Mapping the interactions of dengue virus NS1 protein with human liver proteins using a yeast two-hybrid system: identification of C1q as an interacting partner

Abstract

Dengue constitutes a global health concern. The clinical manifestation of this disease varies from mild febrile illness to severe hemorrhage and/or fatal hypovolemic shock. Flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein that is displayed on the surface of infected cells but is absent in viral particles. NS1 accumulates at high levels in the plasma of dengue virus (DENV)-infected patients, and previous reports highlight its involvement in immune evasion, dengue severity, liver dysfunction and pathogenesis. In the present study, we performed a yeast two-hybrid screen to search for DENV2 NS1-interacting partners using a human liver cDNA library. We identified fifty genes, including human complement component 1 (C1q), which was confirmed by coimmunoprecipitation, ELISA and immunofluorescence assays, revealing for the first time the direct binding of this protein to NS1. Furthermore, the majority of the identified genes encode proteins that are secreted into the plasma of patients, and most of these proteins are classified as acute-phase proteins (APPs), such as plasminogen, haptoglobin, hemopexin, α-2-HS-glycoprotein, retinol binding protein 4, transferrin, and C4. The results presented here confirm the direct interaction of DENV NS1 with a key protein of the complement system and suggest a role for this complement protein in the pathogenesis of DENV infection.

Figure 1. Plasmid linkage assays for transformants identified using DENV2 NS1 as bait in the yeast two-hybrid screening.

Transformants containing the bait and prey plasmids were visualized by their growth on double drop-out media (SD–Leu–Trp; column A). Putative interacting partners were visualized by their growth on triple (SD–His–Leu–Trp; column B) and quadruple drop-out media (SD–Ade–His–Leu–Trp; column C) and by the blue color staining of the colony-lift filter assay (column D) indicating HIS3, ADE2 and lacZ reporter gene activation, respectively. AH109 yeast cells cotransformed with the plasmids pGBKT7-53 (murine p53 fused to the GAL4 DNA-binding domain) and pGADT7-T (SV40 large T-antigen fused to the GAL4 activation domain) served as positive controls (C+). AH109 cotransformed with the plasmids pGBKT7-NS1 and pGADT7-AD (C1), pGBKT7-NS1 and pGADT7-T (C2), pGBKT7 and pGADT7 (C3), pGBKT7 and pGADT7-T (C4), pGBKT7-Lam (laminin C) and pGADT7 (C5), and pGBKT7-Lam and pGADT7-T (C6) served as negative controls. The gene name for each acronym is detailed in Table 1.

Figure 2. Cellular localization of DENV2 NS1 interacting-partners identified by yeast two-hybrid screening.

Figure 3. Coimmunoprecipitation of human C1q and NS1 proteins.

(A) Purified NS1 from the supernatants of DENV-infected BHK cells and purified human C1q protein were immunoprecipitated with anti-NS1 polyclonal antibody, and the eluted fractions 1 and 2 (E1 and E2) were subjected to Western blot analysis. Bands of approximately 30 and 50 kDa corresponding to C1q and NS1, respectively, were observed in the elution fraction of the coimmunoprecipitation. (B) C1q was capable of binding anti-NS1 antibody, although the band intensity in the DENV lane E1 was more than four-fold intense than that in the control lane E1. The IgG₁-coated resin eluted a similar amount of C1q as the control. Error bars indicate the standard deviation from three independent experiments, and the p value denotes significant differences from the control.

Figure 4. DENV NS1 directly binds human C1q in an ELISA assay.

(A) Microtiter plates were coated with purified human C1q (10 µg/mL). After incubation with increasing concentrations of purified DENV NS1 that was purified from the supernatant of BHK cells, bound NS1 was detected using a specific conformational monoclonal anti-NS1 antibody. (B) Microtiter plates were coated with purified human C1q (10 µg/mL). After incubation with increasing concentrations of purified DENV NS1 that was purified from E. coli cells, bound NS1 was detected with a specific polyclonal anti-NS1 antibody. Error bars indicate standard deviation from three independent experiments, and asterisks indicate significant difference from the control mock or BSA. *p<0.05, **p<0.01, ***p<0.001.

Figure 5. Colocalization of NS1 and C1q proteins by confocal microscopy.

DENV2-infected THP-1 cells were labeled by incubation with polyclonal anti-NS1 (red stained) or monoclonal anti-C1q (green stained) antibodies. NS1 and C1q proteins were localized in vesicle-like structures in the cytoplasm, which is characteristic of secretory proteins. When the images were merged, distinct yellow regions were revealed, indicating colocalization of NS1 with C1q in these areas (detail). The subcellular localization of C1q was also analyzed in mock-infected cells, and it appeared at an identical position as that observed in DENV-infected cells, whereas no NS1 protein was detected in these cells. The cells were also incubated with DAPI for nuclear staining.