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

Abstract

Dengue virus (DENV) is a mosquito-borne flavivirus that causes dengue fever in humans, worldwide. Using in vitro cell lines derived from and, 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 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.

Role of RNA-binding proteins during the late stages of Flavivirus replication cycle

“…Progeny virus particles are considered the sole mechanism for the dissemination of flaviviruses. Nevertheless, cell-derived extracellular vesicles carrying Flavivirus vRNA have recently been shown to be infectious [111][112][113][114][115][116], thus representing an alternate mode of virus dissemination. The pathway leading to the secretion of vRNA independently of virus particles is unknown, but parallels can be drawn with the export of cellular RNAs.…”

“…The intersection between cytoplasmic ribonucleoprotein complexes and endosomes might be a step prior the export of RNA molecules via exosomes propagation [142] [143]. In particular, cells infected with HCV [144][145][146], DENV [111][112][113][114], ZIKV [115] and TBEV [116] release exosomes that are infectious to recipient cells. These infectious exosomes carry full-length vRNA but do not always contain the structural viral proteins associated with virions, thus suggesting that in Flavivirus-infected cells, vRNA molecules are sorted into exosomes independently of the assembly of fully mature particles.…”

Message in a vesicle – trans-kingdom intercommunication at the vector–host interface

“…Similarly, other viruses hijack extracellular vesicles from the arthropod vector for their transmission. For instance, the Dengue virus serotype 2 (DENV2) uses extracellular vesicles derived from mosquitoes to infect mammalian cells (Vora et al, 2018). During Dengue virus infection, mosquito-derived vesicles carry viral proteins and a full-length DENV2 genome.…”

“…During Dengue virus infection, mosquito-derived vesicles carry viral proteins and a full-length DENV2 genome. Importantly, DENV2 transmission is dependent on vesicle secretion by the vector, which may occur through the interaction between the tetraspanin domain-containing glycoprotein Tsp29Fb, a mosquito homolog of the exosomal marker CD63, and the viral E protein (Vora et al, 2018). Although these findings were primarily obtained in vitro, they clearly suggest that arthropod extracellular vesicles may transmit viruses.…”

“…Although we do not fully understand how arthropod vectors aid in the establishment of illnesses, an emerging principle is that pathogens exploit extracellular vesicles secreted by their vectors. Indeed, independent groups have shown that vectorborne microbes can change the protein and miRNA content of vector and host vesicles by adding pathogen-derived components that assist in microbial spreading (Sampaio et al, 2018;Vora et al, 2018;Zhou et al, 2018). Dissection of these underlying mechanisms is clearly a priority, and there are outstanding questions that should be explored.…”