Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

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Dengue Nonstructural Protein 1 Maintains Autophagy through Retarding Caspase-Mediated Cleavage of Beclin-1

International Journal of Molecular Sciences ◽

10.3390/ijms21249702 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9702

Author(s):

Zi-Yi Lu ◽

Miao-Huei Cheng ◽

Chia-Yi Yu ◽

Yee-Shin Lin ◽

Trai-Ming Yeh ◽

...

Keyword(s):

Cell Apoptosis ◽

Nonstructural Protein ◽

Antiviral Drug ◽

Cellular Responses ◽

Denv Infection ◽

Beclin 1 ◽

Protein Inhibition ◽

Nonstructural Protein 1 ◽

Significant Public Health ◽

Related Proteins

Dengue virus (DENV) infection is a significant public health threat in tropical and subtropical regions; however, there is no specific antiviral drug. Accumulated studies have revealed that DENV infection induces several cellular responses, including autophagy and apoptosis. The crosstalk between autophagy and apoptosis is associated with the interactions among components of these two pathways, such as apoptotic caspase-mediated cleavage of autophagy-related proteins. Here, we show that DENV-induced autophagy inhibits early cell apoptosis and hence enhances DENV replication. Later, the apoptotic activities are elevated to suppress autophagy through cleavage of Beclin-1, an essential autophagy-related protein. Inhibition of cleavage of Beclin-1 by a pan-caspase inhibitor, Z-VAD, increases both autophagy and viral replication. Regarding the mechanism, we further found that DENV nonstructural protein 1 (NS1) is able to interact with Beclin-1 during DENV infection. The interaction between Beclin-1 and NS1 attenuates Beclin-1 cleavage and facilitates autophagy to prevent cell apoptosis. Our study suggests a novel mechanism whereby NS1 preserves Beclin-1 for maintaining autophagy to antagonize early cell apoptosis; however, elevated caspases trigger apoptosis by degrading Beclin-1 in the late stage of infection. These findings suggest implications for anti-DENV drug design.

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Anti-dengue virus nonstructural protein 1 antibodies contribute to platelet phagocytosis by macrophages

Thrombosis and Haemostasis ◽

10.1160/th15-06-0498 ◽

2016 ◽

Vol 115 (03) ◽

pp. 646-656 ◽

Cited By ~ 15

Author(s):

Ya-Ting Chu ◽

Chiou-Feng Lin ◽

Chih-Peng Chang ◽

Trai-Ming Yeh ◽

Robert Anderson ◽

...

Keyword(s):

Dengue Virus ◽

Platelet Adhesion ◽

Molecular Mimicry ◽

Nonstructural Protein ◽

Denv Infection ◽

Dengue Disease ◽

Nonstructural Protein 1 ◽

Tnf Α ◽

Β3 Integrin ◽

Lines Of Evidence

SummaryThrombocytopenia is an important clinical manifestation of dengue disease. The hypotheses concerning the pathogenesis of thrombocytopenia include decreased production and increased destruction or consumption of platelets. We previously suggested a mechanism of molecular mimicry in which antibodies (Abs) directed against dengue virus (DENV) nonstructural protein 1 (NS1) cross-react with platelets. Furthermore, several lines of evidence show activation of endothelial cells (ECs) and macrophages are related to dengue disease severity. Previous studies also suggested that Ab-opsonised platelets facilitate the engulfment of platelets by macrophages. Here we show that TNF-α-activated ECs upregulate adhesion molecule expression to enhance the binding of platelets and macrophages and lead to anti-DENV NS1 Ab-mediated platelet phagocytosis. We further demonstrate that the interaction between macrophages and TNF-α-activated ECs requires binding of FcγR with the Fc region of platelet-bound anti-DENV NS1 Abs. Importantly, the binding of anti-DENV NS1 Abs to platelets did not interfere with platelet adhesion to ECs. The adhesion molecules ICAM-1 and β3 integrin expressed on ECs as well as the FcγR expressed on macrophages were critical in anti-DENV NS1 Ab-mediated platelet phagocytosis on activated ECs. Moreover, anti-DENV NS1 Abs dramatically enhanced platelet engulfment by macrophages in a murine model of DENV infection. Our study provides evidence for a novel role for anti-DENV NS1 Abs in the pathogenesis of thrombocytopenia in dengue disease by enhancing platelet phagocytosis by macrophages.

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Epidemiological survey and screening strategy for dengue virus in blood donors from Yunnan Province

BMC Infectious Diseases ◽

10.1186/s12879-021-05810-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Ling Li ◽

Ying Li ◽

Shaofang Lu ◽

Jing Dong ◽

Haixia Xu ◽

...

Keyword(s):

Dengue Virus ◽

Southern China ◽

Nonstructural Protein ◽

Denv Infection ◽

Screening Strategy ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Donor Screening ◽

Blood Center ◽

Nonstructural Protein 1

Abstract Background Dengue virus (DENV) infection is increasingly common in southern China and can be transmitted through blood transfusion but is not currently part of donor screening throughout the region. We assessed DENV prevalence among donors at the Xishuangbanna Blood Center, Yunnan, to support development of DENV screening strategies. Methods Blood samples were collected randomly between June 2019 and August 2019. These were screened for anti-DENV IgG and IgM using enzyme-linked immunosorbent assay (ELISA). Then, all reactive samples and some randomly-chosen non-reactive samples were used to detect DENV RNAs using real-time polymerase-chain-reaction (RT-PCR) assays. After RT-PCR, samples were further tested for soluble nonstructural protein 1 (NS1) using the colloidal gold method. Donors demographics were also collected and assessed. Results Over the study period, 2254 donor samples were collected and tested for anti-DENV IgG and IgM by ELISA. This revealed 598 anti-DENV IgG and/or IgM reactive samples, a serological prevalence of 26.53%. Of these, 26 were RT-PCR positive and/or NS1 positive. Significant differences in DENV prevalence were noted by occupation (P = 0.001), education (P < 0.001), and ethnicity (P = 0.026). Conclusion The prevalence of DENV in Xishuangbanna Blood Center was higher than most other blood centers that have implemented DENV donor screening. Our study provides first-hand data about the prevalence of DENV and allows the development of a screening strategy for clinical use.

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Epidemiological Survey and Screening Strategy for Dengue Virus in Blood Donors from Yunnan Province

10.21203/rs.3.rs-50346/v3 ◽

2021 ◽

Author(s):

LING LI ◽

YING LI ◽

Shaofang Lu ◽

Jing Dong ◽

Haixia Xu ◽

...

Keyword(s):

Dengue Virus ◽

Southern China ◽

Nonstructural Protein ◽

Denv Infection ◽

Screening Strategy ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Donor Screening ◽

Blood Center ◽

Nonstructural Protein 1

Abstract BACKGROUND Dengue virus (DENV) infection is increasingly common in southern China and can be transmitted through blood transfusion but is not currently part of donor screening throughout the region. We assessed DENV prevalence among donors at the Xishuangbanna Blood Center, Yunnan, to support development of DENV screening strategies.METHODS Blood samples were collected randomly between June 2019 and August 2019. These were screened for anti-DENV IgG and IgM using enzyme-linked immunosorbent assay (ELISA). Then, all reactive samples and some randomly-chosen non-reactive samples were used to detect DENV RNAs using real-time polymerase-chain-reaction (RT-PCR) assays. After RT-PCR, samples were further tested for soluble nonstructural protein 1 (NS1) using the colloidal gold method. Donors demographics were also collected and assessed.RESULTS Over the study period, 2,254 donor samples were collected and tested for anti-DENV IgG and IgM by ELISA. This revealed 598 anti-DENV IgG and/or IgM reactive samples, a serological prevalence of 26.53%. Of these, 26 were RT-PCR positive and/or NS1 positive. Significant differences in DENV prevalence were noted by occupation (P=0.001), education (P<0.001), and ethnicity (P=0.026).CONCLUSION The prevalence of DENV in Xishuangbanna Blood Center was higher than most other blood centers that have implemented DENV donor screening. Our study provides first-hand data about the prevalence of DENV and allows the development of a screening strategy for clinical use.

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The Good, the Bad, and the Shocking: The Multiple Roles of Dengue Virus Nonstructural Protein 1 in Protection and Pathogenesis

Annual Review of Virology ◽

10.1146/annurev-virology-101416-041848 ◽

2018 ◽

Vol 5 (1) ◽

pp. 227-253 ◽

Cited By ~ 38

Author(s):

Dustin R. Glasner ◽

Henry Puerta-Guardo ◽

P. Robert Beatty ◽

Eva Harris

Keyword(s):

Dengue Virus ◽

Nonstructural Protein ◽

Direct Action ◽

Adaptive Immune Response ◽

Denv Infection ◽

Multiple Roles ◽

Severe Dengue ◽

Potential Contribution ◽

Dengue Disease ◽

Nonstructural Protein 1

Dengue virus (DENV) is the most prevalent medically important mosquito-borne virus in the world. Upon DENV infection of a host cell, DENV nonstructural protein 1 (NS1) can be found intracellularly as a monomer, associated with the cell surface as a dimer, and secreted as a hexamer into the bloodstream. NS1 plays a variety of roles in the viral life cycle, particularly in RNA replication and immune evasion of the complement pathway. Over the past several years, key roles for NS1 in the pathogenesis of severe dengue disease have emerged, including direct action of the protein on the vascular endothelium and triggering release of vasoactive cytokines from immune cells, both of which result in endothelial hyperpermeability and vascular leak. Importantly, the adaptive immune response generates a robust response against NS1, and its potential contribution to dengue vaccines is also discussed.

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Comparison of Two Commercially Available Dengue Virus (DENV) NS1 Capture Enzyme-Linked Immunosorbent Assays Using a Single Clinical Sample for Diagnosis of Acute DENV Infection

Clinical and Vaccine Immunology ◽

10.1128/cvi.00140-08 ◽

2008 ◽

Vol 15 (10) ◽

pp. 1513-1518 ◽

Cited By ~ 87

Author(s):

Kovi Bessoff ◽

Mark Delorey ◽

Wellington Sun ◽

Elizabeth Hunsperger

Keyword(s):

Dengue Virus ◽

Real Time ◽

Virus Isolation ◽

Nonstructural Protein ◽

Clinical Sample ◽

Denv Infection ◽

Acute Stage ◽

Rt Pcr ◽

Nonstructural Protein 1 ◽

Immunosorbent Assays

ABSTRACT Dengue virus (DENV) nonstructural protein 1 (NS1) has shown promise as a novel diagnostic marker of acute DENV infection. Current techniques used to diagnose acute DENV infection, including virus isolation and reverse transcription-PCR (RT-PCR), are costly and difficult to perform, while traditional serological assays have low sensitivities during the acute stage of infection. Two commercially available NS1 antigen capture enzyme-linked immunosorbent assays (ELISAs), the Platelia dengue NS1Ag test (Bio-Rad Laboratories, Marnes La Coquette, France) and the Pan-E dengue early ELISA test (Panbio Diagnostics, Brisbane, Australia), were evaluated against a well-characterized panel of 208 real-time RT-PCR- and virus isolation-positive sera, as well as 45 real-time RT-PCR- and serologically negative sera from patients with other acute febrile illnesses. The overall sensitivities were 64.9% (95% confidence interval [CI95], 58.2 to 71.1%) for the Panbio test and 83.2% (CI95, 77.5 to 87.7%) for the Bio-Rad test, with interserotype variation, especially for DENV serotype 4. Predictive models were constructed to identify factors that had a significant influence on a test's outcome with respect to this panel of samples in order to identify the conditions in which the test will be most effective as a diagnostic tool. The immunoglobulin G titer was found to be the only covariate that significantly influenced results in the Bio-Rad test, while serotype and the day postonset were found to significantly influence results in the Panbio test. We concluded that the NS1 capture ELISA is a useful tool that can improve testing algorithms to diagnose DENV infection in single samples from acute and early convalescent cases.

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Combination of Nonstructural Protein 1-Based Enzyme-Linked Immunosorbent Assays Can Detect and Distinguish Various Dengue Virus and Zika Virus Infections

Journal of Clinical Microbiology ◽

10.1128/jcm.01464-18 ◽

2018 ◽

Vol 57 (2) ◽

Cited By ~ 2

Author(s):

Jasmine Tyson ◽

Wen-Yang Tsai ◽

Jih-Jin Tsai ◽

Carlos Brites ◽

Ludvig Mässgård ◽

...

Keyword(s):

Dengue Virus ◽

Zika Virus ◽

Nonstructural Protein ◽

Density Ratio ◽

Denv Infection ◽

Virus Infections ◽

Denv Serotypes ◽

Nonstructural Protein 1 ◽

Optical Density Ratio ◽

Immunosorbent Assays

ABSTRACTThe recent outbreaks of Zika virus (ZIKV) and associated birth defects in regions of dengue virus (DENV) endemicity emphasize the need for sensitive and specific serodiagnostic tests. We reported previously that enzyme-linked immunosorbent assays (ELISAs) based on the nonstructural protein 1 (NS1) of DENV serotype 1 (DENV1) and ZIKV can distinguish primary DENV1, secondary DENV, and ZIKV infections. Whether ELISAs based on NS1 proteins of other DENV serotypes can discriminate various DENV and ZIKV infections remains unknown. We herein developed DENV2, DENV3, and DENV4 NS1 IgG ELISAs to test convalescent- and postconvalescent-phase samples from reverse transcription-PCR-confirmed cases, including 25 primary DENV1, 24 primary DENV2, 10 primary DENV3, 67 secondary DENV, 36 primary West Nile virus, 38 primary ZIKV, and 35 ZIKV with previous DENV infections as well as 55 flavivirus-naive samples. Each ELISA detected primary DENV infection with a sensitivity of 100% for the same serotype and 23.8% to 100% for different serotypes. IgG ELISA using a mixture of DENV1-4 NS1 proteins detected different primary and secondary DENV infections with a sensitivity of 95.6% and specificity of 89.5%. The ZIKV NS1 IgG ELISA detected ZIKV infection with a sensitivity of 100% and specificity of 82.9%. On the basis of the relative optical density ratio, the combination of DENV1-4 and ZIKV NS1 IgG ELISAs distinguished ZIKV with previous DENV and secondary DENV infections with a sensitivity of 91.7% to 94.1% and specificity of 87.0% to 95.0%. These findings have important applications to serodiagnosis, serosurveillance, and monitoring of both DENV and ZIKV infections in regions of endemicity.

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Establishment and characterization of dengue virus type 2 nonstructural protein 1 specific T cell lines

Comparative Immunology Microbiology and Infectious Diseases ◽

10.1016/j.cimid.2010.01.002 ◽

2010 ◽

Vol 33 (6) ◽

pp. e75-e80 ◽

Cited By ~ 1

Author(s):

Yang Xiao-meng ◽

Jiang Li-fang ◽

Tang Yun-xia ◽

Yin Yue ◽

Liu Wen-quan ◽

...

Keyword(s):

T Cell ◽

Dengue Virus ◽

Cell Lines ◽

Virus Type ◽

Nonstructural Protein ◽

Nonstructural Protein 1 ◽

T Cell Lines ◽

Dengue Virus Type 2

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NITD-688, a pan-serotype inhibitor of the dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models

Science Translational Medicine ◽

10.1126/scitranslmed.abb2181 ◽

2021 ◽

Vol 13 (579) ◽

pp. eabb2181

Author(s):

Stephanie A. Moquin ◽

Oliver Simon ◽

Ratna Karuna ◽

Suresh B. Lakshminarayana ◽

Fumiaki Yokokawa ◽

...

Keyword(s):

Dengue Virus ◽

Nonstructural Protein ◽

Antiviral Drug ◽

Pharmacokinetic Studies ◽

Resistance Mutations ◽

Binding Studies ◽

Log Reduction ◽

Elimination Half ◽

Preclinical Animal Models

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.

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