A C-terminal fragment of Aedes albopictus salivary protein 34k2 facilitates DENV-2 replication and modulates host inflammation

Mosquito salivary proteins facilitate arboviral transmission through diverse biological mechanisms, including mechanical transmission, immune regulation. While the Aedes albopictus 34k2 salivary protein enhances DENV-2 infection, its precise functional domain remains unclear. This study aimed to delineate the structural basis of 34k2-mediated DENV-2 enhancement and elucidate its role in viral binding and host immune modulation. Structural prediction (AlphaFold3) and molecular docking (ZDOCK) localized binding interfaces. Two truncated variants were constructed: an N-terminal fragment (rAlb-34k2-I) and a C-terminal fragment (rAlb-34k2-II). Binding affinity was evaluated using surface plasmon resonance (SPR). Viral replication and infectivity were assessed via immunofluorescence, plaque assays, and qRT-PCR in RAW264.7 macrophages and THP-1 monocytes. Immunomodulatory effects of rAlb-34k2-II and rAlb-34k2 were analyzed using qPCR and ELISA in vitro, alongside an in vivo murine footpad injection model. Docking identified the viral-binding site within the C-terminal region. SPR confirmed that rAlb-34k2-II bound DENV-2 virions with high affinity (KD = 2.88 nM), whereas rAlb-34k2-I showed no measurable binding. Pre-incubation with rAlb-34k2-II significantly enhanced DENV-2 replication and infectious plaque production in immune cells. Furthermore, both rAlb-34k2-II and the full-length rAlb-34k2 protein significantly upregulated the transcription of pro-inflammatory cytokines (IL-6, IL-1β) and chemokines (CCL2, CXCL2) in vitro. This immune response was characterized by a distinct kinetic pattern: a transient early spike in inflammatory cytokines followed by sustained expression of chemokines. Consistent with these in vivo findings, the full-length 34k2 protein induced a similar pro-inflammatory profile in the local murine footpad tissue. The C-terminal fragment (residues 148–322) of Ae. albopictus 34k2 constitutes a high-affinity viral-binding domain that potentiates DENV-2 replication and modulates host inflammation. Targeting this specific salivary–viral interface represents a promising rational strategy for developing competitive decoy peptides or next-generation salivary-based vaccines to interrupt arboviral transmission.