were excluded by Southern blotting. The existence of patients with clinical and histological features of HH, but without mutations in HLA-H gene, suggests that in Italy the disease is more heterogeneous than reported in northern Europe.

Chromosomes, Human, Pair 6↗
PubMed2006

Mutation analysis of hCDC4 in AML cells identifies a new intronic polymorphism.

hCDC4 (FBW7, FBXW7) is a new potential tumor suppressor gene which provides substrate specificity for SCF (Skp-Cullin-F-box) ubiquitin ligases and thereby regulates the degradation of potent oncogenes such as cyclin E, Myc, c-Jun and Notch. Mutations in the hCDC4 gene have been found in several solid tumors such as pancreas, colorectal or endometrial cancer. We carried out a mutation analysis of the hCDC4 gene in 35 samples of patients with Acute Myeloid Leukemia (AML) to elucidate a possible role of hCDC4 mutations in this disease. By direct DNA sequencing and digestion with Surveyor nuclease one heterozygous mutation in the 5' untranslated region of exon 1, transcript variant 3 was detected. Additionally, we could identify a new intronic SNP downstream of exon 10. The new variation was present in 20% of AML samples and was furthermore confirmed in a panel of 51 healthy individuals where it displayed a frequency of 14%. In conclusion we provide first data that in contrast to several solid tumors, mutations in the hCDC4 gene may not play a pivotal role in the pathogenesis of AML. Furthermore, we describe a new intronic polymorphism with high frequency in the intron sequence of the hCDC4 gene.

Journal Article↗
PubMedDate not supplied

Mutation analysis of human LEFTY A and LEFTY B genes in children with Ivemark syndrome.

The search for genes responsible for the abnormal development of the left-right (L/R) asymmetry has been conducted but no definite results have been reported. Recently, two human homologus mouse lefty1 genes, LEFTY A and LEFTY B, were analyzed for mutations in patients with the L/R anomalies. However, only two mutations were found in a survey of 126 patients. We collected genomic DNA from 10 children with Ivemark syndrome, a disease with anomalies in L/R asymmetry. Mutation analysis of LEFTY A and LEFTY B genes using single strand conformation polymorphism and direct sequencing was performed, but no mutations were found. This indicates that the L/R asymmetry anomaly in Ivemark syndrome may not be caused by the mutation of LEFTY A and LEFTY B genes. Other genes responsible for the anomalies of L/R asymmetry should be further investigated.

Abnormalities, Multiple↗
PubMed2003

Combined microsatellite and FGFR3 mutation analysis enables a highly sensitive detection of urothelial cell carcinoma in voided urine.

PURPOSE: Fibroblast growth factor receptor 3 (FGFR3) mutations were reported recently at a high frequency in low-grade urothelial cell carcinoma (UCC). We investigated the feasibility of combining microsatellite analysis (MA) and the FGFR3 status for the detection of UCC in voided urine. EXPERIMENTAL DESIGN: In a prospective setting, 59 UCC tissues and matched urine samples were obtained, and subjected to MA (23 markers) and FGFR3 mutation analysis (exons 7, 10, and 15). In each case, a clinical record with tumor and urine features was provided. Fifteen patients with a negative cystoscopy during follow-up served as controls. RESULTS: A mutation in the FGFR3 gene was found in 26 (44%) UCCs of which 22 concerned solitary pTaG1/2 lesions. These mutations were absent in the 15 G3 tumors. For the 6 cases with leukocyturia, 46 microsatellite alterations were found in the tumor. Only 1 of these was also detected in the urine. This was 125 of 357 for the 53 cases without leukocyte contamination. The sensitivity of MA on voided urine was lower for FGFR3-positive UCC (15 of 21; 71%) as compared with FGFR3 wild-type UCC (29 of 32; 91%). By including the FGFR3 mutation, the sensitivity of molecular cytology increased to 89% and was superior to the sensitivity of morphological cytology (25%) for every clinical subdivision. The specificity was 14 of 15 (93%) for the two (molecular and morphological) cytological approaches. CONCLUSIONS: Molecular urine cytology by MA and FGFR3 mutation analysis enables a highly sensitive and specific detection of UCC. The similarity of molecular profiles in tumor and urine corroborate their clonal relation.

Adult↗
PubMed1997

p53 gene mutation analysis in porokeratosis and porokeratosis-associated squamous cell carcinoma.

In this and previous studies, we have shown p53 overexpression immunohistochemically in 14 of 17 porokeratotic specimens obtained from 14 lesions of nine cases, and in all six specimens of squamous cell carcinoma (SCC) arising on porokeratotic lesions of two cases. We screened mutations in exons 5 to 10 of the p53 gene in all these specimens by polymerase chain reaction-single strand conformation polymorphism analysis. Mutations of the p53 gene were detected in two of the six SCCs but not in any of the 17 porokeratotic specimens. These two mutations were C to T transitions at codons 146 and 175 in exon 5, which were a nonsense mutation at a dipyrimidine site and a missense mutation at a CG site, respectively. To our knowledge, neither of these mutations has been identified in skin cancers before. Our observations indicate that mutations of the p53 gene are not the major molecular etiology for porokeratosis, but are related to its skin carcinogenesis, and that p53 overexpression in porokeratosis is not due to p53 gene mutations.

Adult↗
PubMedDate not supplied

Mutational analysis of the enzyme IIIGlc of the phosphoenolpyruvate phosphotransferase system in Escherichia coli.

The phosphoenolpyruvate phosphotransferase system (PTS) component EIIIGlc is responsible for transport and phosphorylation of glucose via EIIGlc. It also regulates the catabolism of other carbon sources, such as lactose and maltose, by modulating both the intracellular concentrations of the corresponding inducers and of cAMP. Mutational analysis of EIIIGlc was performed in order to identify crucial residues mediating the interactions between EIIIGlc and its target proteins. Such mutations were isolated by in vitro hydroxylamine mutagenesis of the cloned EIIIGlc gene, crr. Five mutated EIIIGlc impaired in the function of inducer exclusion were obtained. However, these mutations did not abolish the function of EIIIGlc in the transport and phosphorylation of glucose, nor in activation of adenylate cyclase. A single amino acid change was found for each mutation, which is located in a restricted area of the polypeptide chain: Gly47-->Ser47 for the HA2 and HA5 mutations, Ala76-->Thr76 for HA4 mutation and Ser78-->Phe78 for HA3 mutation, indicative of quaternary interactions between the corresponding region of EIIIGlc and its target protein(s).

Adenylyl Cyclases↗
PubMed1999

Mutational analysis of three tumor suppressor genes in two models of rat hepatocarcinogenesis.

An albumin-simian virus 40 (SV40) large T-antigen (T-Ag) transgenic model and a chemically induced model of multistage hepatocarcinogenesis were created in our laboratory to study the molecular mechanisms involved in the genesis and progression of neoplasia in the rat liver. In the study presented here, these two models of rat hepatocarcinogenesis were used to perform a comparative mutational analysis of three tumor suppressor genes involved in hepatic neoplastic growth. By using polymerase chain reaction-single strand conformation polymorphism analysis and sequencing, exons 5-8 of the p53 tumor suppressor gene and a region between nt 4325 and 4479 of the rat mannose 6-phosphate/insulin-like growth factor 2 receptor (M6p/Igf2r) coding sequence were screened. The latter is homologous to the human M6P/IGF2r coding sequence which is mutated in human hepatocellular carcinoma. A complete single strand conformation polymorphism analysis of the entire coding region of the rat adenomatous polyposis coli (Apc) gene was also performed for the first time in rat tumorigenic samples. Twenty-six chemically induced rat hepatocellular carcinomas, 21 neoplasms from the livers of SV40 T-Ag animals, and five immortalized hepatic cell lines from the transgenic rats were evaluated. None of the hepatic tumors exhibited mutations in the regions analyzed. The albumin-SV40 T-Ag transgenic cell line L-60, derived from normal hepatic tissue, had two mutations in contiguous codons of exon 5 of the p53 gene: a GGT --> GTT missense transversion in codon 183 and a silent mutation in codon 184. The transversion, which may affect the DNA binding domain of the p53 protein, probably originated during cell culture and may have been positively selected because it gave a growth advantage to the mutated cells. The studied region of the M6p/Igf2r gene was not found to be mutated in these two models of rat hepatocarcinogenesis. Although M6p/Igf2r, Apc, and p53 have been shown to be mutated in a variety of human hepatic proliferative diseases, our results indicate that aberrations in these genes may not be necessary for liver carcinogenesis in the rat.

Amino Acid Sequence↗
PubMed1994

Mutation analysis of the Bruton's tyrosine kinase gene in X-linked agammaglobulinemia: identification of a mutation which affects the same codon as is altered in immunodeficient xid mice.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease in man, reflecting an arrest in differentiation of pre-B cells to mature B cell stages. The gene defective in XLA has been identified as a cytoplasmic protein tyrosine kinase, named btk (Bruton's tyrosine kinase). Here we report the characterization of mutations in the btk gene of five unrelated XLA families. Amplified products were generated from cDNA, cloned and sequenced. Three single point mutations and two small insertions were identified. One of the point mutations and the two insertions created stop codons that would lead to truncated btk proteins. In one XLA patient we found a single basepair substitution that altered the highly conserved Arg288 within the SH2 domain and would therefore abrogate interactions with substrate phosphotyrosines. In another XLA patient a single basepair substitution was observed that altered the conserved Arg28 residue in the N-terminal unique region of unknown function. This residue is also mutated in the xid mouse, which has a different, less severe, B cell deficiency. We conclude that a similar mutation in the btk gene leads in man to an almost complete arrest at an early stage of B cell differentiation, but in the mouse to only limited B cell abnormalities.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1992

Mutational analysis of the Escherichia coli phosphate-specific transport system, a member of the traffic ATPase (or ABC) family of membrane transporters. A role for proline residues in transmembrane helices.

The Escherichia coli Pst system is a periplasmic phosphate permease. A mutational analysis of the requirement for function of specific charged residues or proline residues in the two hydrophobic subunits (PstC and PstA) has been carried out. No residues, among 19 charged residues altered, were found to be essential for phosphate uptake, although some alterations resulted in partial effects. Evidence was obtained that the 3 residues, R220 in the PstA protein and R237 and E241 in the PstC protein, previously shown to be required for phosphate transport (Cox, G. B., Webb, D., Godovac-Zimmermann, J., and Rosenberg, H. (1988) J. Bacteriol. 170, 2283-2286; Cox, G. B., Webb, D., and Rosenberg, H. (1989) J. Bacteriol. 171, 1531-1534), interact with each other. A feature of the proposed structures of the PstA and PstC proteins was 2 pairs of proline residues in putative transmembrane helices 3 and 4. While individual substitutions of these proline residues by leucine resulted in loss of phosphate transport activity substitution by alanine only had partial effects. However, if the proline to alanine changes were paired then, depending on the particular subunit, markedly different effects were obtained. The double mutation in the PstA protein resulted in a permanently "closed" system, whereas the double mutation in the PstC protein resulted in a permanently "open" transport system.

ATP-Binding Cassette Transporters↗
PubMed2005

Mutation analysis of the ATP7B gene and genotype/phenotype correlation in 227 patients with Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder of copper transport. WD patients are presenting with a wide range of heterogeneous clinical syndromes including hepatic, neurological, or psychiatric presentations. The disease is caused by mutations in the ATP7B gene. This study presents the results of comprehensive mutation analysis in 227 WD patients from 200 unrelated families (173 from Czech Republic and 27 from Slovakia). More than 80% of all mutant alleles were identified, using a combination of PCR/RFLP, DGGE, TTGE, DHPLC, and sequencing. A total of 40 different mutations and 18 polymorphisms were detected on 400 independent mutant chromosomes. The most common molecular defect was H1069Q (57% of all 400 studied alleles). Each of the other 39 mutations was present in no more than 4% of WD alleles and 23 mutations were found in only one WD allele each (0.25%). Thirteen novel mutations were identified, including seven missense mutations (L641S, T737R, D918E, T1033S, G1111D, D1271N, and G1355C), four small deletions (19_20delCA, 1518_1522del5, 3140delA, and 3794_3803del10), and two splice-site mutations (2446-2A>G, 2865+1G>A). We did not find a significant correlation between H1069Q homozygosity and age of onset, and clinical and biochemical manifestation. Our data provide evidence that the H1069Q mutation-the most common molecular defect of the ATP7B gene in the Caucasian population-originates from Central/Eastern Europe. Screening of five prevalent mutations is predicted to reveal 70% of all mutant alleles presented in WD patients. This will provide a good starting point for early clinical classification of WD in our population.

Adenosine Triphosphatases↗
PubMed2007

RPGR mutation analysis and disease: an update.

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are the most common single cause of retinitis pigmentosa, accounting for up to 15 to 20% of cases in Caucasians. A total of 240 different RPGR mutations have been reported, including 24 novel ones in this work, which are associated with X-linked retinitis pigmentosa (XLRP) (95%), cone, cone-rod dystrophy, or atrophic macular atrophy (3%), and syndromal retinal dystrophies with ciliary dyskinesia and hearing loss (2%). All disease-causing mutations occur in one or more RPGR isoforms containing the carboxyl-terminal exon open reading frame 15 (ORF15), which are widely expressed but show their highest expression in the connecting cilia of rod and cone photoreceptors. Of reported RPGR mutations, 55% occur in a glutamic acid-rich domain within exon ORF15, which accounts for only 31% of the protein. RPGR forms complexes with a variety of other proteins and appears to have a role in microtubular organization and transport between photoreceptor inner and outer segments.

Animals↗
PubMed1996

Mutational analysis of a neutralization epitope on the dengue type 2 virus (DEN2) envelope protein: monoclonal antibody resistant DEN2/DEN4 chimeras exhibit reduced mouse neurovirulence.

The antigenic site of dengue type 2 virus (DEN2)-neutralizing monoclonal antibody (mab) 3H5 was investigated by mutational analysis. Sequence comparisons indicated that much of the 12-amino-acid sequence extending from position 386 to 397 of the DEN2 envelope glycoprotein (E) previously thought to represent the DEN2-specific mab 3H5 binding site was also present in some dengue type 1, 3, or 4 virus strains. However, the region occupied by the Glu-Pro-Gly sequence at upstream positions 383 to 385 was completely conserved among DEN2 strains, but divergent in other serotype viruses, suggesting that this sequence might be part of the antigenic site of mab 3H5. We investigated this possibility by employing the previously constructed chimeric DEN2(PreM-E)/DEN4 cDNA clone to produce viable mutants bearing DEN2 PreM and E sequences that could be analyzed for binding to and neutralization by mab 3H5. We constructed 13 such DEN2 mutants that contained a single amino acid substitution in the region between positions 383 and 393 of DEN2 E. Each single substitution in the region spanning positions 386 through 393 of DEN2 yielded a virus that was as reactive with mab 3H5 as the parental chimeric virus. These results are consistent with the extent of sequence conservation in the region. In contrast, 5 of 6 mutants that sustained an amino acid substitution at position 383, 384, or 385 failed to react with mab 3H5 as detected by immunofluorescence assay and failed to be neutralized by the mab. Interestingly, each of the 5 mab-resistant DEN2 mutants also exhibited reduced mouse neurovirulence compared to parental chimeric DEN2 when inoculated intracerebrally. These observations suggest that the Glu-Pro-Gly sequence at positions 383-386 of the DEN2 E is a component of the site against which mab 3H5 is directed. In the recently determined three-dimensional structure of the related tick-borne encephalitis virus E, the Glu-Pro-Gly sequence would be located on the lateral surface of the immunoglobulin-like domain that is proposed to bind to the host cell receptor.

Aedes↗