Characterization of Dengue Virus Resistance to Brequinar in Cell Culture

ABSTRACT Brequinar is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for de novo pyrimidine biosynthesis. Here we report that brequinar has activity against a broad spectrum of viruses. The compound not only inhibits flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Powassan virus) but also suppresses a plus-strand RNA alphavirus (Western equine encephalitis virus) and a negative-strand RNA rhabdovirus (vesicular stomatitis virus). Using dengue virus serotype 2 (DENV-2) as a model, we found that brequinar suppressed the viral infection cycle mainly at the step of RNA synthesis. Supplementing the culture medium with pyrimidines (cytidine or uridine) but not purines (adenine or guanine) could be used to reverse the inhibitory effect of the compound. Continuous culturing of DENV-2 in the presence of brequinar generated viruses that were partially resistant to the inhibitor. Sequencing of the resistant viruses revealed two amino acid mutations: one mutation (M260V) located at a helix in the domain II of the viral envelope protein and another mutation (E802Q) located at the priming loop of the nonstructural protein 5 (NS5) polymerase domain. Functional analysis of the mutations suggests that the NS5 mutation exerts resistance through enhancement of polymerase activity. The envelope protein mutation reduced the efficiency of virion assembly/release; however, the mutant virus became less sensitive to brequinar inhibition at the step of virion assembly/release. Taken together, the results indicate that (i) brequinar blocks DENV RNA synthesis through depletion of intracellular pyrimidine pools and (ii) the compound may also exert its antiviral activity through inhibition of virion assembly/release.

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Folding of the Ig-Like Domain of the Dengue Virus Envelope Protein Analyzed by High-Hydrostatic-Pressure NMR at a Residue-Level Resolution

Biomolecules ◽

10.3390/biom9080309 ◽

2019 ◽

Vol 9 (8) ◽

pp. 309 ◽

Cited By ~ 4

Author(s):

Saotome ◽

Doret ◽

Kulkarni ◽

Yang ◽

Barthe ◽

...

Keyword(s):

Hydrostatic Pressure ◽

Dengue Virus ◽

Envelope Protein ◽

High Hydrostatic Pressure ◽

Public Health Problem ◽

Viral Envelope ◽

Specific Information ◽

Pressure Jump ◽

Viral Envelope Protein ◽

Domain Iii

Dengue fever is a mosquito-borne endemic disease in tropical and subtropical regions, causing a significant public health problem in Southeast Asia. Domain III (ED3) of the viral envelope protein contains the two dominant putative epitopes and part of the heparin sulfate receptor binding region that drives the dengue virus (DENV)’s fusion with the host cell. Here, we used high-hydrostatic-pressure nuclear magnetic resonance (HHP-NMR) to obtain residue-specific information on the folding process of domain III from serotype 4 dengue virus (DEN4-ED3), which adopts the classical three-dimensional (3D) ß-sandwich structure known as the Ig-like fold. Interestingly, the folding pathway of DEN4-ED3 shares similarities with that of the Titin I27 module, which also adopts an Ig-like fold, but is functionally unrelated to ED3. For both proteins, the unfolding process starts by the disruption of the N- and C-terminal strands on one edge of the ß-sandwich, yielding a folding intermediate stable over a substantial pressure range (from 600 to 1000 bar). In contrast to this similarity, pressure-jump kinetics indicated that the folding transition state is considerably more hydrated in DEN4-ED3 than in Titin I27.

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Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique

Journal of General Virology ◽

10.1099/vir.0.062562-0 ◽

2014 ◽

Vol 95 (10) ◽

pp. 2155-2165 ◽

Cited By ~ 3

Author(s):

Peng-Yeh Lai ◽

Chia-Tse Hsu ◽

Shao-Hung Wang ◽

Jin-Ching Lee ◽

Min-Jen Tseng ◽

...

Keyword(s):

Dengue Virus ◽

Envelope Protein ◽

Linear Array ◽

Polyclonal Antibodies ◽

Academic Research ◽

Neutralizing Antibody ◽

Viral Envelope ◽

Host Cells ◽

Viral Envelope Protein ◽

Domain Iii

Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.

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Two Distinct Sets of NS2A Molecules Are Responsible for Dengue Virus RNA Synthesis and Virion Assembly

Journal of Virology ◽

10.1128/jvi.02882-14 ◽

2014 ◽

Vol 89 (2) ◽

pp. 1298-1313 ◽

Cited By ~ 52

Author(s):

Xuping Xie ◽

Jing Zou ◽

Chunya Puttikhunt ◽

Zhiming Yuan ◽

Pei-Yong Shi

Keyword(s):

Dengue Virus ◽

Rna Synthesis ◽

Nonstructural Protein ◽

Viral Rna ◽

Infection Cycle ◽

Wild Type ◽

Replication Complex ◽

Virion Assembly ◽

Virus Rna ◽

Viral Replication Complex

ABSTRACTFlavivirus nonstructural protein 2A (NS2A) plays important roles in both viral RNA synthesis and virion assembly. The molecular details of how the NS2A protein modulates the two distinct events have not been defined. To address this question, we have performed a systematic mutagenesis of NS2A using dengue virus (DENV) serotype 2 (DENV-2) as a model. We identified two sets of NS2A mutations with distinct defects during a viral infection cycle. One set of NS2A mutations (D125A and G200A) selectively abolished viral RNA synthesis. Mechanistically, the D125A mutation abolished viral RNA synthesis through blocking the N-terminal cleavage of the NS2A protein, leading to an unprocessed NS1-NS2A protein; this result suggests that amino acid D125 (far downstream of the N terminus of NS2A) may contribute to the recognition of host protease at the NS1-NS2A junction. The other set of NS2A mutations (G11A, E20A, E100A, Q187A, and K188A) specifically impaired virion assembly without significantly affecting viral RNA synthesis. Remarkably, mutants defective in virion assembly could be rescued by supplying intranswild-type NS2A molecules expressed from a replicative replicon, by wild-type NS2A protein expressed alone, by a mutant NS2A (G200A) that is lethal for viral RNA synthesis, or by a different mutant NS2A that is defective in virion assembly. In contrast, none of the mutants defective in viral RNA synthesis could be rescued bytrans-complementation. Collectively, the results indicate that two distinct sets of NS2A molecules are responsible for DENV RNA synthesis and virion assembly.IMPORTANCEDengue virus (DENV) represents the most prevalent mosquito-borne human pathogen. Understanding the replication of DENV is essential for development of vaccines and therapeutics. Here we characterized the function of DENV-2 NS2A using a systematic mutagenesis approach. The mutagenesis results revealed two distinct sets of NS2A mutations: one set of mutations that result in defects in viral RNA synthesis and another set of mutations that result in defects in virion assembly.trans-Complementation analysis showed that mutants defective in viral RNA synthesis could not be rescued by wild-type NS2A; in contrast, mutants defective in virion assembly could be successfully rescued by wild-type NS2A or even by a mutant NS2A that is incompetent to support viral RNA synthesis. These results support a model in which two distinct sets of NS2A molecules are responsible for DENV RNA synthesis (located in the viral replication complex) and virion assembly (located in the virion assembly/budding site). The study confirms and extends our understanding of the two critical roles of flavivirus NS2A in viral RNA synthesis and virion assembly.

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Computational Analysis of Dengue Virus Envelope Protein (E) Reveals an Epitope with Flavivirus Immunodiagnostic Potential in Peptide Microarrays

International Journal of Molecular Sciences ◽

10.3390/ijms20081921 ◽

2019 ◽

Vol 20 (8) ◽

pp. 1921 ◽

Cited By ~ 9

Author(s):

Greta Bergamaschi ◽

Enrico M. A. Fassi ◽

Alessandro Romanato ◽

Ilda D'Annessa ◽

Maria Teresa Odinolfi ◽

...

Keyword(s):

Dengue Virus ◽

Envelope Protein ◽

Epitope Prediction ◽

Viral Envelope ◽

Epidemiological Surveillance ◽

Internal Dynamic ◽

Viral Envelope Protein ◽

Virus Envelope ◽

Serological Tests ◽

Specificity And Sensitivity

The mosquito-borne viral disease caused by the Dengue virus is an expanding global threat. Diagnosis in low-resource-settings and epidemiological surveillance urgently requires new immunoprobes for serological tests. Structure-based epitope prediction is an efficient method to design diagnostic peptidic probes able to reveal specific antibodies elicited in response to infections in patients’ sera. In this study, we focused on the Dengue viral envelope protein (E); computational analyses ranging from extensive Molecular Dynamics (MD) simulations and energy-decomposition-based prediction of potentially immunoreactive regions identified putative epitope sequences. Interestingly, one such epitope showed internal dynamic and energetic properties markedly different from those of other predicted sequences. The epitope was thus synthesized as a linear peptide, modified for chemoselective immobilization on microarrays and used in a serological assay to discriminate Dengue-infected individuals from healthy controls. The synthetic epitope probe showed a diagnostic performance comparable to that of the full antigen in terms of specificity and sensitivity. Given the high level of sequence identity among different flaviviruses, the epitope was immune-reactive towards Zika-infected sera as well. The results are discussed in the context of the quest for new possible structure-dynamics-based rules for the prediction of the immunoreactivity of selected antigenic regions with potential pan-flavivirus immunodiagnostic capacity.

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A novel mosquito ubiquitin targets viral envelope protein for degradation and reduces virion production during dengue virus infection

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/j.bbagen.2016.05.033 ◽

2016 ◽

Vol 1860 (9) ◽

pp. 1898-1909 ◽

Cited By ~ 15

Author(s):

Andrea Troupin ◽

Berlin Londono-Renteria ◽

Michael J. Conway ◽

Erin Cloherty ◽

Samuel Jameson ◽

...

Keyword(s):

Virus Infection ◽

Dengue Virus ◽

Envelope Protein ◽

Viral Envelope ◽

Viral Envelope Protein ◽

Dengue Virus Infection ◽

Virion Production

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Two different genes coding for processable and nonprocessable forms of a viral envelope protein can account for the apparent hormonal stimulation of protein processing in W7MG1 lymphoma cells.

Molecular Endocrinology ◽

10.1210/mend.6.3.1316543 ◽

1992 ◽

Vol 6 (3) ◽

pp. 459-467

Author(s):

R M Bedgood ◽

I Bahner ◽

D B Kohn ◽

M R Stallcup

Keyword(s):

Envelope Protein ◽

Protein Processing ◽

Viral Envelope ◽

Viral Envelope Protein ◽

Lymphoma Cells ◽

Hormonal Stimulation ◽

Stimulation Of

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The Role of Viral Envelope Protein Glycosylation in Pathogenesis of Infl uenza A Virus

Glycobiology and Human Diseases ◽

10.1201/b20120-7 ◽

2016 ◽

pp. 107-118

Keyword(s):

Envelope Protein ◽

Viral Envelope ◽

Protein Glycosylation ◽

Viral Envelope Protein

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The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

Journal of Virology ◽

10.1128/jvi.73.2.1293-1301.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 1293-1301 ◽

Cited By ~ 21

Author(s):

Kazunori Inabe ◽

Masako Nishizawa ◽

Shigeru Tajima ◽

Kazuyoshi Ikuta ◽

Yoko Aida

Keyword(s):

Viral Entry ◽

Envelope Protein ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Transient Transfection ◽

Viral Envelope ◽

Tyrosine Residue ◽

Wild Type ◽

Viral Envelope Protein

ABSTRACT The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.

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