As mosquitoes are prominent vectors for flaviviruses, specific interactions between the virus and arthropod likely enhance pathogen survival. In the mammalian host, C-type lectins such as DC-SIGN and the mannose receptor augment viral entry into specific DCs and macrophages (Tassaneetrithep et al., 2003
; Davis et al., 2006
; Miller et al., 2008
). Our results show that a secreted mosquito C-type lectin, mosGCTL-1, binds to WNV in a calcium-dependent manner, and enhances viral infection. A mosquito homologue of human CD45 (mosPTP-1) recruits mosGCTL-1 to facilitate viral attachment to cells. Based on our findings, we envision a model whereby WNV that is inoculated into mosquitoes binds to secreted mosGCTL-1 in the hemolymph, thereby forming a complex in the extracellular milieu which has the ability to interact with the membrane protein, mosPTP-1, to facilitate cellular entry. The virus rapidly replicates in the mosquito thorax. This induces additional mosGCTL-1 expression, which accelerates formation of the mosGCTL-1/WNV complex -- enabling WNV to invade different mosquito tissues, and enhancing viral spread throughout the mosquito body. This mechanism, which involves WNV associating with mosGCTL-1 and then being captured by mosPTP-1 onto the cell surface in mosquitoes, suggests that an extracellular soluble protein is an important receptor for flavivirus in arthropods.
mosGCTL-1 shares homology with human MBL. In mammals, MBL is a pattern recognition molecule that recognizes carbohydrate moieties on invading microbes (Neth et al., 2002
). As examples, MBL interacts with HIV envelope protein (gp120) (Saifuddin et al., 2000
) and HBV surface antigen (HBsAg) (Chong et al., 2005
), and has a role in the opsonization of HIV (Ezekowitz et al., 1989
). In these processes, MBL associates with serine proteases, MASPs, and activates the complement system (Neth et al., 2002
). Homologues of the proteins that associate with mammalian MBL have not been found in A. aegypti
), suggesting that the A. aegypti
mosGCTL-1 may have different physiological functions than mammalian MBL. Invertebrates lack antibody- and interferon-based immune responses (Cheng et al., 2009
). Since lectin expression is significantly up regulated by microbial infection, these molecules are presumed to participate in non-self recognition and pathogen resistance (Wilson et al., 1999
; Tanji et al., 2006
). Indeed, recent studies have shown that a complement-like system exists in the hemolymph of Anopheles gambiae
and mediates parasite killing (Blandin et al., 2004
; Povelones, et al. 2009
). It is possible that mosGCTL-1 and other subtypes in this family, similar to their mammalian homologues, may normally recognize most pathogens and be involved in the arthropod complement-like system. Nevertheless, our studies showed that the expression of mosGCTL-1 is induced by WNV infection. The induced mosGCTL-1 that then binds to virus, amplifies WNV infection. Overall, these suggest a critical role for mosGCTL-1 in WNV infection of mosquitoes.
In the mammalian host, the association between MBL and the CD45 external domain primarily occurs in immature T cells, and affect the development of thymocytes (Baldwin and Ostergaard, 2001
). Mammalian CD45 is expressed on the hemopoietic-originated nucleated cells (Thomas, 1989
); however, the mosquito CD45 homologue, mosPTP-1, does not appear to be restricted to particular cells. As a transmembrane protein, mosPTP-1 was abundantly detected in the salivary glands and hemolymph of mosquitoes. The pattern of mosPTP-1 expression correlated with the distribution of WNV in A. aegypti
. In our model, after binding to mosGCTL-1, WNV binds to membrane bound mosPTP-1. This implies that mosGCTL-1 and mosPTP-1 are recruited as receptors to facilitate cellular invasion by WNV.
Mosquito control is a common strategy to influence WNV numbers in nature (van der Meulen et al., 2005
; Dauphin and Zientara, 2007
). The increase in viral spread and fatalities over the last decade (Reisen and Brault, 2007
; Lindsey et al., 2009
) suggests that additional strategies could assist in combating WNV. For arthropod-borne microbes, vector ligands that interact with pathogens are potential targets for interfering with the successful acquisition of the microbe from the vertebrate host. As an example, blocking the tick gut receptor for the Lyme disease agent limits the colonization of ticks by Borrelia. burgdorferi
(Pal et al., 2004
). Our studies show that blocking mosGCTL-1 within A. aegypti
reduced the vector competence for WNV and interrupted the infective cycle of WNV. These results indicate that it is theoretically possible to develop a transmission-blocking vaccine to interfere with the migration of WNV from vertebrates to mosquito, thereby restricting viral dissemination in the environment.
In summary, we identified a lectin-based pathway to facilitate flaviviral entry, in which mosGCTL-1 and mosPTP-1 are cascade receptors in WNV infection of A. aegypti and C. quinquefasciatus. Characterization of mosquito ligands for WNV enhances our understanding of flavivirus-arthropod interactions, and may aid in the development of strategies to target selected points in the flaviviral life cycle and interfere with these pathogens in nature.