In Vitro and In Vivo Stability of P884T, a Mutation that Relocalizes Dengue Virus 2 Non-structural Protein 5

AbstractDengue is the most common virus disease transmitted to humans by mosquitoes. The dengue virus NS1 is a multifunctional protein that form part of replication complexes. In addition, NS1 is also secreted, as a hexamer, to the extracellular milieu. Circulating NS1 has been associated with dengue pathogenesis by several different mechanisms. Cell binding and internalization of soluble NS1 result in the disruption of tight junctions and in down regulation of the innate immune response. In this work, we report that the HDL scavenger receptor B1 (SRB1) in human hepatic cells, and a scavenger receptor B1-like in mosquito C6/36 cells act as cell surface binding receptor for dengue virus NS1. The presence of the SRB1 on the plasma membrane of C6/36 cells, as well as in Huh-7 cells, was demonstrated by confocal microcopy. Internalization of NS1 can be efficiently blocked by anti-SRB1 antibodies and previous incubation of the cells with HDL significantly reduces NS1 internalization. In addition, the transient expression of SRB1 in Vero cells, which lack the receptor, renders these cells susceptible to NS1 entry. Direct interaction between soluble NS1 and the SRB1 in Huh7 and C6/36 cells was demonstrated in vivo by proximity ligation assays an in vitro by surface plasmon resonance. Finally, data is presented indicating that the SRB1 also act as cell receptor for zika virus NS1. These results demonstrate that dengue virus NS1, a bona fide lipoprotein, usurps the HDL receptor for cell entry and offers explanations for the altered serum lipoprotein homeostasis observed in dengue patients.

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Interaction of Proteus mirabilis Urease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology

Journal of Bacteriology ◽

10.1128/jb.183.4.1423-1433.2001 ◽

2001 ◽

Vol 183 (4) ◽

pp. 1423-1433 ◽

Cited By ~ 28

Author(s):

Susan R. Heimer ◽

Harry L. T. Mobley

Keyword(s):

Proteus Mirabilis ◽

Accessory Proteins ◽

Structural Protein ◽

Yeast Two Hybrid ◽

Nickel Ions ◽

Catalytically Active ◽

Tract Infections ◽

Two Hybrid

ABSTRACT Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.

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Diterpenes/Diterpenoids and Their Derivatives as Potential Bioactive Leads against Dengue Virus: A Computational and Network Pharmacology Study

Molecules ◽

10.3390/molecules26226821 ◽

2021 ◽

Vol 26 (22) ◽

pp. 6821

Author(s):

Rasel Ahmed Khan ◽

Rajib Hossain ◽

Abolghasem Siyadatpanah ◽

Khattab Al-Khafaji ◽

Abul Bashar Ripon Khalipha ◽

...

Keyword(s):

Dengue Virus ◽

Dengue Fever ◽

Computational Study ◽

Viral Proteins ◽

Network Pharmacology ◽

Binding Affinities ◽

Ns1 Protein ◽

Structural Protein ◽

E Protein

Dengue fever is a dangerous infectious endemic disease that affects over 100 nations worldwide, from Africa to the Western Pacific, and is caused by the dengue virus, which is transmitted to humans by an insect bite of Aedes aegypti. Millions of citizens have died as a result of dengue fever and dengue hemorrhagic fever across the globe. Envelope (E), serine protease (NS3), RNA-directed RNA polymerase (NS5), and non-structural protein 1 (NS1) are mostly required for cell proliferation and survival. Some of the diterpenoids and their derivatives produced by nature possess anti-dengue viral properties. The goal of the computational study was to scrutinize the effectiveness of diterpenoids and their derivatives against dengue viral proteins through in silico study. Methods: molecular docking was performed to analyze the binding affinity of compounds against four viral proteins: the envelope (E) protein, the NS1 protein, the NS3 protein, and the NS5 protein. Results: among the selected drug candidates, triptolide, stevioside, alepterolic acid, sphaeropsidin A, methyl dodovisate A, andrographolide, caesalacetal, and pyrimethamine have demonstrated moderate to good binding affinities (−8.0 to −9.4 kcal/mol) toward the selected proteins: E protein, NS3, NS5, and NS1 whereas pyrimethamine exerts −7.5, −6.3, −7.8, and −6.6 kcal/mol with viral proteins, respectively. Interestingly, the binding affinities of these lead compounds were better than those of an FDA-approved anti-viral medication (pyrimethamine), which is underused in dengue fever. Conclusion: we can conclude that diterpenoids can be considered as a possible anti-dengue medication option. However, in vivo investigation is recommended to back up the conclusions of this study.

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Viperin is anti-viral in vitro but is dispensable for restricting dengue virus replication or induction of innate and inflammatory responses in vivo

Journal of General Virology ◽

10.1099/jgv.0.001669 ◽

2021 ◽

Vol 102 (10) ◽

Author(s):

Wisam-Hamzah Al Shujairi ◽

Luke P. Kris ◽

Kylie van der Hoek ◽

Evangeline Cowell ◽

Gustavo Bracho-Granado ◽

...

Keyword(s):

Dengue Virus ◽

Immune Cell ◽

Inflammatory Responses ◽

Denv Infection ◽

Brain Morphology ◽

Moderate Increase ◽

Tnf Α ◽

Significant Difference

Viperin has antiviral function against many viruses, including dengue virus (DENV), when studied in cells in culture. Here, the antiviral actions of viperin were defined both in vitro and in a mouse in vivo model of DENV infection. Murine embryonic fibroblasts (MEFs) derived from mice lacking viperin (vip−/−) showed enhanced DENV infection, accompanied by increased IFN-β and induction of ISGs; IFIT1 and CXCL-10 but not IRF7, when compared to wild-type (WT) MEFs. In contrast, subcutaneous challenge of immunocompetent WT and vip−/− mice with DENV did not result in enhanced infection. Intracranial infection with DENV resulted in body weight loss and neurological disease with a moderate increase in mortality in vip−/− compared with WT mice, although this was not accompanied by altered brain morphology, immune cell infiltration or DENV RNA level in the brain. Similarly, DENV induction of IFN-β, IFIT1, CXCL-10, IRF7 and TNF-α was not significantly different in WT and vip−/− mouse brain, although there was a modest but significant increase in DENV induction of IL-6 and IfI27la in the absence of viperin. NanoString nCounter analysis confirmed no significant difference in induction of a panel of inflammatory genes in WT compared to vip−/− DENV-infected mouse brains. Further, polyI:C stimulation of bone marrow-derived macrophages (BMDMs) induced TNF-α, IFN-β, IL-6 and Nos-2, but responses were not different in BMDMs generated from WT or vip−/− mice. Thus, while there is significant evidence of anti-DENV actions of viperin in some cell types in vitro, for DENV infection in vivo a lack of viperin does not affect systemic or brain susceptibility to DENV or induction of innate and inflammatory responses.

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A Glance at the Nuclear Envelope Spectrin Repeat Protein 3

BioMed Research International ◽

10.1155/2019/1651805 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8

Author(s):

Liwei Liao ◽

Rongmei Qu ◽

Jun Ouang ◽

Jingxing Dai

Keyword(s):

Nuclear Envelope ◽

Active Role ◽

Structural Protein ◽

Linc Complex ◽

Repeat Protein ◽

Spectrin Repeat ◽

Physiological Importance ◽

Functional Perspective

Nuclear envelope spectrin repeat protein 3 (nesprin-3) is an evolutionarily-conserved structural protein, widely-expressed in vertebrate cells. Along with other nesprin family members, nesprin-3 acts as an essential component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Naturally, nesprin-3 shares many functions with LINC, including the localization of various cellular structures and bridging of the nucleoskeleton and cytoskeleton, observed in vitro. When nesprin-3 was knocked down in vivo, using zebrafish and mouse models, however, the animals were minimally affected. This paradoxical observation should not limit the physiological importance of nesprin-3, as recently, nesprin-3 has reignited the interest of the research community in studies on cancer cells migration. Moreover, nesprin-3 also plays an active role in certain developmental conditions such as adipogenesis and spermatogenesis, although more studies are needed. Meanwhile, the various protein binding partners of nesprin-3 should also be emphasized, as they are necessary for maintaining the structure of nesprin-3 and enabling it to carry out its various physiological and pathological functions. Nesprin-3 promises to further our understanding of these complex cellular events. Therefore, this review will focus on nesprin-3, examining it from a genetic, structural, and functional perspective. The final part of the review will in turn address the limitations of existing research and the future perspectives for the study of nesprin-3.

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The Role of Tissue Oxygen Tension in Dengue Virus Replication

Cells ◽

10.3390/cells7120241 ◽

2018 ◽

Vol 7 (12) ◽

pp. 241 ◽

Cited By ~ 8

Author(s):

Efseveia Frakolaki ◽

Panagiota Kaimou ◽

Maria Moraiti ◽

Katerina Kalliampakou ◽

Kalliopi Karampetsou ◽

...

Keyword(s):

Dengue Virus ◽

Oxygen Tension ◽

Atmospheric Oxygen ◽

Rna Replication ◽

Metabolic Reprogramming ◽

Anaerobic Glycolysis ◽

Low Oxygen ◽

Hypoxia Response

Low oxygen tension exerts a profound effect on the replication of several DNA and RNA viruses. In vitro propagation of Dengue virus (DENV) has been conventionally studied under atmospheric oxygen levels despite that in vivo, the tissue microenvironment is hypoxic. Here, we compared the efficiency of DENV replication in liver cells, monocytes, and epithelial cells under hypoxic and normoxic conditions, investigated the ability of DENV to induce a hypoxia response and metabolic reprogramming and determined the underlying molecular mechanism. In DENV-infected cells, hypoxia had no effect on virus entry and RNA translation, but enhanced RNA replication. Overexpression and silencing approaches as well as chemical inhibition and energy substrate exchanging experiments showed that hypoxia-mediated enhancement of DENV replication depends on the activation of the key metabolic regulators hypoxia-inducible factors 1α/2α (HIF-1α/2α) and the serine/threonine kinase AKT. Enhanced RNA replication correlates directly with an increase in anaerobic glycolysis producing elevated ATP levels. Additionally, DENV activates HIF and anaerobic glycolysis markers. Finally, reactive oxygen species were shown to contribute, at least in part through HIF, both to the hypoxia-mediated increase of DENV replication and to virus-induced hypoxic reprogramming. These suggest that DENV manipulates hypoxia response and oxygen-dependent metabolic reprogramming for efficient viral replication.

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Cell-Fusing Agent Virus Reduces Arbovirus Dissemination in Aedes aegypti Mosquitoes In Vivo

Journal of Virology ◽

10.1128/jvi.00705-19 ◽

2019 ◽

Vol 93 (18) ◽

Cited By ~ 22

Author(s):

Artem Baidaliuk ◽

Elliott F. Miot ◽

Sebastian Lequime ◽

Isabelle Moltini-Conclois ◽

Fanny Delaigue ◽

...

Keyword(s):

Aedes Aegypti ◽

Dengue Virus ◽

Cell Line ◽

Content Type ◽

Cells In Culture ◽

Mosquito Cells ◽

Wide Range ◽

Natural Hosts

ABSTRACT Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo. For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo. Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of human-pathogenic flaviviruses. IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly, A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. aegypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission.

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Dengue virus-induced ER stress is required for autophagy activation, viral replication, and pathogenesis both in vitro and in vivo

Scientific Reports ◽

10.1038/s41598-017-18909-3 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 34

Author(s):

Ying-Ray Lee ◽

Szu-Han Kuo ◽

Ching-Yen Lin ◽

Po-Jung Fu ◽

Yee-Shin Lin ◽

...

Keyword(s):

Dengue Virus ◽

Viral Replication ◽

Er Stress

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Protective Capacity of the Human Anamnestic Antibody Response during Acute Dengue Virus Infection

Journal of Virology ◽

10.1128/jvi.01096-16 ◽

2016 ◽

Vol 90 (24) ◽

pp. 11122-11131 ◽

Cited By ~ 9

Author(s):

Meihui Xu ◽

Roland Züst ◽

Ying Xiu Toh ◽

Jennifer M. Pfaff ◽

Kristen M. Kahle ◽

...

Keyword(s):

Virus Infection ◽

Antibody Response ◽

Dengue Virus ◽

Secondary Infection ◽

Immune Memory ◽

Protective Capacity ◽

Dengue Virus Infection ◽

Immune Correlates

ABSTRACT Half of the world's population is exposed to the risk of dengue virus infection. Although a vaccine for dengue virus is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue virus infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue virus-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue virus-reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype-cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting the survival of infected mice. Our data suggest that the cross-reactive anamnestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo . IMPORTANCE Antibodies can protect from symptomatic dengue virus infection. However, it is not easy to assess which classes of antibodies provide protection because in vitro assays are not always predictive of in vivo protection. During a repeat infection, dengue virus-specific immune memory cells are reactivated and large amounts of antibodies are produced. By studying antibodies cloned from patients with heterologous secondary infection, we tested the protective value of the serotype-cross-reactive “recall” or “anamnestic” response. We found that results from in vitro neutralization assays did not always correlate with the ability of the antibodies to reduce viremia in a mouse model. In addition, a decrease of viremia in mice did not necessarily improve survival. The most protective antibodies were stable at pH 5, suggesting that antibody binding in the endosomes, after the antibody-virus complex is internalized, might be important to block virus spread in the organism.

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Epitope Determinants of a Chimpanzee Dengue Virus Type 4 (DENV-4)-Neutralizing Antibody and Protection against DENV-4 Challenge in Mice and Rhesus Monkeys by Passively Transferred Humanized Antibody

Journal of Virology ◽

10.1128/jvi.01420-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 12766-12774 ◽

Cited By ~ 54

Author(s):

Ching-Juh Lai ◽

Ana P. Goncalvez ◽

Ruhe Men ◽

Claire Wernly ◽

Olivia Donau ◽

...

Keyword(s):

Dengue Virus ◽

Virus Type ◽

High Titer ◽

Neutralizing Antibody ◽

Denv Infection ◽

Passive Transfer ◽

Escape Mutant ◽

Protective Capacity

ABSTRACT The chimpanzee monoclonal antibody (MAb) 5H2 is specific for dengue virus type 4 (DENV-4) and neutralizes the virus at a high titer in vitro. The epitope detected by the antibody was mapped by sequencing neutralization escape variants of the virus. One variant contained a Lys174-Glu substitution and another contained a Pro176-Leu substitution in domain I of the DENV-4 envelope protein (E). These mutations reduced binding affinity for the antibody 18- to >100-fold. Humanized immunoglobulin G (IgG) 5H2, originally produced from an expression vector, has been shown to be a variant containing a nine-amino-acid deletion in the Fc region which completely ablates antibody-dependent enhancement of DENV replication in vitro. The variant MAb, termed IgG 5H2 ΔD, is particularly attractive for exploring its protective capacity in vivo. Passive transfer of IgG 5H2 ΔD at 20 μg/mouse afforded 50% protection of suckling mice against challenge with 25 50% lethal doses of mouse neurovirulent DENV-4 strain H241. Passive transfer of antibody to monkeys was conducted to demonstrate proof of concept for protection against DENV challenge. Monkeys that received 2 mg/kg of body weight of IgG 5H2 ΔD were completely protected against 100 50% monkey infectious doses (MID50) of DENV-4, as indicated by the absence of viremia and seroconversion. A DENV-4 escape mutant that contained a Lys174-Glu substitution identical to that found in vitro was isolated from monkeys challenged with 106 MID50 of DENV-4. This substitution was also present in all naturally occurring isolates belonging to DENV-4 genotype III. These studies have important implications for possible antibody-mediated prevention of DENV infection.

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