The dengue virus non-structural protein 1 (NS1) is secreted efficiently from infected mosquito cells

ABSTRACTDengue virus (DENV) is a mosquito-borne virus of the familyFlaviviridae.The RNA viral genome encodes for a polyprotein that is co-translationally processed into three structural proteins and seven non-structural proteins. The non-structural protein 1 (NS1) is a multifunctional viral protein actively secreted in vertebrate and mosquito cells during DENV infection. In mosquito cells, NS1 is secreted in a caveolin-1 (CAV-1) dependent manner by an unconventional pathway. The caveolin chaperone complex (CCC) is a cytoplasmic complex formed by caveolin-1 and the chaperones FKBP52, Cy40 and CyA which is responsible for cholesterol traffic inside the cell. In this work, we demonstrate that in infected mosquito cells, DENV NS1 is secreted by an early and unconventional route that bypasses the Golgi apparatus in close association with the CCC. Treatment of mosquito cells with classic secretion inhibitors such as brefeldin A, golgicide A and Fli-06 showed no effect on NS1 secretion, but significant reductions in recombinant luciferase secretion and virion release. Silencing the expression of CAV1, FKBP52 with siRNAs or the inhibition of CyA by cyclosporine A resulted in significant decrease in NS1 secretion without affecting virion release. Using co-localization, co-inmunoprecipitation and proximity ligation assays, NS1 was found to co-localize and interact with all the protein components of the CCC in mosquito infected cells. In addition, CAV-1 and FKBP52 expression was found augmented in DENV infected cells. Finally, the treatment of ZIKV infected mosquito cells with brefeldin A and golgicide A showed no effect on NS1 secretion, while affecting virion release. ZIKV NS1 was also found to co-localize with CAV-1 in infected mosquito cells. These results suggest that in mosquito cells, ZIKV NS1 follows the same secretory pathway observed for DENV NS1. The association of NS1 with the cholesterol transporter CCC agrees with the lipoprotein nature of secreted hexameric NS1.AUTHOR SUMMARYDengue protein NS1 is secreted in infected mosquito and vertebrate cells. In humans, secreted NS1 have been associated with pathogenesis. In mosquito cells, NS1 follows an unconventional secretion pathway that is dependent on Caveolin-1. This work shows that in mosquito cells, NS1 secretion is associated to the chaperone caveolin complex, a complex formed by caveolin-1 and several chaperones, in charge of cholesterol transport within the cells. Reduction of the expression or the activity of chaperone caveolin complex in mosquito infected cells, diminished the secretion of NS1 without affecting virion release. Direct interaction between NS1 and the chaperone caveolin complex proteins was demonstrated by several assays. Moreover, increased expression of the caveolin-1 and co-chaperone FKBP52 during dengue infection was found, presumably in response to the higher requirements of these proteins during dengue virus infection. Results obtained with ZIKV infected mosquito cells suggest that also ZIKV NS1 is released following an unconventional secretory route in association with the chaperone caveolin complex. The functions of secreted NS1 within mosquito are unclear. However, giving the importance of the soluble NS1 in the vertebrate host, manipulation of the NS1 secretory route may prove a valuable strategy for dengue mosquito control and patient treatment.

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Ultrastructural Characterization and Three-Dimensional Architecture of Replication Sites in Dengue Virus-Infected Mosquito Cells

Journal of Virology ◽

10.1128/jvi.00118-14 ◽

2014 ◽

Vol 88 (9) ◽

pp. 4687-4697 ◽

Cited By ~ 113

Author(s):

J. Junjhon ◽

J. G. Pennington ◽

T. J. Edwards ◽

R. Perera ◽

J. Lanman ◽

...

Keyword(s):

Dengue Virus ◽

Three Dimensional ◽

Infected Mosquito ◽

Mosquito Cells ◽

Ultrastructural Characterization ◽

Replication Sites

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Secretion of Nonstructural Protein 1 of Dengue Virus from Infected Mosquito Cells: Facts and Speculations

Journal of Virology ◽

10.1128/jvi.00275-18 ◽

2018 ◽

Vol 92 (14) ◽

Cited By ~ 4

Author(s):

Ana C. Alcalá ◽

Laura A. Palomares ◽

Juan E. Ludert

Keyword(s):

Dengue Virus ◽

Nonstructural Protein ◽

Infected Mosquito ◽

Nonstructural Protein 1 ◽

Mosquito Cells

ABSTRACTDengue virus nonstructural protein 1 (NS1) is a multifunctional glycoprotein. For decades, the notion in the field was that NS1 is secreted exclusively from vertebrate cells and not from mosquito cells. However, recent evidence shows that mosquito cells also secrete NS1 efficiently. In this review, we discuss the evidence for secretion of NS1 of dengue virus, and of other flaviviruses, from mosquito cells, differences between NS1 secreted from mosquito and NS1 secreted from vertebrate cells, and possible roles of soluble NS1 in the insect flavivirus vector.

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Life as a Vector of Dengue Virus: The Antioxidant Strategy of Mosquito Cells to Survive Viral Infection

Antioxidants ◽

10.3390/antiox10030395 ◽

2021 ◽

Vol 10 (3) ◽

pp. 395

Author(s):

Chih-Chieh Cheng ◽

Eny Sofiyatun ◽

Wei-June Chen ◽

Lian-Chen Wang

Keyword(s):

Dengue Virus ◽

Viral Infection ◽

Denv Infection ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Infected Mosquito ◽

Mosquito Vector ◽

Evolutionary Pathway ◽

Antioxidant Genes ◽

Mosquito Cells

Dengue fever is a mosquito-borne viral disease of increasing global importance. The disease has caused heavy burdens due to frequent outbreaks in tropical and subtropical areas of the world. The dengue virus (DENV) is generally transmitted between human hosts via the bite of a mosquito vector, primarily Aedes aegypti and Ae. albopictus as a minor species. It is known that the virus needs to alternately infect mosquito and human cells. DENV-induced cell death is relevant to the pathogenesis in humans as infected cells undergo apoptosis. In contrast, mosquito cells mostly survive the infection; this allows infected mosquitoes to remain healthy enough to serve as an efficient vector in nature. Overexpression of antioxidant genes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutaredoxin (Grx), thioredoxin (Trx), and protein disulfide isomerase (PDI) have been detected in DENV2-infected mosquito cells. Additional antioxidants, including GST, eukaryotic translation initiation factor 5A (eIF5a), and p53 isoform 2 (p53-2), and perhaps some others, are also involved in creating an intracellular environment suitable for cell replication and viral infection. Antiapoptotic effects involving inhibitor of apoptosis (IAP) upregulation and subsequent elevation of caspase-9 and caspase-3 activities also play crucial roles in the ability of mosquito cells to survive DENV infection. This article focused on the effects of intracellular responses in mosquito cells to infection primarily by DENVs. It may provide more information to better understand virus/cell interactions that can possibly elucidate the evolutionary pathway that led to the mosquito becoming a vector.

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Direct nucleotide sequencing using total cellular RNA from dengue-virus-infected mosquito cells

Research in Virology ◽

10.1016/s0923-2516(06)80103-2 ◽

1992 ◽

Vol 143 ◽

pp. 193-197 ◽

Cited By ~ 2

Author(s):

I.M. Kautner ◽

S.-K. Lam

Keyword(s):

Dengue Virus ◽

Infected Mosquito ◽

Nucleotide Sequencing ◽

Mosquito Cells

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Monitoring Mitochondrial Function in Aedes albopictus C6/36 Cell Line during Dengue Virus Infection

Insects ◽

10.3390/insects12100934 ◽

2021 ◽

Vol 12 (10) ◽

pp. 934

Author(s):

María E. Santana-Román ◽

Paola Maycotte ◽

Salvador Uribe-Carvajal ◽

Cristina Uribe-Alvarez ◽

Nayeli Alvarado-Medina ◽

...

Keyword(s):

Dengue Virus ◽

Cell Line ◽

Aedes Albopictus ◽

Mitochondrial Function ◽

Mitochondrial Fission ◽

Hemorrhagic Fever ◽

Denv Infection ◽

Infected Mosquito ◽

Mosquito Cells ◽

Induced Changes

Aedes aegypti and Aedes albopictus mosquitoes are responsible for dengue virus (DENV) transmission in tropical and subtropical areas worldwide, where an estimated 3 billion people live at risk of DENV exposure. DENV-infected individuals show symptoms ranging from sub-clinical or mild to hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The interactions between viruses and host mitochondria are crucial for virus replication and pathogenicity. DENV infection in vertebrate cells modulates mitochondrial function and dynamics to facilitate viral proliferation. Here, we describe that DENV also regulates mitochondrial function and morphology in infected C6/36 mosquito cells (derived from Aedes albopictus). Our results showed that DENV infection increased ROS (reactive oxygen species) production, modulated mitochondrial transmembrane potential and induced changes in mitochondrial respiration. Furthermore, we offer the first evidence that DENV causes translocation of mitofusins to mitochondria in the C6/36 mosquito cell line. Another protein Drp-1 (Dynamin-related protein 1) did not localize to mitochondria in DENV-infected cells. This observation therefore ruled out the possibility that the abovementioned alterations in mitochondrial function are associated with mitochondrial fission. In summary, this report provides some key insights into the virus–mitochondria crosstalk in DENV infected mosquito cells.

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A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells

Cell Reports ◽

10.1016/j.celrep.2018.12.068 ◽

2019 ◽

Vol 26 (3) ◽

pp. 529-535.e3 ◽

Cited By ~ 9

Author(s):

Gholamreza Haqshenas ◽

Gerard Terradas ◽

Prasad N. Paradkar ◽

Jean-Bernard Duchemin ◽

Elizabeth A. McGraw ◽

...

Keyword(s):

Dengue Virus ◽

Insulin Receptor ◽

Zika Virus ◽

Infected Mosquito ◽

Mosquito Cells

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The dengue virus non-structural protein 1 (NS1) interacts with the putative epigenetic regulator DIDO1 to promote flavivirus replication.

10.1101/2021.09.01.458517 ◽

2021 ◽

Author(s):

gerson caraballo ◽

Romel Rosales ◽

Mercedes Viettri ◽

Siyuan Ding ◽

Harry B Greenberg ◽

...

Keyword(s):

Dengue Virus ◽

Viral Replication ◽

Ribosomal Proteins ◽

Denv Infection ◽

Structural Protein ◽

Transcription Analysis ◽

Multifunctional Protein ◽

Rna Surveillance ◽

Mosquito Cells ◽

Expression Pathways

Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. Approximately 14% of the 817 identified proteins coincide with interactomes obtained in vertebrate cells, including ontology groups of the oligosaccharide transferase complex, the chaperonin containing TCP-1, and nuclear import and export, vesicle localization and ribosomal proteins. Notably, other protein pathways such as epigenetic regulation and RNA silencing, not previously reported in vertebrate cells, were also found in the NS1 interactome in mosquito cells. Due to the novel interaction observed for NS1 and DIDO1 (Death Inducer-Obliterator 1), we further explored the role of DIDO1 in viral replication. Interactions between NS1 and DIDO1were corroborated in infected C6/36 and Aag2 cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression in C6/36 and Aag2 cells results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected C6/36 silenced for DIDO1, revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response and necessary for flavivirus replication. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication and add to the understanding of NS1 as a multifunctional protein.

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Releasing Intracellular NS1 from Mosquito Cells for the Detection of Dengue Virus-Infected Mosquitoes

Viruses ◽

10.3390/v12101105 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1105

Author(s):

Lie Cheng ◽

Wei-Liang Liu ◽

Hsing-Han Li ◽

Matthew P. Su ◽

Shih-Cheng Wu ◽

...

Keyword(s):

Dengue Virus ◽

Mosquito Control ◽

Detection Threshold ◽

Disease Outbreaks ◽

Rapid Test ◽

Infected Mosquito ◽

Mosquito Cells ◽

Crucial Component ◽

Sensitivity Tests ◽

Triton X

Dengue virus (DENV), the pathogen that causes dengue fever, is mainly transmitted by Aedes aegypti. Surveillance of infected mosquitoes is a major component of integrated mosquito control methods for reducing the risk of vector-born disease outbreaks. However, a specialized rapid test for DENV detection in mosquitoes is not currently available. Utilizing immunoblotting, we found that the secretion of NS1 from both a DENV-infected mosquito cell line and mosquito bodies was below the detection threshold. However, when Triton X-100 was used to lyse infected mosquitoes, intracellular NS1 was released, and could then be effectively detected by the NS1 rapid test. The distribution of DENV NS1 in intrathoracically infected mosquitoes was different from that of orally infected mosquitoes. Next, we performed sensitivity tests by bisecting mosquitoes longitudinally; one half of each mosquito was subjected to the NS1 rapid test while the other half was used for qPCR confirmation. This modified test had a sensitivity of nearly 90% from five days post-infection onwards, while DENV had escaped from the midgut barrier. This adapted test offers a valuable, easy-to-use tool for mosquito surveillance, which is a crucial component of DENV disease control.

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