Mechanisms of viral capture.

The liver appears to be the main mediator of viral vascular clearance. However, the specific mechanisms of removing virions from the circulation is distinct and virus-specific. The removal of AdV particles is mainly performed by KCs; however, some of the receptors shown to interact with AdV can also be expressed by LSECs (SR-F1 and SR-A1). In addition to SRs (SR-F1, SR-A1, and SR-A6), nAb, and CRIg also promote clearance of AdV from the bloodstream. For arthritogenic alphaviruses (CHIKV, RRV, and ONNV), clearance is mediated specifically by SR-A6 (MARCO) and KCs. However, particles that have a single point mutation to replace a lysine residue on the E2 glycoprotein (K200X for CHIKV and ONNV; K251X for RRV) evade capture. For the flaviviruses DENV and WNV, the type of virion glycosylation present affects clearance mediated by MBL. Specifically, MBL binds the high-mannose glycosylated virus particles, but not virions decorated with complex glycosylation. However, MBL is not the only mediator of DENV and WNV clearance, and it is clear another, as-yet-unknown mechanism also exists. This figure was created with BioRender.com. AdV, adenovirus; CHIKV, chikungunya virus; DENV, dengue virus; KC, Kupffer cell; LSEC, liver sinusoidal endothelial cell; MBL, mannose-binding lectin; nAb, natural antibodies; ONNV, o’nyong’nyong virus; RRV, Ross River virus; WNV, West Nile virus.</p

Host mechanisms of viral clearance.

Host mechanisms of viral clearance.</p

Macrophages of the spleen.

Splenic macrophages also participate in the capture of circulating virus particles. There are 3 major splenic macrophage populations (MMM, MZM, and RpM), and they localize to distinct regions of the spleen. These macrophage subsets can be identified by their localization and the indicated key cellular markers. While the mechanisms by which specific splenic macrophage populations mediate viral clearance are not well understood, they are critical in activating immune responses to circulating viruses. This figure was created with BioRender.com. MMM, marginal zone metallophilic macrophage; MZM, marginal zone macrophage; RpM, red pulp macrophage.</p

The liver sinusoid.

There are 2 key cell types located in the liver sinusoid that have been shown to contribute to viral vascular clearance. Although in vitro studies suggest that LSECs, which form the liver endothelium, interact with certain viruses (e.g., AdV), KCs, which are the liver’s main tissue-resident macrophages, are responsible for clearing diverse circulating viruses (e.g., CHIKV and AdV) in vivo. In addition, KCs are important in controlling pathogenesis of viruses like LCMV. This figure was created with BioRender.com. AdV, adenovirus; CHIKV, chikungunya virus; HA, hepatic artery; IFN, interferon; KC, Kupffer cell; LCMV, lymphocytic choriomeningitis virus; LSEC, liver sinusoidal endothelial cell; PRR, pathogen recognition receptor; PV, portal vein; VACV, vaccinia virus.</p

Documented surface-expressed pattern recognition receptors of LSECs and KCs.

Documented surface-expressed pattern recognition receptors of LSECs and KCs.</p

E2 and VENUS are equally detectable in CHIKV-infected EVA cells at 24 hpi.

EVA cells were inoculated with 106 PFU of CHIKV-VENKL for 24hpi, harvested, fixed, permeabilized, and stained with anti-E2 CHIKV (CHK-11) at decreasing (2-fold) serial dilutions. Cells were analyzed for E2 and VENUS expression by flow cytometry. (A) Representative flow cytometry plots. (B) Quantification of cell frequency for E2 and VENUS positive cells (red = infected, black = mock). (EPS)</p

CD8⁺ T cells are activated by CHIKV-infected cells when peptide processing and ER transport are bypassed.

(A-B) EVA cells were inoculated with 106 PFU CHIKV-VENKL or CHIKV-SIINFEKLβ2m. At 24 hpi, EVA cells were cocultured with a 1:1 mixture of 106 bystander and 106 OT-I SIINFEKL-specific CD8+ T cells for 6 h. Bystander CD8+ T cells were distinguished from OT-I CD8+ T cells by gating on CD8+, KbOVA257-264 tetramer positive or negative cells, and both populations were assessed for the expression of CD69, CD25, IRF4 and CD8 by flow cytometry. (A) Representative flow cytometry plots. (B) Quantification of CD25, CD69, IRF4 (intracellular), and CD8 cell surface expression (upper graphs) and frequency (lower graphs) for OT-I cells above bystander CD8+ T cells. Data are representative of two independent experiments (n = 6). P-values were determined by unpaired student’s t-test. *, PPPP<0.0001.</p

Determinants in nsP2 promote impairment of MHC-I antigen presentation by CHIKV-infected cells.

(A) Schematic depicting the location of nsP2 V-loop mutations (QMS; blue) compared with wildtype nsP2 (ATL; red). (B) WT and Ifnar1-/- murine embryonic fibroblasts (MEFs) were inoculated with CHIKV-VENKL or CHIKVQMS-VENKL at an MOI of 0.1 FFU/cell. At 0 (input), 1, 24, 48, and 72 hpi, the amount of infectious virus present in culture supernatants was quantified by focus formation assay. (C-H) EVA cells were mock-inoculated or inoculated with CHIKV-VENKL or CHIKVQMS-VENKL. At 24 hpi, cells were assessed for cell surface expression of H2-Kb, and SIINFEKL-loaded H2-Kb (H2-Kb-SIINFEKL) by flow cytometry. (C) Representative flow cytometry plots depicting H2-Kb-SIINFEKL and H2-Kb cell surface expression on live, CD45- and live, CD45-, VENUS+ cells from mock-, CHIKV-VENKL-, and CHIKVQMS-VENKL-inoculated EVA cells. (D) Percentage of VENUS+ cells among live, CD45-, EVA cells. (E) CD29 cell surface expression on live, CD45-, VENUS+ EVA cells. (F) Quantification of H2-Db and H2-Kb surface expression on live, CD45-, VENUS+ EVA cells. (G) Percentage of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells on live, CD45-, VENUS+ EVA cells. (H) pMHC-I presentation efficiency on live, CD45-, VENUS+ EVA cells. Data are representative of 4 experiments (n = 12). P values were determined by unpaired student’s t-test (D-H). **, PPP<0.0001.</p

Frequency of VENUS⁺ cells after transfection of EVA cell cultures.

EVA cells were co-transfected with the designated WT or mutant pCMV-nsP2 plasmids or an identical control plasmid lacking the methionine start codon for nsP2 (vector) and pCMV-VENKL. At 24 h post-transfection, cells were evaluated by flow cytometry for expression of VENUS. (A) Representative flow cytometry plots for vector (black, circle), nsP2 (red, circle) and nsP2QMS (blue, circle) and frequency of VENUS+ cells among live, CD45- cells. (B) Representative flow cytometry plots for vector (black, circle), nsP2 (red, circle), nsP2K192A (red, triangle), nsP2C478S (red, square) and nsP2QMS (blue, circle) and frequency of VENUS+ cells among live, CD45- cells. (EPS)</p

Gating strategy for CHIKV-VENKL- vs CHIKV^QMS-VENKL-infected EVA cells.

Representative flow cytometry plots demonstrating gating strategy for CD45-, CD29+, VENUS+ fibroblasts from EVA cell cultures. Cell surface expression of CD29 was assessed within singlets, CD45-, VENUS+ or VENUS- gates. (EPS)</p