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 - Funding Information:
ACKNOWLEDGMENTS. We thank Drs. Michel Ledizet (for 4G2 monoclonal antibody), John Catravas and Ms. Betsy Gregory (for providing growing human umbilical vein endothelial cells and required cell culture media), and Dr. John F. Anderson [for the generous gift of wild-type DENV2 El Salvador strain (TVP2176)]. H.S. thanks the kind support from BEI Resources. The following reagents were obtained through BEI Resources, National Institute of Allergy and Infectious Diseases, NIH: (i) monoclonal anti-Flavivirus group antigen, clone D1-4G2-4-15 (produced in vitro), NR-50327; (ii) dengue virus type 3, DENV-3/US/BID-V1619/2005, NR-43283; (iii) monoclonal anti-dengue virus type 3 envelope protein, clone E2 (produced in vitro), NR-15511. This work was supported by start-up funds from Old Dominion University (to H.S.).
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 -