TY - JOUR
T1 - Arthropod EVs mediate dengue virus transmission through interaction with a tetraspanin domain containing glycoprotein Tsp29Fb
AU - Vora, Ashish
AU - Zhou, Wenshuo
AU - Londono-Renteria, Berlin
AU - Woodson, Michael
AU - Sherman, Michael B.
AU - Colpitts, Tonya M.
AU - Neelakanta, Girish
AU - Sultana, Hameeda
N1 - Publisher Copyright:
© 2018 National Academy of Sciences. All rights reserved.
PY - 2018/7/10
Y1 - 2018/7/10
N2 - 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.
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.
KW - Arthropod EVs
KW - Dengue
KW - HSP70
KW - Transmission
KW - Tsp29Fb
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U2 - 10.1073/pnas.1720125115
DO - 10.1073/pnas.1720125115
M3 - Article
C2 - 29946031
AN - SCOPUS:85049644313
SN - 0027-8424
VL - 115
SP - E6604-E6613
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 28
ER -