Dengue virus NS3 serine protease. Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects.
The mosquito-borne dengue viruses are widespread human pathogens causing dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, placing 40% of the world's population at risk with no effective treatment. The viral genome is a positive strand RNA that encodes a single polyprotein precursor. Processing of the polyprotein precursor into mature proteins is carried out by the host signal peptidase and by NS3 serine protease, which requires NS2B as a cofactor. We report here the crystal structure of the NS3 serine protease domain at 2.1 A resolution. This structure of the protease combined with modeling of peptide substrates into the active site suggests identities of residues involved in substrate recognition as well as providing a structural basis for several mutational effects on enzyme activity. This structure will be useful for development of specific inhibitors as therapeutics against dengue and other flaviviral proteases. (+info)
Variation in oral susceptibility to dengue type 2 virus of populations of Aedes aegypti from the islands of Tahiti and Moorea, French Polynesia.
Twenty three samples of Aedes aegypti populations from the islands of Tahiti and Moorea (French Polynesia) were tested for their oral susceptibility to dengue type 2 virus. The high infection rates obtained suggest that the artificial feeding protocol used was more efficient than those previously described. Statistical analysis of the results allowed us to define two distinct geographic areas on Tahiti with respect to the susceptibility of Ae. aegypti: the east coast, with homogeneous infection rates, and the west coast, with heterogeneous infection rates. No geographic differences could be demonstrated on Moorea. The possible mechanisms of this phenomenon are discussed in connection with recent findings on the variability of susceptibility of Ae. aegypti to insecticides. (+info)
Replication of dengue type 2 virus in Culex quinquefasciatus (Diptera: Culicidae).
We were able to infect Culex quinquefasciatus by the parenteral route with dengue virus type 2. The percentage of mosquitoes infected was dose dependent and we obtained a rate of 45.6% infected Cx. quinquefasciatus when a 10(5.9) MID50 (mosquito infectious dose for 50% of the individuals as measured in Aedes aegypti) of dengue virus type 2 per mosquito was used. Infection was detected by an immunofluorescent assay performed on mosquito head squashes 14 days after infection. The replication of dengue virus in Cx. quinquefasciatus was either at a very low level of magnitude or generated a large number of noninfectious particles since the triturated bodies of infected Cx. quinquefasciatus did not infect Ae. aegypti mosquitoes when inoculated parenterally. We were unable to infect Cx. quinquefasciatus females orally with an artificial meal that infected 100% of Ae. aegypti females. These findings lead us to agree with the consensus that Cx. quinquefasciatus should not be considered a biological vector of dengue viruses. (+info)
Bacterial lipopolysaccharide inhibits dengue virus infection of primary human monocytes/macrophages by blockade of virus entry via a CD14-dependent mechanism.
Monocytes/macrophages (MO/Mphi) are the major target cells for both dengue virus (DV) and bacterial lipopolysaccharide (LPS), and the aim of this study was to define their interactions. We had found that LPS markedly suppressed DV infection of primary human MO/Mphi when it was added to cultures prior to or together with, but not after, viral adsorption. The inhibitory effect of LPS was direct and specific and was not mediated by LPS-induced secretion of cytokines and chemokines such as tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12, alpha interferon, MIP-1alpha, and RANTES. In fact, productive DV infection was not blocked but was just postponed by LPS, with a time lag equal to one viral replication cycle. Time course studies demonstrated that LPS was only effective in suppressing DV infection of MO/Mphi that had not been previously exposed to the virus. At various time points after viral adsorption, the level of unbound viruses that remained free in the culture supernatants of LPS-pretreated cultures was much higher than that of untreated controls. These observations suggest that the LPS-induced suppression of DV replication was at the level of virus attachment and/or entry. Blockade of the major LPS receptor, CD14, with monoclonal antibodies MY4 or MoS39 failed to inhibit DV infection but could totally abrogate the inhibitory effect of LPS. Moreover, human serum could significantly enhance the LPS-induced DV suppression in a CD14-dependent manner, indicating that the "binding" of LPS to CD14 was critical for the induction of virus inhibition. Taken together, our results suggest that LPS blocked DV entry into human MO/Mphi via its receptor CD14 and that a CD14-associated cell surface structure may be essential for the initiation of a DV infection. (+info)
The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids.
NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity. (+info)
Effects of dengue fever during pregnancy in French Guiana.
To determine the effects of dengue fever (DF) during pregnancy, pregnant women presenting with a dengue-like syndrome at a hospital in Saint-Laurent du Maroni, French Guiana, from 1 January 1992 to 1 April 1998 were studied. The diagnosis of DF was made by serological tests, virus isolation on AP 61 mosquito cells, and/or reverse transcriptase polymerase chain reaction analysis. Twenty-two women had either probable or confirmed DF. Dengue virus serotype 2 was detected in four cases, and dengue virus serotype 1 was detected in one. Three fetuses died following the onset of the disease, and three cases of prematurity occurred. All infants appeared normal during physical examination, and no neonatal DF was diagnosed. In conclusion, DF in pregnant women did not cause any infant abnormality, but it may have been responsible for fetal death. The rate of fetal death associated with DF (13.6%) was much higher than the mean rate for the gynecology unit at the hospital (1.9%). However, these differences were not significant, and consequently these preliminary results need to be confirmed. (+info)
Bystander target cell lysis and cytokine production by dengue virus-specific human CD4(+) cytotoxic T-lymphocyte clones.
Dengue hemorrhagic fever, the severe form of dengue virus infection, is believed to be an immunopathological response to a secondary infection with a heterologous serotype of dengue virus. Dengue virus capsid protein-specific CD4(+) cytotoxic T-lymphocyte (CTL) clones were shown to be capable of mediating bystander lysis of non-antigen-presenting target cells. After activation by anti-CD3 or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Lysis of HepG2 cells suggests a potential role for CD4(+) CTL in the liver involvement observed during dengue virus infection. Three CD4(+) CTL clones were demonstrated to lyse cognate, antigen-presenting target cells by a mechanism that primarily involves perforin, while bystander lysis occurred through Fas/Fas ligand interactions. In contrast, one clone used a Fas/Fas ligand mechanism to lyse both cognate and bystander targets. Cytokine production by the CTL clones was also examined. In response to stimulation with D2 antigen, CD4(+) T-cell clones produced gamma interferon, tumor necrosis factor alpha (TNF-alpha) and TNF-beta. The data suggest that CD4(+) CTL clones may contribute to the immunopathology observed upon secondary dengue virus infections through direct cytolysis and/or cytokine production. (+info)
Evaluation of the MRL diagnostics dengue fever virus IgM capture ELISA and the PanBio Rapid Immunochromatographic Test for diagnosis of dengue fever in Jamaica.
We evaluated two new commercial dengue diagnostic tests, the MRL Diagnostics Dengue Fever Virus IgM Capture ELISA and the PanBio Rapid Immunochromatographic Test, on serum samples collected during a dengue epidemic in Jamaica. The MRL ELISA method correctly identified 98% (78 of 80) of the samples as dengue positive, while the PanBio test identified 100% (80 of 80). Both tests were 100% (20 samples of 20) specific. (+info)