Arthropod EVs mediate dengue virus transmission through interaction with a tetraspanin domain containing glycoprotein Tsp29Fb

Ashish Vora, Wenshuo Zhou, Berlin Londono-Renteria, Michael Woodson, Michael Sherman, Tonya M. Colpitts, Girish Neelakanta, Hameeda Sultana

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Dengue virus (DENV) is a mosquito-borne flavivirus that causes dengue fever in humans, worldwide. Using in vitro cell lines derived from Aedes albopictus and Aedes aegypti, the primary vectors of DENV, we report that DENV2/DENV3-infected cells secrete extracellular vesicles (EVs), including exosomes, containing infectious viral RNA and proteins. A full-length DENV2 genome, detected in arthropod EVs, was infectious to naïve mosquito and mammalian cells, including human-skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed mosquito EVs with a size range from 30 to 250 nm. Treatments with RNase A, Triton X-100, and 4G2 antibody-bead binding assays showed that infectious DENV2-RNA and proteins are contained inside EVs. Viral plaque formation and dilution assays also showed securely contained infectious viral RNA and proteins in EVs are transmitted to human cells. Up-regulated HSP70 upon DENV2 infection showed no role in viral replication and transmission through EVs. In addition, qRT-PCR and immunoblotting results revealed that DENV2 up-regulates expression of a mosquito tetraspanin-domain–containing glycoprotein, designated as Tsp29Fb, in A. aegypti mosquitoes, cells, and EVs. RNAi-mediated silencing and antibody blocking of Tsp29Fb resulted in reduced DENV2 loads in both mosquito cells and EVs. Immunoprecipitation showed Tsp29Fb to directly interact with DENV2 E-protein. Furthermore, treatment with GW4869 (exosome-release inhibitor) affected viral burden, direct interaction of Tsp29Fb with E-protein and EV-mediated transmission of viral RNA and proteins to naïve human cells. In summary, we report a very important finding on EV-mediated transmission of DENV2 from arthropod to mammalian cells through interactions with an arthropod EVs-enriched marker Tsp29Fb.

Original languageEnglish (US)
Pages (from-to)E6604-E6613
JournalProceedings of the National Academy of Sciences of the United States of America
Volume115
Issue number28
DOIs
StatePublished - Jul 10 2018

Fingerprint

Dengue Virus
Arthropods
Glycoproteins
Culicidae
Viral RNA
Viral Proteins
Exosomes
Aedes
Extracellular Vesicles
Cryoelectron Microscopy
Flavivirus
Pancreatic Ribonuclease
Proteins
Blocking Antibodies
Dengue
Octoxynol
RNA Interference
Viral Load
Keratinocytes
Immunoprecipitation

Keywords

  • Arthropod EVs
  • Dengue
  • HSP70
  • Transmission
  • Tsp29Fb

ASJC Scopus subject areas

  • General

Cite this

Arthropod EVs mediate dengue virus transmission through interaction with a tetraspanin domain containing glycoprotein Tsp29Fb. / Vora, Ashish; Zhou, Wenshuo; Londono-Renteria, Berlin; Woodson, Michael; Sherman, Michael; Colpitts, Tonya M.; Neelakanta, Girish; Sultana, Hameeda.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 115, No. 28, 10.07.2018, p. E6604-E6613.

Research output: Contribution to journalArticle

Vora, Ashish ; Zhou, Wenshuo ; Londono-Renteria, Berlin ; Woodson, Michael ; Sherman, Michael ; Colpitts, Tonya M. ; Neelakanta, Girish ; Sultana, Hameeda. / Arthropod EVs mediate dengue virus transmission through interaction with a tetraspanin domain containing glycoprotein Tsp29Fb. In: Proceedings of the National Academy of Sciences of the United States of America. 2018 ; Vol. 115, No. 28. pp. E6604-E6613.
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abstract = "Dengue virus (DENV) is a mosquito-borne flavivirus that causes dengue fever in humans, worldwide. Using in vitro cell lines derived from Aedes albopictus and Aedes aegypti, the primary vectors of DENV, we report that DENV2/DENV3-infected cells secrete extracellular vesicles (EVs), including exosomes, containing infectious viral RNA and proteins. A full-length DENV2 genome, detected in arthropod EVs, was infectious to na{\"i}ve mosquito and mammalian cells, including human-skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed mosquito EVs with a size range from 30 to 250 nm. Treatments with RNase A, Triton X-100, and 4G2 antibody-bead binding assays showed that infectious DENV2-RNA and proteins are contained inside EVs. Viral plaque formation and dilution assays also showed securely contained infectious viral RNA and proteins in EVs are transmitted to human cells. Up-regulated HSP70 upon DENV2 infection showed no role in viral replication and transmission through EVs. In addition, qRT-PCR and immunoblotting results revealed that DENV2 up-regulates expression of a mosquito tetraspanin-domain–containing glycoprotein, designated as Tsp29Fb, in A. aegypti mosquitoes, cells, and EVs. RNAi-mediated silencing and antibody blocking of Tsp29Fb resulted in reduced DENV2 loads in both mosquito cells and EVs. Immunoprecipitation showed Tsp29Fb to directly interact with DENV2 E-protein. Furthermore, treatment with GW4869 (exosome-release inhibitor) affected viral burden, direct interaction of Tsp29Fb with E-protein and EV-mediated transmission of viral RNA and proteins to na{\"i}ve human cells. In summary, we report a very important finding on EV-mediated transmission of DENV2 from arthropod to mammalian cells through interactions with an arthropod EVs-enriched marker Tsp29Fb.",
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AU - Zhou, Wenshuo

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AU - Woodson, Michael

AU - Sherman, Michael

AU - Colpitts, Tonya M.

AU - Neelakanta, Girish

AU - Sultana, Hameeda

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AB - Dengue virus (DENV) is a mosquito-borne flavivirus that causes dengue fever in humans, worldwide. Using in vitro cell lines derived from Aedes albopictus and Aedes aegypti, the primary vectors of DENV, we report that DENV2/DENV3-infected cells secrete extracellular vesicles (EVs), including exosomes, containing infectious viral RNA and proteins. A full-length DENV2 genome, detected in arthropod EVs, was infectious to naïve mosquito and mammalian cells, including human-skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed mosquito EVs with a size range from 30 to 250 nm. Treatments with RNase A, Triton X-100, and 4G2 antibody-bead binding assays showed that infectious DENV2-RNA and proteins are contained inside EVs. Viral plaque formation and dilution assays also showed securely contained infectious viral RNA and proteins in EVs are transmitted to human cells. Up-regulated HSP70 upon DENV2 infection showed no role in viral replication and transmission through EVs. In addition, qRT-PCR and immunoblotting results revealed that DENV2 up-regulates expression of a mosquito tetraspanin-domain–containing glycoprotein, designated as Tsp29Fb, in A. aegypti mosquitoes, cells, and EVs. RNAi-mediated silencing and antibody blocking of Tsp29Fb resulted in reduced DENV2 loads in both mosquito cells and EVs. Immunoprecipitation showed Tsp29Fb to directly interact with DENV2 E-protein. Furthermore, treatment with GW4869 (exosome-release inhibitor) affected viral burden, direct interaction of Tsp29Fb with E-protein and EV-mediated transmission of viral RNA and proteins to naïve human cells. In summary, we report a very important finding on EV-mediated transmission of DENV2 from arthropod to mammalian cells through interactions with an arthropod EVs-enriched marker Tsp29Fb.

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