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Biomedical subjects

C J Lai

Publications and source records attributed to C J Lai.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1996

A frizzled homolog functions in a vertebrate Wnt signaling pathway.

BACKGROUND: Wnts are secreted proteins implicated in cell-cell interactions during embryogenesis and tumorigenesis, but receptors involved in transducing Wnt signals have not yet been definitively identified. Members of a large family of putative transmembrane receptors homologous to the frizzled protein in Drosophila have been identified recently in both vertebrates and invertebrates, raising the question of whether they are involved in transducing signals for any known signaling factors. RESULTS: To test the potential involvement of frizzled homologs in Wnt signaling, we examined the effects of overexpressing rat frizzled-1 (Rfz-1) on the subcellular distribution of Wnts and of dishevelled, a cytoplasmic component of the Wnt signalling pathway. We demonstrate that ectopic expression of Rfz-1 recruits the dishevelled proten-as well as Xenopus Wnt-8 (Xwnt-8), but not the functionally distinct Xwnt-5A-to the plasma membrane. Moreover, Rfz-1 is sufficient to induce the expression of two Xwnt-8-responsive genes, siamois and Xnr-3, in Xenopus explants in a manner which is antagonized by glycogen synthase kinase-3, which also antagonizes Wnt signaling. When Rfz-1 and Xwnt-8 are expressed together in this assay, we observe greater induction of these genes, indicating that Rfz-1 can synergize with a Wnt. CONCLUSIONS: The results demonstrate that a vertebrate frizzled homolog is involved in Wnt signaling in a manner which discriminates between functionally distinct Wnts, which involves translocation of the dishevelled protein to the plasma membrane, and which works in a synergistic manner with Wnts to induce gene expression. These data support the likely function of frizzled homologs as Wnt receptors, or as components of a receptor complex.

Adaptor Proteins, Signal Transducing↗
PubMed1996

A monoclonal IgG anticardiolipin antibody from a patient with the antiphospholipid syndrome is thrombogenic in mice.

Antiphospholipid antibodies, including anticardiolipin antibodies (ACA), are strongly associated with recurrent thrombosis in patients with the antiphospholipid syndrome (APS). To date, reports about the binding specificities of ACA and their role(s) in causing and/or sustaining thrombosis in APS are conflicting and controversial. The plasmas of patients with APS, usually containing a mixture of autoantibodies, vary in binding specificity for different phospholipids/cofactors and vary in in vitro lupus anticoagulant activity. Although in vivo assays that allow assessment of the pathogenic procoagulant activity of patient autoantibodies have recently been developed, the complex nature of the mixed species prevented determination of the particular species responsible for in vivo thrombosis. We have generated two human IgG monoclonal ACA from an APS patient with recurrent thrombosis. Both bound to cardiolipin in the presence of 10% bovine serum, but not in its absence, and both were reactive against phosphatidic acid, but were nonreactive against purified human beta-2 glycoprotein 1, DNA, heparan sulfate, or four other test antigens. Both monoclonal autoantibodies lacked lupus anticoagulant activity and did not inhibit prothrombinase activity. Remarkably, one of the monoclonal antibodies has thrombogenic properties when tested in an in vivo mouse model. This finding provides the first direct evidence that a particular antiphospholipid antibody specificity may contribute to in vivo thrombosis.

Adult↗
PubMed1996

Oxidized aldose reductase: in vivo factor not in vitro artifact.

Characterization of aldose reductase purified from human placenta confirms that activation, as first analyzed in detail for the bovine enzyme, also occurs in humans. Routinely between 5 and 20% of the aldose reductase activity freshly purified from human placenta exhibits kinetic properties and insensitivity to aldose reductase inhibitors (ARIs) characteristic of the activated or oxidized enzyme form, as determined using a sensitive Sorbinil titration assay. In confirmation of previous studies, the amount of aldose reductase activity and the ratio of aldose to aldehyde reductase activity show wide patient to patient variability, with aldose reductase accounting for between 30 and 95% of the total aldo-keto reductase activity. The kinetic behavior described for enzyme isolated from human tissues (e.g., biphasic Dixon plots for ARI inhibition) can be reproduced exactly using mixtures of native and oxidized recombinant human aldose reductase and is not restricted to DL-glyceraldehyde. Measurement of substrate (NADPH versus NADPD and solvent (H2O versus D2O) deuterium isotope effects indicates that the ARI-resistant form is altered in a manner that perturbs the relative rates of steps along the normal reaction pathway. These results suggest that not only the level of enzyme activity, but also the extent of activation of human aldose reductase in vivo, may be an important factor in determining susceptibility to diabetic complications and responsiveness to ARI therapy.

Aldehyde Reductase↗
PubMed1996

Posttraumatic gluteal epidermal cyst with ischiorectal and presacral extension.

An unusual case of rapid-growing posttraumatic gluteal epidermal cyst with ischiorectal and presacral extension is reported. Pertinent clinical history and magnetic resonance imaging allowed accurate preoperative diagnosis. Cautious complete excision was important to prevent recurrence. To our knowledge, posttraumatic gluteal epidermal cyst with this unusual clinical presentation has not been described previously.

Adult↗
PubMed1996

Dengue type 4 virus mutants containing deletions in the 3' noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys.

The dengue type 4 virus (DEN4) genome contains a 384-nucleotide (nt) 3' noncoding sequence in which the last 81 nt, predicted to form a secondary structure, are thought to be essential for virus replication. Immediately upstream of the secondary structure, short RNA sequences that are conserved among mosquito-borne flaviviruses have been identified. A series of deletions that range from 30 to 262 nt were introduced into this upstream region of full-length DEN4 cDNA to create viable deletion mutants, some of which might prove to be useful for inclusion in a live attenuated virus vaccine. When studied by an infectious-center assay, most full-length RNA transcripts of the deletion constructs exhibited reduced infectivity when transfected into simian LLC-MK2 cells compared with the full-length RNA transcripts of wild-type parental virus. Deletion mutations that extended as far as the 5' boundary of the 3' noncoding region and whose 3' boundary did not extend beyond the last 113 nt of the 3' end were viable. With the exception of mutant 3'd 303-183, which contained a deletion of nt 303 to 183 from the 3' terminus, deletion mutants produced plaques that appeared late on simian LLC-MK2 cells or exhibited a small-plaque morphology on mosquito C6/36 cells compared with the wild-type virus. These mutants also replicated less efficiently and attained a lower titer in LLC-MK2 cells than parental wild-type virus. Significantly, mutant 3'd 303-183 grew to a high titer and was least restricted in growth. Mutant 3'd 303-183 and four other moderately to severely restricted mutants were selected for evaluation of infectivity and immunogenicity in rhesus monkeys. There was a suggestion that occurrence and duration of viremia were reduced for some of the deletion mutants compared with the wild-type virus. However, more convincing evidence for attenuation of some of the mutants was provided by an analysis of antibody response to infection. Mutant 3'd 303-183 induced an antibody response equivalent to that stimulated by wild-type virus, whereas other mutants induced low to moderate levels of antibodies, as measured by radioimmunoprecipitation and virus neutralization. The immunogenicity of these 3' DEN4 deletion mutants in monkeys appeared to correlate with their efficiency of growth in simian LLC-MK2 cells. One or more mutants described in this paper may prove to be useful for immunization of humans against disease caused by dengue virus.

Animals↗
PubMed1996

Monkeys immunized with intertypic chimeric dengue viruses are protected against wild-type virus challenge.

Dengue epidemics caused by the four dengue virus serotypes continue to pose a major public health problem in most tropical and subtropical regions. A safe and effective vaccine against dengue is still not available. The current strategy for dengue immunization favors the use of a vaccine containing each of the four serotypes. We previously employed full-length dengue type 4 virus (DEN4) cDNA to construct a viable intertypic dengue virus of type 1 or type 2 antigenic specificity that contained the genes for the capsid-premembrane-envelope (C-pre-M-E) structural proteins of DEN1 or pre-M and E structural proteins of DEN2 substituting for the corresponding DEN4 genes. Chimeras DEN1/DEN4 and DEN2/DEN4, which express the nonstructural proteins of DEN4 and the C-pre-M-E structural proteins of DEN1 or the pre-M-E structural proteins of DEN2, and therefore the antigenicity of type 1 or type 2, were used to immunize rhesus monkeys. Other monkeys were inoculated with parental DEN1, DEN2, or cDNA-derived DEN4. Three of four monkeys immunized with DEN1/DEN4 developed neutralizing antibodies against DEN1 and were protected against subsequent DEN1 challenge. All four monkeys immunized with DEN2/DEN4 developed antibodies against DEN2 and were protected against subsequent DEN2 challenge. DEN1- and DEN2-immunized monkeys were protected against homologous virus challenge, but DEN4-immunized animals became viremic on cross-challenge with DEN1 or DEN2. In a second experiment, eight monkeys were immunized with equal mixtures of DEN1/DEN4 and DEN2/DEN4. Each of these monkeys developed neutralizing antibodies against both DEN1 and DEN2 and were protected against subsequent challenge with DEN1 or DEN2. Chimeric dengue viruses similar to those described here could be used to express serotype-specific antigens in a live attenuated tetravalent human vaccine.

Animals↗
PubMed1996

Effects of report order, identification method, and stimulus characteristics on multidimensional stimulus identification.

An experiment was conducted to investigate the effect of order of report, identification method, redundant color coding, and stimulus location under dual task conditions on multidimensional stimulus-identification performance. Analysis showed that order of report and identification method did affect speed and accuracy of identification. Subjects reacted faster and more accurately if this order of reporting stimulus-dimension values was appropriate. Physical identification was also faster and more accurate than identification of meaning, but there was no effect for redundant color coding, stimulus location, and difficulty of the dual task on identification. The implications of the results for design of visual displays were discussed.

Adult↗
PubMed1996

Effects of practice on identification of multidimensional stimuli.

This study investigated the effects of practice on the identification of multidimensional stimuli. Subjects were instructed to perform a two dimensional identification task. The stimuli could be identified either physically or connotatively. There were two orders of reporting the two dimensions of a stimulus. Analysis showed that method of identification and order of report significantly affected speed of identification. Physical identification was faster than identification of meaning. An appropriate order of report shortened response times, and increased practice reduced response times. Practice in multidimensional identification seemed to follow the same laws and trends of unidimensional performance. The effects of practice on multimensional identification could be represented by straight lines if response times were plotted against the logarithm of the number of trials as noted in unidimensional performance. Further, practice attenuated initial differences in response times between physical identification and identification of meaning.

Adult↗
PubMed1996

Inhibitory influence of lung vagal C-fiber afferents on the delayed ventilatory response to inhaled wood smoke in rats.

We studied the influences of lung vagal afferents on the delayed ventilatory response to inhaled wood smoke in 35 Sprague-Dawley rats. Wood smoke (approximately 6 ml) was inhaled via tracheostomy when animals breathed spontaneously. Immediately after inhalation, wood smoke evoked a slowing of respiration or an augmented inspiration, followed by a delayed tachypnea. Owing to the increase in respiratory frequency, the delayed ventilatory response usually resulted in an increase in minute ventilation (VI). The smoke-induced increase in VI was elevated after perineural capsaicin treatment of both cervical vagi (PCT, n = 12), but was unaltered during cooling of both vagi to 6.7 degrees C (VGC, n = 9); PCT selectively blocked the conduction of unmyelinated C-fibers, whereas VGC differentially blocked the conduction of myelinated fibers. Furthermore, the smoke-induced increase in VI after PCT was unaffected by an additional application of VGC (n = 12), whereas it was magnified by an additional application of PCT during VGC (n = 9). This magnification was not changed after a subsequent bilateral vagotomy (n = 21). Removal of smoke particulates did not affect the increase in VI evoked by smoke inhalation (n = 14). These results suggest that 1) the smoke-induced delayed ventilatory response is restrained by the inhibitory input from lung vagal C-fibers, whereas it was not influenced by the input from irritant receptors and 2) the gas phase of wood smoke is responsible for eliciting this delayed ventilatory response.

Afferent Pathways↗
PubMed1995

Human aldose reductase: rate constants for a mechanism including interconversion of ternary complexes by recombinant wild-type enzyme.

We have used transient kinetic data for partial reactions of recombinant human aldose reductase and simulations of progress curves for D-xylose reduction with NADPH and for xylitol oxidation with NADP+ to estimate rate constants for the following mechanism at pH 8.0: E<-->E.NADPH<-->*E.NADPH<-->*E.NADPH.RCHO<-->*E.NADP+.RCH2OH <-->*E.NADP+<--> E.NADP+<-->E. The mechanism includes kinetically significant conformational changes of the two binary E.nucleotide complexes which correspond to the movement of a crystallographically identified nucleotide-clamping loop involved in nucleotide exchange. The magnitude of this conformational clamping is substantial and results in a 100- and 650-fold lowering of the nucleotide dissociation constant in the productive *E.NADPH and *E.NADP+ complexes, respectively. The transient reduction of D-xylose displays burst kinetics consistent with the conformational change preceding NADP+ release (*E.NADP+-->E.NADP+) as the rate-limiting step in the forward direction. The maximum burst rate also displays a large deuterium isotope effect (Dkburst = 3.6-4.1), indicating that hydride transfer contributes significantly to rate limitation of the sequence of steps up to and including release of xylitol. In the reverse reaction, no burst of NADPH production is observed because the hydride transfer step is overall 85% rate-limiting. Even so, the conformational change preceding NADPH release (*E.NADPH-->E.NADPH) still contributes 15% to the rate limitation for reaction in this direction. The estimated rate constant for hydride transfer from NADPH to the aldehyde of D-xylose (130 s-1) is only 5- to 10-fold lower than the corresponding rate constant determined for NADH-dependent carbonyl reduction catalyzed by lactate or liver alcohol dehydrogenase. Hydride transfer from alcohol to NADP+ (0.6 s-1), however, is at least 100- to 1000-fold slower than NAD(+)-dependent alcohol oxidation mediated by these two enzymes, resulting in a bound-state equilibrium constant for aldose reductase which greatly favors the forward reaction. The proposed kinetic model provides a basic set of rate constants for interpretation of kinetic results obtained with aldose reductase mutants generated for the purpose of examining structure-function relationships of different components of the native enzyme.

Aldehyde Reductase↗
PubMed1995

Human aldose reductase: subtle effects revealed by rapid kinetic studies of the C298A mutant enzyme.

Transient kinetic data for D-xylose reduction with NADPH and NADPD and for xylitol oxidation with NADP+ catalyzed by recombinant C298A mutant human aldose reductase at pH 8 have been used to obtain estimates for each of the rate constants in the complete reaction mechanism as outlined for the wild-type enzyme in the preceding paper (Grimshaw et al., 1995a). Analysis of the resulting kinetic model shows that the nearly 9-fold increase in Vxylose/Et for C298A mutant enzyme relative to wild-type human aldose reductase is due entirely to an 8.7-fold increase in the rate constant for the conformational change that converts the tight (Ki NADP+ = 0.14 microM) binary *E.NADP+ complex to the weak (Kd NADP+ = 6.8 microM) E.NADP+ complex from which NADP+ is released. Evaluation of the rate expressions derived from the kinetic model for the various steady-state kinetic parameters reveals that the 37-fold increase in Kxylose seen for C298A relative to wild-type aldose reductase is largely due to this same increase in the net rate of NADP+ release; the rate constant for xylose binding accounts for only a factor of 5.5. A similar 17-fold increase in the rate constant for the conformational change preceding NADPH release does not, however, result in any increase in Vxylitol/Et, because hydride transfer is largely rate-limiting for reaction in this direction.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Reductase↗
PubMed1995

Human aldose reductase: pK of tyrosine 48 reveals the preferred ionization state for catalysis and inhibition.

Detailed analyses of the pH variation of kinetic parameters for the forward aldehyde reduction and reverse alcohol oxidation reactions mediated by recombinant human aldose reductase, for inhibitor binding, and for kinetic isotope effects on aldehyde reduction have revealed that the pK value for the active site acid-base catalyst group Tyr48 is quite sensitive to the oxidation state of the bound nucleotide (NADPH or NADP+) and to the presence or absence of the Cys298 sulfhydryl moiety. Thus, the Tyr48 residue of C298A mutant enzyme displays a pK value that ranges from 7.6 in the productive *E.NADP+ complex that binds and reacts with alcohols to 8.7 in the productive *E.NADPH complex that binds and reacts with aldehyde substrates. For wild-type enzyme, Tyr48 in the latter complex displays a lower pK value of about 8.25. Assignment of the pK values was facilitated by the recognition and quantitation of the degree of stickiness of several aldehyde substrates in the forward reaction. The unusual pH dependence for Valdehyde/Et and DValdehyde, which decrease roughly 20-fold through a wave and remain constant at high pH, respectively, is shown to arise from the pH-dependent decrease in the net rate of NADP+ release. The results described are fully consistent with the chemical mechanism for aldose reductase catalysis proposed previously (Bohren et al., 1994) and, furthermore, establish that binding of the spirohydantoin class of aldose reductase inhibitors, e.g., sorbinil, occurs via a reverse protonation scheme in which the ionized inhibitor binds preferentially to the *E.NADP+ complex with Tyr48 present as the protonated hydroxyl form. The latter finding allows us to propose a unified model for high-affinity aldose reductase inhibitor binding that focuses on the transition state-like nature of the *E-Tyr48-OH.NADP+.inhibitor- complex.

Aldehyde Reductase↗
PubMed1995

Growth-restricted dengue virus mutants containing deletions in the 5' noncoding region of the RNA genome.

The dengue type 4 virus (DEN4) RNA genome contains a 101-nt 5' noncoding (NC) sequence which is predicted to form a stable secondary structure. DEN4 cDNA from which infectious RNA can be transcribed was used to engineer deletions in the 5' NC region for functional analysis of RNA structure and for isolation of DEN4 mutants that could be evaluated as candidates for use in a live attenuated vaccine. Eleven distinct deletions in the region of the DEN4 genome between nts 18 and 98 were constructed; each mutation was predicted to alter or disrupt the local base-parings in the 5' NC RNA structure. An infectious virus was not recovered from the RNA transcripts of five of these deletion mutants. Significantly, four of the five apparently lethal deletions were located in a 5- to 6-nt base-paired region of a predicted long stem or adjacent to it. In contrast, with one exception, mutants which yielded infectious virus had deletions which were located in a loop or short stem region. The effect of the deletions on the efficiency of translation of viral RNA transcripts was examined in vitro. The RNA transcripts of deletion constructs which did not yield viable virus were translated at an efficiency ranging from 40 to 160% that of wild-type virus transcripts. The translation efficiency of infectious RNA transcripts also varied. Deletion mutants recovered from RNA transcripts that exhibited low to moderate efficiency of translation had a small plaque morphology and exhibited reduced growth in simian LLC-MK2 and mosquito C6/36 cells compared to the wild-type virus. Among the 11 mutant constructs, deletion of nts 82-87 caused the greatest reduction in translation efficiency. Nevertheless, an infectious virus was recovered from LLC-MK2 cells transfected with the RNA transcripts of mutant d(82-87). The progeny of this mutant produced small plaques on LLC-MK2 cells and grew to low titer in these cells. Unlike wild-type DEN4 or other DEN4 deletion mutants tested, mutant d(82-87) failed to produce plaques on C6/36 cells and was also replication-defective in Aedes aegypti and Aedes albopictus following intrathoracic inoculation.

Aedes↗
PubMed1995

Dengue virus-specific, HLA-B35-restricted, human CD8+ cytotoxic T lymphocyte (CTL) clones. Recognition of NS3 amino acids 500 to 508 by CTL clones of two different serotype specificities.

Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas of the world. We analyzed dengue virus-specific CD8+ CD4- CTL at the clonal level to further understand the role of CD8+ CTL in dengue virus infections. Dengue virus-specific CD8+ CTL clones were established from lymphocytes of a dengue 4-immune adult. Three patterns of dengue serotype specificities were identified: 1) specific for dengue 4, 2) cross-reactive for dengue 2 and dengue 4 (subcomplex-specific); and 3) cross-reactive for all four dengue virus serotypes. Three dengue 4-specific clones and one dengue 2/dengue 4 cross-reactive clone were further analyzed. All four of the clones were HLA-B35 restricted and recognized NS3. The epitopes were mapped to amino acids (aa) 483 to 618 of NS3. The epitope was then defined by using synthetic peptides. Three dengue 4-specific clones and one dengue 2/dengue 4 cross-reactive clone recognized the same peptide (TPEGIIPTL) encompassing aa 500 to 508 of dengue 4 NS3. The peptide encompassing aa 500-508 of dengue 2 NS3 was recognized by a dengue 2/dengue 4 cross-reactive clone but was not recognized by the dengue 4-specific clones. Dengue 4-specific and dengue 2/dengue 4 cross-reactive clones used different TCR. These results indicate that CD8+ CTL clones that use different TCR and demonstrate two distinct serotype specificities recognize the same 9-mer peptide in the context of HLA-B35.

Adult↗
PubMed1995

Specification of the anteroposterior neural axis through synergistic interaction of the Wnt signaling cascade with noggin and follistatin.

Embryological data and the activities of the neural-inducing factors noggin and follistatin are consistent with the hypothesis that the nervous system is initially induced with an anterior character, with subsequent signals imparting posterior pattern. We report that Xwnt3a is a candidate for involvement in anteroposterior neural patterning, as it synergizes with the neural-inducing factors noggin and follistatin to increase the expression of posterior neural genes. Furthermore we show that beta-catenin, an intracellular protein implicated in the Wnt signal transduction cascade, mimics the activity of Xwnt3a. These data suggest that the generation of pattern within the vertebrate nervous system may rely on synergism between a Wnt signaling pathway and multiple neural-inducing factors.

Animals↗
PubMed1995

Processing of Japanese encephalitis virus non-structural proteins: NS2B-NS3 complex and heterologous proteases.

Processing of Japanese encephalitis (JE) virus non-structural (NS) proteins expressed by recombinant vaccinia viruses was analysed to characterize the responsible viral protease. Analysis of the processing of polyprotein NS2A-2B-3' containing the N-terminal 322 amino acids of NS3 revealed products consistent with cleavages at the predicted intergenic junctions as well as at one or possibly two sites within NS2A. Cleavage at the alternate site(s) containing the cleavage sequence motif within NS2A could possibly explain the production of the NS1' protein in JE virus-infected cells. Polyprotein NS2A-d2B-3' containing a large deletion within NS2B was cleavage-defective, despite the presence of the proposed NS3 protease domain. Cleavage of NS2A-d2B-3' was restored if NS2B or NS2A-2B was supplied in trans, providing evidence that NS2B is strictly required for NS3 proteolytic activity. NS2B- or NS3-specific sera raised against the bacterial TrpE fusion protein co-precipitated NS2B and NS3 or NS3' from the lysate of JE virus or recombinant virus-infected cells. Thus both protease components are associated as a complex, presumably representing the active JE virus protease. JE virus and the analogous dengue 4 (DEN-4) protease components were employed to examine the activity of heterologous proteases. The defective cleavage of JE virus NS2A-d2B-3' was complemented by heterologous DEN-4 NS2B, whereas the defective cleavage of DEN-4 NS2A-d2B-3' was not corrected by heterologous JE virus NS2B. This suggests that the heterologous JE virus NS2B-DEN-4 NS3 protease is not active, despite the considerable sequence conservation of NS2B and NS3 between the two viruses. The cleavage activity was restored by replacement of the C-terminal 80 amino acids of JE virus NS2B with the corresponding DEN-4 sequence, consistent with the notion that the C-terminal region contains amino acid residues for interaction with DEN-4 NS3.

Amino Acid Sequence↗