Primary infection of BALB/c mice with a dengue virus type 4 strain leads to kidney injury

BACKGROUND Dengue is a disease caused by dengue virus (DENV-1 through -4). Among the four serotypes, DENV-4 remains the least studied. Acute kidney injury is a potential complication of dengue generally associated with severe dengue infection. OBJECTIVES The goal of this study was to investigate the alterations caused by experimental dengue infection in the kidney of adult BALB/c mice. METHODS In this study, BALB/c mice were infected through the intravenous route with a DENV-4 strain, isolated from a human patient. The kidneys of the mice were procured and subject to histopathological and ultrastructural analysis. FINDINGS The presence of the viral antigen was confirmed through immunohistochemistry. Analysis of tissue sections revealed the presence of inflammatory cell infiltrate throughout the parenchyma. Glomerular enlargement was a common find. Necrosis of tubular cells and haemorrhage were also observed. Analysis of the kidney on a transmission electron microscope allowed a closer look into the necrotic tubular cells, which presented nuclei with condensed chromatin, and loss of cytoplasm. MAIN CONCLUSIONS Even though the kidney is probably not a primary target of dengue infection in mice, the inoculation of the virus in the blood appears to damage the renal tissue through local inflammation.

Phenolic Profile and Antioxidant Properties in Extracts of Developing Açaí (Euterpe oleracea Mart.) Seeds.

We investigated changes in the phenolic profile and antioxidant properties in the extracts of developing seeds of açaí (Euterpe oleracea). Four developmental stages were evaluated, with earlier stages displaying higher antioxidant activity and polyphenols content, while mass spectrometry analysis identified procyanidins (PCs) as the major components of the extracts in all stages. B-type PCs varied from dimers to decamers, with A-type linkages in a smaller number. Extracted PCs decreased in average length from 20.5 to 10.1 along seed development. PC composition indicated that (-)-epicatechin corresponded to over 95% of extension units in all stages, while (+)-catechin presence as the starter unit increased from 42 to 78.8% during seed development. This variation was correlated to the abundance of key enzymes for PC biosynthesis during seed development. This study is the first to report PC content and composition variations during açaí seed development, which can contribute to studies on the plant's physiology and biotechnological applications.

Cytokines and inflammatory mediators: Markers involved in interstitial damage to the pancreas in two dengue fever cases associated with acute pancreatitis.

Dengue viral (DENV) infections can lead to acute pancreatitis and associated tissue damage. This study examined the pancreas from two fatal cases of DENV for histopathological changes as well as for the detection of cytokines, and other inflammatory mediators. Tissue sections were prepared for examination by ultrastructural and histopathological techniques. Sections from the pancreas of non-infected individuals were prepared in parallel as a control. The presence of viral replication in macrophages was detected by co-staining for the proteins NS3 and CD68 by immunofluorescence. Immunohistochemistry was used to detect cells that expressed cytokines and inflammatory mediators to characterize the inflammatory response. Edema, acinar necrosis and fibrosis areas associated with a mononuclear infiltrate were found in infected tissues. The major site of virus replication appeared to be macrophages based on their exclusive presentation of the viral protein NS3. Pancreatic tissues from the infected individuals also displayed increased levels of high mobility group box-1, caspase-3, gelatinase B and tumor necrosis factor alpha compared to controls. The presence of virus replicating macrophages in the pancreas was associated with multiple changes in tissue structure that included elevated levels of cytokines and inflammatory markers that may differentiate acute pancreatitis due to DENV infections from other causes.

ApoA1 Neutralizes Proinflammatory Effects of Dengue Virus NS1 Protein and Modulates Viral Immune Evasion.

Dengue is a mosquito-borne infectious disease that is highly endemic in tropical and subtropical countries. Symptomatic patients can rapidly progress to severe conditions of hemorrhage, plasma extravasation, and hypovolemic shock, which leads to death. The blood tests of patients with severe dengue typically reveal low levels of high-density lipoprotein (HDL), which is responsible for reverse cholesterol transport (RCT) and regulation of the lipid composition in peripheral tissues. It is well known that dengue virus (DENV) depends on membrane cholesterol rafts to infect and to replicate in mammalian cells. Here, we describe the interaction of DENV nonstructural protein 1 (NS1) with apolipoprotein A1 (ApoA1), which is the major protein component of HDL. NS1 is secreted by infected cells and can be found circulating in the serum of patients with the onset of symptoms. NS1 concentrations in plasma are related to dengue severity, which is attributed to immune evasion and an acute inflammatory response. Our data show that the DENV NS1 protein induces an increase of lipid rafts in noninfected cell membranes and enhances further DENV infection. We also show that ApoA1-mediated lipid raft depletion inhibits DENV attachment to the cell surface. In addition, ApoA1 is able to neutralize NS1-induced cell activation and to prevent NS1-mediated enhancement of DENV infection. Furthermore, we demonstrate that the ApoA1 mimetic peptide 4F is also capable of mediating lipid raft depletion to control DENV infection. Taken together, our results suggest the potential of RCT-based therapies for dengue treatment. These results should motivate studies to assess the importance of RCT in DENV infection in vivo. IMPORTANCE DENV is one of the most relevant mosquito-transmitted viruses worldwide, infecting more than 390 million people every year and leading to more than 20 thousand deaths. Although a DENV vaccine has already been approved, its potential side effects have hampered its use in large-scale immunizations. Therefore, new treatment options are urgently needed to prevent disease worsening or to improve current clinical management of severe cases. In this study, we describe a new interaction of the NS1 protein, one of the major viral components, with a key component of HDL, ApoA1. This interaction seems to alter membrane susceptibility to virus infection and modulates the mechanisms triggered by DENV to evade the immune response. We also propose the use of a mimetic peptide named 4F, which was originally developed for atherosclerosis, as a potential therapy for relieving DENV symptoms.

Enzymatic and structural properties of human glutamine:fructose-6-phosphate amidotransferase 2 (hGFAT2).

Glycoconjugates play a central role in several cellular processes, and alteration in their composition is associated with numerous human pathologies. Substrates for cellular glycosylation are synthesized in the hexosamine biosynthetic pathway, which is controlled by the glutamine:fructose-6-phosphate amidotransfera-se (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer; however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in Escherichia coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the expected ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much more slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerize fructose-6-phosphate into glucose-6-phosphate even in the presence of equimolar amounts of glutamine, which results in unproductive glutamine hydrolysis. Structural analysis of a three-dimensional model of rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in the glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations suggest that this loop is the most flexible portion of the protein and plays a key role on conformational states of hGFAT2. Thus, our study provides the first comprehensive set of data on the structure, kinetics, and mechanics of hGFAT2, which will certainly contribute to further studies on the (patho)physiology of hGFAT2.

Inflammatory signaling in dengue-infected platelets requires translation and secretion of nonstructural protein 1.

Emerging evidence identifies major contributions of platelets to inflammatory amplification in dengue, but the mechanisms of infection-driven platelet activation are not completely understood. Dengue virus nonstructural protein-1 (DENV NS1) is a viral protein secreted by infected cells with recognized roles in dengue pathogenesis, but it remains unknown whether NS1 contributes to the inflammatory phenotype of infected platelets. This study shows that recombinant DENV NS1 activated platelets toward an inflammatory phenotype that partially reproduced DENV infection. NS1 stimulation induced translocation of α-granules and release of stored factors, but not of newly synthesized interleukin-1ß (IL-1ß). Even though both NS1 and DENV were able to induce pro-IL-1ß synthesis, only DENV infection triggered caspase-1 activation and IL-1ß release by platelets. A more complete thromboinflammatory phenotype was achieved by synergistic activation of NS1 with classic platelet agonists, enhancing α-granule translocation and inducing thromboxane A2 synthesis (thrombin and platelet-activating factor), or activating caspase-1 for IL-1ß processing and secretion (adenosine triphosphate). Also, platelet activation by NS1 partially depended on toll-like receptor-4 (TLR-4), but not TLR-2/6. Finally, the platelets sustained viral genome translation and replication, but did not support the release of viral progeny to the extracellular milieu, characterizing an abortive viral infection. Although DENV infection was not productive, translation of the DENV genome led to NS1 expression and release by platelets, contributing to the activation of infected platelets through an autocrine loop. These data reveal distinct, new mechanisms for platelet activation in dengue, involving DENV genome translation and NS1-induced platelet activation via platelet TLR4.

Dengue virus nonstructural 3 protein interacts directly with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and reduces its glycolytic activity.

Dengue is an important mosquito-borne disease and a global public health problem. The disease is caused by dengue virus (DENV), which is a member of the Flaviviridae family and contains a positive single-stranded RNA genome that encodes a single precursor polyprotein that is further cleaved into structural and non-structural proteins. Among these proteins, the non-structural 3 (NS3) protein is very important because it forms a non-covalent complex with the NS2B cofactor, thereby forming the functional viral protease. NS3 also contains a C-terminal ATPase/helicase domain that is essential for RNA replication. Here, we identified 47 NS3-interacting partners using the yeast two-hybrid system. Among those partners, we highlight several proteins involved in host energy metabolism, such as apolipoprotein H, aldolase B, cytochrome C oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH directly binds full-length NS3 and its isolated helicase and protease domains. Moreover, we observed an intense colocalization between the GAPDH and NS3 proteins in DENV2-infected Huh7.5.1 cells, in NS3-transfected BHK-21 cells and in hepatic tissue from a fatal dengue case. Taken together, these results suggest that the human GAPDH-DENV NS3 interaction is involved in hepatic metabolic alterations, which may contribute to the appearance of steatosis in dengue-infected patients. The interaction between GAPDH and full-length NS3 or its helicase domain in vitro as well as in NS3-transfected cells resulted in decreased GAPDH glycolytic activity. Reduced GAPDH glycolytic activity may lead to the accumulation of metabolic intermediates, shifting metabolism to alternative, non-glycolytic pathways. This report is the first to identify the interaction of the DENV2 NS3 protein with the GAPDH protein and to demonstrate that this interaction may play an important role in the molecular mechanism that triggers hepatic alterations.

Isosorbide-based peptidomimetics as inhibitors of hepatitis C virus serine protease.

Hepatitis C infection is a cause of chronic liver diseases such as cirrhosis and carcinoma. The current therapy for hepatitis C has limited efficacy and low tolerance. The HCV encodes a serine protease which is critical for viral replication, and few protease inhibitors are currently on the market. In this paper, we describe the synthesis and screening of novel isosorbide-based peptidomimetic inhibitors, in which the compounds 1d, 1e, and 1i showed significant inhibition of the protease activity in vitro at 100µM. The compound 1e also showed dose-response (IC50=36±3µM) and inhibited the protease mutants D168A and V170A at 100µM, indicating it as a promising inhibitor of the HCV NS3/4A protease. Our molecular modeling studies suggest that the activity of 1e is associated with a change in the interactions of S2 and S4 subsites, since that the increased flexibility favors a decrease in activity against D168A, whereas the appearance of a hydrophobic cavity in the S4 subsite increase the inhibition against V170A strain.

The Complement System in Flavivirus Infections.

The incidence of flavivirus infections has increased dramatically in recent decades in tropical and sub-tropical climates worldwide, affecting hundreds of millions of people each year. The Flaviviridae family includes dengue, West Nile, Zika, Japanese encephalitis, and yellow fever viruses that are typically transmitted by mosquitoes or ticks, and cause a wide range of symptoms, such as fever, shock, meningitis, paralysis, birth defects, and death. The flavivirus genome is composed of a single positive-sense RNA molecule encoding a single viral polyprotein. This polyprotein is further processed by viral and host proteases into three structural proteins (C, prM/M, E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) that are involved in viral replication and pathogenicity. The complement system has been described to play an important role in flavivirus infection either by protecting the host and/or by influencing disease pathogenesis. In this mini-review, we will explore the role of complement system inhibition and/or activation against infection by the Flavivirus genus, with an emphasis on dengue and West Nile viruses.

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins.

Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. IMPORTANCE: Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system.

Recombinant dengue 2 virus NS3 protein conserves structural antigenic and immunological properties relevant for dengue vaccine design.

The NS3 protein is a multifunctional non-structural protein of flaviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2. The full-length NS3 gene was cloned and expressed as a His-tagged fusion protein in Escherichia coli. The pNS3 protein was purified by two chromatography steps. The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This purified protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-α in mice splenocytes, suggesting a predominantly Th-1-type T cell response. Immunized BALB/c mice with the purified NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specific recognition of native dengue protein by Western blotting and immunofluorescence techniques. The successfully purified recombinant protein was able to preserv the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations.

pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities.

The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication.

Synthetic 1,4-pyran naphthoquinones are potent inhibitors of dengue virus replication.

Dengue virus infection is a serious public health problem in endemic areas of the world where 2.5 billion people live. Clinical manifestations of the Dengue infection range from a mild fever to fatal cases of hemorrhagic fever. Although being the most rapidly spreading mosquito-borne viral infection in the world, until now no strategies are available for effective prevention or control of Dengue infection. In this scenario, the development of compounds that specifically inhibit viral replication with minimal effects to the human hosts will have a substantial effect in minimizing the symptoms of the disease and help to prevent viral transmission in the affected population. The aim of this study was to screen compounds with potential activity against dengue virus from a library of synthetic naphthoquinones. Several 1,2- and 1,4-pyran naphthoquinones were synthesized by a three-component reaction of lawsone, aldehyde (formaldehyde or arylaldehydes) and different dienophiles adequately substituted. These compounds were tested for the ability to inhibit the ATPase activity of the viral NS3 enzyme in in vitro assays and the replication of dengue virus in cultured cells. We have identified two 1,4-pyran naphthoquinones, which inhibited dengue virus replication in mammal cells by 99.0% and three others that reduced the dengue virus ATPase activity of NS3 by two-fold in in vitro assays.

The dengue vector Aedes aegypti contains a functional high mobility group box 1 (HMGB1) protein with a unique regulatory C-terminus.

The mosquito Aedes aegypti can spread the dengue, chikungunya and yellow fever viruses. Thus, the search for key molecules involved in the mosquito survival represents today a promising vector control strategy. High Mobility Group Box (HMGB) proteins are essential nuclear factors that maintain the high-order structure of chromatin, keeping eukaryotic cells viable. Outside the nucleus, secreted HMGB proteins could alert the innate immune system to foreign antigens and trigger the initiation of host defenses. In this work, we cloned and functionally characterized the HMGB1 protein from Aedes aegypti (AaHMGB1). The AaHMGB1 protein typically consists of two HMG-box DNA binding domains and an acidic C-terminus. Interestingly, AaHMGB1 contains a unique alanine/glutamine-rich (AQ-rich) C-terminal region that seems to be exclusive of dipteran HMGB proteins. AaHMGB1 is localized to the cell nucleus, mainly associated with heterochromatin. Circular dichroism analyses of AaHMGB1 or the C-terminal truncated proteins revealed α-helical structures. We showed that AaHMGB1 can effectively bind and change the topology of DNA, and that the AQ-rich and the C-terminal acidic regions can modulate its ability to promote DNA supercoiling, as well as its preference to bind supercoiled DNA. AaHMGB1 is phosphorylated by PKA and PKC, but not by CK2. Importantly, phosphorylation of AaHMGB1 by PKA or PKC completely abolishes its DNA bending activity. Thus, our study shows that a functional HMGB1 protein occurs in Aedes aegypt and we provide the first description of a HMGB1 protein containing an AQ-rich regulatory C-terminus.

Dengue virus capsid protein binding to hepatic lipid droplets (LD) is potassium ion dependent and is mediated by LD surface proteins.

Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of -19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at +13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C protein-functionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na(+)/K(+)-ATPase in DENV-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C protein ligand on the surface of LDs.

The major surface carbohydrates of the Echinococcus granulosus cyst: mucin-type O-glycans decorated by novel galactose-based structures.

The cestodes constitute important but understudied human and veterinary parasites. Their surfaces are rich in carbohydrates, on which very little structural information is available. The tissue-dwelling larva (hydatid cyst) of the cestode Echinococcus granulosus is outwardly protected by a massive layer of carbohydrate-rich extracellular matrix, termed the laminated layer. The monosaccharide composition of this layer suggests that its major carbohydrate components are exclusively mucin-type O-glycans. We have purified these glycans after their release from the crude laminated layer and obtained by MS and NMR the complete structure of 10 of the most abundant components. The structures, between two and six residues in length, encompass a limited number of biosynthetic motifs. The mucin cores 1 and 2 are either nondecorated or elongated by a chain of Galpbeta1-3 residues. This chain can be capped by a single Galpalpha1-4 residue, such capping becoming more dominant with increasing chain size. In addition, the core 2 N-acetylglucosamine residue is in cases substituted with the disaccharide Galpalpha1-4Galpbeta1-4, giving rise to the blood P(1)-antigen motif. Larger, also related, glycans exist, reaching at least 18 residues in size. The glycans described are related but larger than those previously described from an Echinococcus multilocularis mucin [Hulsmeier, A. J., et al. (2002) J. Biol. Chem. 277, 5742-5748]. Our results reveal that the E. granulosus cyst exposes to the host only a few different major carbohydrate motifs. These motifs are composed essentially of galactose units and include the elongation by (Galpbeta1-3)(n) and the capping by Galpalpha1-4, novel in animal mucin-type O-glycans.

Interaction of the Dengue virus fusion peptide with membranes assessed by NMR: The essential role of the envelope protein Trp101 for membrane fusion.

Dengue virus (DV) infection depends on a step of membrane fusion, which occurs in the acidic environment of the endosome. This process is mediated by virus surface envelope glycoprotein, in which the loop between residues D98-G112 is considered to be crucial, acting as a fusion peptide. Here, we have characterized functionally and structurally the interaction between the DV fusion peptide and different model membranes by fluorescence and NMR. Its interaction was strongest in dodecylphosphocholine (DPC) micelles and anionic phosphatidylcholine/phosphatidylglycerol vesicles, the only vesicle that was fused by DV fusion peptide. The three-dimensional structure of DV fusion peptide bound to DPC micelles was solved by solution homonuclear NMR with an r.m.s.d. of 0.98 A. The most striking result obtained from the solution structure was the hydrophobic triad formed by residues W101, L107, and F108, pointing toward the same direction, keeping the segment between G102 and G106 in a loop conformation. The interaction of DV fusion peptide with phosphatidylcholine/phosphatidylglycerol vesicles was also mapped by transfer-nuclear Overhauser enhancement (NOE) experiments, in which the majority of the NOE cross-peaks were from the hydrophobic triad, corroborating the DPC-bound structure. Substitution of the residue W101 by an alanine residue completely abolished membrane binding and, thus, fusion by the peptide and its NOE cross-peaks. In conclusion, the 15-residue DV fusion peptide has intrinsic ability to promote membrane fusion, most likely due to the hydrophobic interaction among the residues W101, L107, and F108, which maintains its loop in the correct spatial conformation.

Interaction between dengue virus fusion peptide and lipid bilayers depends on peptide clustering.

Dengue fever is one of the most widespread tropical diseases in the world. The disease is caused by a virus member of the Flaviviridae family, a group of enveloped positive sense single-stranded RNA viruses. Dengue virus infection is mediated by virus glycoprotein E, which binds to the cell surface. After uptake by endocytosis, this protein induces the fusion between viral envelope and endosomal membrane at the acidic environment of the endosomal compartment. In this work, we evaluated by steady-state and time-resolved fluorescence spectroscopy the interaction between the peptide believed to be the dengue virus fusion peptide and large unilamellar vesicles, studying the extent of partition, fusion capacity and depth of insertion in membranes. The roles of the bilayer composition (neutral and anionic phospholipids), ionic strength and pH of the medium were also studied. Our results indicate that dengue virus fusion peptide has a high affinity to vesicles composed of anionic lipids and that the interaction is mainly electrostatic. Both partition coefficient and fusion index are enhanced by negatively charged phospholipids. The location determined by differential fluorescence quenching using lipophilic probes demonstrated that the peptide is in an intermediate depth in the hemilayers, in-between the bilayer core and its surface. Ultimately, these data provide novel insights on the interaction between dengue virus fusion peptide and its target membranes, namely, the role of oligomerization and specific types of membranes.

Structural characterization of partially folded intermediates of apomyoglobin H64F.

We present a detailed investigation of unfolded and partially folded states of a mutant apomyoglobin (apoMb) where the distal histidine has been replaced by phenylalanine (H64F). Previous studies have shown that substitution of His64, located in the E helix of the native protein, stabilizes the equilibrium molten globule and native states and leads to an increase in folding rate and a change in the folding pathway. Analysis of changes in chemical shift and in backbone flexibility, detected via [1H]-15N heteronuclear nuclear Overhauser effect measurements, indicates that the phenylalanine substitution has only minor effects on the conformational ensemble in the acid- and urea-unfolded states, but has a substantial effect on the structure, dynamics, and stability of the equilibrium molten globule intermediate formed near pH 4. In H64F apomyoglobin, additional regions of the polypeptide chain are recruited into the compact core of the molten globule. Since the phenylalanine substitution has negligible effect on the unfolded ensemble, its influence on folding rate and stability comes entirely from interactions within the compact folded or partly folded states. Replacement of His64 with Phe leads to favorable hydrophobic packing between the helix E region and the molten globule core and leads to stabilization of helix E secondary structure and overall thermodynamic stabilization of the molten globule. The secondary structure of the equilibrium molten globule parallels that of the burst phase kinetic intermediate; both intermediates contain significant helical structure in regions of the polypeptide that comprise the A, B, E, G, and H helices of the fully folded protein.

Specificity in DNA recognition by a peptide from papillomavirus E2 protein.

The E2 proteins of papillomavirus specifically bind to double-stranded DNA containing the consensus sequence ACCG-N4-CGGT, where N is any nucleotide. Here, we show the binding and recognition of dissimilar DNA sequences by an 18 amino-acid peptide (alpha1E2), which corresponds to the DNA-recognition helix, alpha-helix-1. Isothermal DNA binding assays performed with the DNA consensus sequence show saturable curves with alpha1E2 peptide, and the alpha1E2 peptide is converted to an ordered conformation upon complexation. Measurements performed with non-specific DNA sequence fail to saturate, a behavior characteristic of non-specific binding. Binding of the alpha1E2 peptide to these DNA sequences display a different counter-ion dependence, indicating a dissimilar, sequence-dependent mechanism of interaction. Quantitative stoichiometric measurements revealed the specificity in alpha1E2 peptide recognition of the ACCG half-site, demonstrating capacity for discrimination of nucleic acid bases sequences without the need of a whole protein architecture.