Dengue-3 virus genomic differences that correlate with in vitro phenotype on a human cell line but not with disease severity

We compared the full genome sequence of nine clinical isolates of dengue virus obtained during an epidemic of dengue-3 in French Polynesia in 1989, from patients with various presentations of disease. The isolates, all belonging to Genotype I, had 25 amino acid substitutions. There was no association with disease severity. When cultured in the K562 human erythroleukemia cell line, the isolates induced a range of cell growth inhibitions that was not associated with the degree of disease severity. By contrast, some substitutions--charge changes in NS1 and NS5, side-chain differences in NS1, loss of the E-153 potential glycosylation site, and 11 nucleotide insertions in the 3'UTR--that have been suggested to result in an increase or attenuation of dengue infection, appeared to be associated with the level of inhibition. These data represent the first documented study of an association between differences in the full dengue-3 genome of clinical isolates and the in vitro phenotype of these isolates on a human cell line.     

Two SCA (stigma/style cysteine-rich adhesin) isoforms show structural differences that correlate with their levels of in vitro pollen tube adhesion activity

Lily pollen tubes grow adhering to an extracellular matrix produced by the transmitting tract epidermis in a hollow style. SCA, a small ( approximately 9.4 kDa), basic protein plus low esterified pectin from this extracellular matrix are involved in the pollen tube adhesion event. The mode of action for this adhesion event is unknown. We partially separated three SCA isoforms from the lily stigma in serial size exclusion column fractions (SCA1, 9370 Da; SCA2, 9384 Da; SCA3, 9484 Da). Peptide sequencing analysis allowed us to determine two amino acid variations in SCA3, compared with SCA1. For SCA2, however, there are more sequence variations yet to be identified. Our structural homology and molecular dynamics modeling results show that SCA isoforms have the plant nonspecific lipid transfer protein-like structure: a globular shape of the orthogonal 4-helix bundle architecture, four disulfide bonds, an internal hydrophobic and solvent-inaccessible cavity, and a long C-terminal tail. The Ala(71) in SCA3, replacing the Gly(71) in SCA1, has no predictable effect on structure. The Arg(26) in SCA3, replacing the Gly(26) in SCA1, is predicted to cause structural changes that result in a significantly reduced volume for the internal hydrophobic cavity in SCA3. The volume of the internal cavity fluctuates slightly during the molecular dynamics simulation, but overall, SCA1 displays a larger cavity than SCA3. SCA1 displays higher activity than SCA3 in the in vitro pollen tube adhesion assay. No differences were found between the two SCAs in a binding assay with pectin. The larger size of the hydrophobic cavity in SCA1 correlates with its higher adhesion activity.     

Oxidation state governs structural transitions in peroxiredoxin II that correlate with cell cycle arrest and recovery

Inactivation of eukaryotic 2-Cys peroxiredoxins (Prxs) by hyperoxidation has been proposed to promote accumulation of hydrogen peroxide (H₂O₂) for redox-dependent signaling events. We examined the oxidation and oligomeric states of PrxI and -II in epithelial cells during mitogenic signaling and in response to fluxes of H₂O₂. During normal mitogenic signaling, hyperoxidation of PrxI and -II was not detected. In contrast, H₂O₂-dependent cell cycle arrest was correlated with hyperoxidation of PrxII, which resulted in quantitative recruitment of ~66- and ~140-kD PrxII complexes into large filamentous oligomers. Expression of cyclin D1 and cell proliferation did not resume until PrxII-SO₂H was reduced and native PrxII complexes were regenerated. Ectopic expression of PrxI or -II increased Prx-SO₂H levels in response to oxidant exposure and failed to protect cells from arrest. We propose a model in which Prxs function as peroxide dosimeters in subcellular processes that involve redox cycling, with hyperoxidation controlling structural transitions that alert cells of perturbations in peroxide homeostasis.     

Dengue viral infections; pathogenesis and epidemiology

Dengue viral infections affect up to 100 million individuals per year. Dengue haemorrhagic fever is a clinical form of disease characterised by intravascular fluid loss. There has been a marked increase in the incidence of this form of the disease over the last few decades, associated with significant mortality, particularly in the paediatric population. A number of theories relating to the pathogenesis of dengue haemorrhagic fever exist that have evolved from the analysis of the epidemiology of this disease. Virological and immunopathological factors are both important but the exact mechanisms for the disease are unknown.     

Protein biophysical properties that correlate with crystallization success in Thermotoga maritima: maximum clustering strategy for structural genomics

Cost and time reduction are two of the driving forces in the development of new strategies for protein crystallization and subsequent structure determination. Here, we report the analysis of the Thermotoga maritima proteome, in which we compare the proteins that were successfully expressed, purified and crystallized versus the rest of the proteome. This set of almost 500 proteins represents one of the largest, internally consistent, protein expression and crystallization datasets available. The analysis shows that individual parameters, such as isoelectric point, sequence length, average hydropathy, low complexity regions (SEG), and combinations of these biophysical properties for crystallized proteins define a distinct subset of the T. maritima proteome. The distribution profiles of the various biophysical properties in the expression/crystallization set are then used to extract rules to improve target selection and improve the efficiency and output of structural genomics, as well as general structural biology efforts.     

During lytic infection herpes simplex virus type 1 is associated with histones bearing modifications that correlate with active transcription

Herpes simplex virus type 1 (HSV-1) is a large (150-kb) double-stranded DNA virus that forms latent infections in neuronal cells of the human peripheral nervous system. Previous work determined that the HSV-1 genome is found in an ordered nucleosomal structure during latent infection. However, during lytic infection, it was unclear whether viral DNA was in a chromatin state. We examined HSV-1 during lytic infection using micrococcal nuclease digestion and chromatin immunoprecipitation. The HSV-1 genome is at least partially nucleosomal, although apparently not in a regular repeating structure. Analysis of histones associated with HSV-1, within both the promoter and the transcribed regions, revealed covalent amino tail modifications similar to those associated with active host mammalian genes. Certain of the modifications were detected in the temporal order expected of the immediate-early, early, and late gene classes. These data suggest that productive infection may be accompanied by acquisition of a permissive chromatin state.     

Qualitative differences in nuclear proteins correlate with neuronal terminal differentiation

1. Protein composition of neuronal nuclei was studied at two stages of brain maturation, i.e., before (embryonic day 16; E16) and after (postnatal day 10; P10) shortening of the nucleosomal repeat length. Glial nuclei were analyzed in parallel as a control. 2. Total nuclear or HCl- and 5% perchloric acid (PCA)-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1 zero with differentiation, although the H1 class undergoes an overall decrease. 4. The chromatin of mature neurons is also enriched in the ubiquinated form of histone H2A (A24), while the high-mobility group (HMG) proteins 1 and 2 seem to decrease slightly relative to core histones. 5. Both quantitative and qualitative differences in the abundance of nonhistone proteins relative to histones accompany neuronal terminal differentiation.     

Plant‐produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice

The global Zika virus (ZIKV) outbreak and its link to foetal and newborn microcephaly and severe neurological complications in adults call for the urgent development of ZIKV vaccines. In response, we developed a subunit vaccine based on the ZIKV envelope (E) protein and investigated its immunogenicity in mice. Transient expression of ZIKV E (zE) resulted in its rapid accumulation in leaves of Nicotiana benthamiana plants. Biochemical analysis revealed that plant‐produced ZIKV E (PzE) exhibited specific binding to a panel of monoclonal antibodies that recognize various zE conformational epitopes. Furthermore, PzE can be purified to >90% homogeneity with a one‐step Ni²⁺ affinity chromatography process. PzE are found to be highly immunogenic, as two doses of PzE elicited both potent zE‐specific antibody and cellular immune responses in mice. The delivery of PzE with alum induced a mixed Th1/Th2 immune response, as the antigen‐specific IgG isotypes were a mixture of high levels of IgG1/IgG2c and splenocyte cultures from immunized mice secreted significant levels of IFN‐gamma, IL‐4 and IL‐6. Most importantly, the titres of zE‐specific and neutralizing antibodies exceeded the threshold that correlates with protective immunity against multiple strains of ZIKV. Thus, our results demonstrated the feasibility of plant‐produced ZIKV protein antigen as effective, safe and affordable vaccines against ZIKV.     

Pretreatment sequence diversity differences in the full-length hepatitis C virus open reading frame correlate with early response to therapy

Pegylated alpha interferon and ribavirin therapy for hepatitis C virus (HCV) genotype 1 infection fails for half of Caucasian American patients (CA) and more often for African Americans (AA). The reasons for these low response rates are unknown. HCV is highly genetically variable, but it is unknown how this variability affects response to therapy. To assess effects of viral diversity on response to therapy, the complete pretreatment genotype 1 HCV open reading frame was sequenced using samples from 94 participants in the Virahep-C study. Sequences from patients with >3.5 log declines in viral RNA levels by day 28 (marked responders) were more variable than those from patients with declines of <1.4 log (poor responders) in NS3 and NS5A for genotype 1a and in core and NS3 for genotype 1b. These correlations remained when all T-cell epitopes were excluded, indicating that these differences were not due to differential immune selection. When the sequences were compared by race of the patients, higher diversity in CA patients was found in E2 and NS2 but only for genotype 1b. Core, NS3, and NS5A can block the action of alpha interferon in vitro; hence, these genetic patterns are consistent with multiple amino acid variations independently impairing the function of HCV proteins that counteract interferon responses in humans, resulting in HCV strains with variable sensitivity to therapy. No evidence was found for novel HCV strains in the AA population, implying that AA patients may be infected with a higher proportion of the same resistant strains that are found in CA patients.     

A comparative study of the alpha-subdomains of bovine and human alpha-lactalbumin reveals key differences that correlate with molten globule stability

The alpha-lactalbumins form stable molten globule states under a range of conditions, with the low pH form being the best characterized. The stability of the molten globule varies among different members of this family, but the origin of the stability difference is not clear. We compare the folding and stability of alpha-subdomain constructs of human and bovine alpha-lactalbumin. Previous studies have demonstrated that the isolated alpha-subdomain of human alpha-lactalbumin folds and forms a molten globule state. The minimum core construct has been defined to include the A, B, and D alpha-helices and the C-terminal 3(10) helix. A construct corresponding to the same region of bovine alpha-lactalbumin is much less structured and less stable than the human alpha-lactalbumin construct. Addition of the C-helix to generate a 75-residue bovine construct does not lead to a significant increase in structure or stability. This construct (AB-CD/3(10)) contains the entire alpha-subdomain of bovine alpha-lactalbumin. Thus molten globule formation in the human protein, but not in the bovine protein, can be rationalized on the basis of a stable alpha-subdomain. Interactions involving more of the protein chain are required to generate a well structured molten globule in the bovine protein. Comparison of AB-CD/3(10) to the molten globule formed by the intact protein and to the protein with the 6-120 disulfide reduced indicates that both the beta-subdomain and the 6-120 disulfide play a role in stabilizing the bovine alpha-lactalbumin molten globule.     

Catalytically active Dengue virus NS3 protease forms aggregates that are separable by size exclusion chromatography

An active form of the Dengue virus protease NS3 (CF40.Gly.NS3pro) was expressed in Escherichia coli. This construct consists of a critical 40 amino acid cofactor domain from NS2B fused to the N-terminal 184 amino acid protease domain of NS3 via a flexible, covalent linker (Gly(4)SerGly(4)). The recombinantly produced protein is soluble and has a hexa-histidine tag engineered at the N-terminus for ease of purification using metal affinity chromatography. However, the presence of lower molecular weight impurities after affinity chromatography indicated the need for additional purification steps. The consistent appearance of these impurities suggested that they may be the products of proteolysis and/or auto-proteolysis. The latter possibility was subsequently excluded by the observation of the same impurities in a purified, catalytically inactive form of the recombinant protease (CF40.Gly.NS3pro.SA). Further analysis indicated that these impurities may represent premature translation termination products. Regardless of their origin, they were shown to form various sized aggregates with full-length CF40.Gly.NS3pro that can be separated by size exclusion chromatography, yielding fractions of active protease of sufficient purity for crystallisation trials. The ultimate goal of these studies is to obtain a crystal structure of a catalytically active form of the Dengue virus NS3 protease for structure-based drug design.     

Exploitation of cellular pathways by Dengue virus

Dengue virus (DENV) is the causative agent of the most prevalent arthropod-borne viral disease, thus representing a significant global health burden. Because of its limited coding capacity, DENV exploits components and pathways of the host cell to assure productive replication. In the past few years, important insights into this intimate interaction between DENV and the host cell have been gained. These include the identification of the ER-associated degradation pathway, autophagy, the unfolded protein response or lipid droplets that all play a crucial role for efficient DENV replication. In addition, strategies used by the virus to combat innate antiviral responses have been unraveled. Improving our understanding of the DENV-host cell relation will facilitate our attempts to develop efficient antiviral strategies.     

Glycyrrhizic acid derivatives as Dengue virus inhibitors

Dengue virus (DENV) is one of the most geographically distributed pathogenic flaviviruses transmitted by mosquitoes Aedes sps. In this study, the structure-antiviral activity relationships of Glycyrrhizic acid (GL) derivatives was evaluated by the inhibitory assays on the cytopathic effect (CPE) and viral infectivity of DENV type 2 (DENV2) in Vero E6 cells. GL (96% purity) had a low cytotoxicity to Vero E6 cells, inhibited DENV2-induced CPE, and reduced the DENV-2 infectivity with the IC50 of 8.1 μM. Conjugation of GL with amino acids or their methyl esters and the introduction of aromatic acylhydrazide residues into the carbohydrate part strongly influenced on the antiviral activity. Among compounds tested GL conjugates with isoleucine 13 and 11-aminoundecanoic acid 17 were found as potent anti-DENV2 inhibitors (IC50 1.2–1.3 μM). Therefore, modification of GL is a perspective way in the search of new antivirals against DENV2 infection.     

Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice

The development of small animal models that elicit human immune responses to dengue virus (DENV) is important since prior immunity is a major risk factor for developing severe dengue disease. This study evaluated anti-DENV human antibody (hAb) responses generated from immortalized B cells after DENV-2 infection in NOD-scid IL2rγ^null mice that were co-transplanted with human fetal thymus and liver tissues (BLT-NSG mice). DENV-specific human antibodies predominantly of the IgM isotype were isolated during acute infection and in convalescence. We found that while a few hAbs recognized the envelope protein produced as a soluble recombinant, a number of hAbs only recognized epitopes on intact virions. The majority of the hAbs isolated during acute infection and in immune mice were serotype-cross-reactive and poorly neutralizing. Viral titers in immune BLT-NSG mice were significantly decreased after challenge with a clinical strain of dengue. DENV-specific hAbs generated in BLT-NSG mice share some of the characteristics of Abs isolated in humans with natural infection. Humanized BLT-NSG mice provide an attractive preclinical platform to assess the immunogenicity of candidate dengue vaccines.     

Sex differences in arterial identity correlate with neointimal hyperplasia after balloon injury

Molecular Biology Reports - Endovascular treatment of atherosclerotic arterial disease exhibits sex differences in clinical outcomes including restenosis. However, sex-specific differences in...     

Dengue virus nonstructural protein 1 is expressed in a glycosyl-phosphatidylinositol-linked form that is capable of signal transduction.

Dengue virus nonstructural protein 1 (NS1) is expressed on the surface of infected cells and is a target of human antibody responses to dengue virus infection. We show here that dengue virus uses the cellular glycosyl-phosphatidylinositol (GPI) linkage pathway to express a GPI-anchored form of NS1 and that GPI anchoring imparts a capacity for signal transduction in response to binding of NS1-specific antibody. This study is the first to identify GPI linkage of a virus-encoded protein. The GPI anchor addition signal for NS1 was identified, by transfection of HeLa cells with dengue cDNA constructs, as a downstream hydrophobic domain in NS2A. GPI linkage of NS1 in both transfected and infected cells was demonstrated by cleavage of NS1 from the surface by PI-specific phospholipase C and by metabolic incorporation of the GPI-specific components ethanolamine and inositol. In common with other GPI-anchored proteins, addition of specific antibody resulted in signal transduction, as evidenced by tyrosine phosphorylation of cellular proteins. Antibody-induced signal transduction by GPI-linked NS1 suggests a mechanism of cellular activation that may contribute to the pathogenesis of human dengue disease. Signal transduction by a GPI-anchored viral antigen interacting with a specific antibody that it induces is a new concept in the pathogenesis of viral disease.