Novel interactions of domain III from the envelope glycoprotein of dengue 2 virus with human plasma proteins.

Blood cells and plasma are important media for the four serotypes of dengue virus (DENV1-4) spreading into an infected person. Thus, interactions with human plasma proteins are expected to be decisive in the course of the viral infection. Affinity purification followed by MS analysis (AP/MS) was used to isolate and identify plasma-derived proteins capable to interact with a recombinant protein comprising the domain III of the envelope protein of DENV2 (DIIIE2). The elution of the AP potently inhibits DENV2 infection. Twenty-nine proteins were identified using a label-free approach as specifically captured by DIIIE2. Of these, a direct interaction with C reactive protein, thrombin and Inter-alpha-inhibitor complexes was confirmed by ELISA. Results provide further evidence of a significant representation of proteins from complement and coagulation cascades on DENV2 interactome in human plasma and stand out the domain III of the viral envelope protein as participant on these interactions. A functional clustering analysis highlights the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. BIOLOGICAL SIGNIFICANCE: Early cycles of dengue virus replication take place in human blood cells. Thus, the characterization of the interactome of dengue virus proteins in human plasma can lead to the identification of pivotal interactions for the infection that can eventually constitute the target for the development of methods to control dengue virus-caused disease. In this work we identified 29 proteins from human plasma that potentially interact with the envelope protein of dengue 2 virus either directly or through co-complex formation. C reactive protein, thrombin and Inter-alpha-inhibitor complexes were validated as interactors of the domain III of the envelope protein of dengue 2. Results highlight the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. This finding together with the participation of domain III of the envelope protein on the interactions with human plasma proteins should contribute to a better understanding of dengue virus interactome in human plasma. Such knowledge can contribute to the development of more effective treatments to infected persons.

Receptor-activated human α2-macroglobulin interacts with the envelope protein of dengue virus and protects virions from temperature-induced inactivation through multivalent binding.

Based on the hypothesis that interactions between virions and serum components may influence the outcome of dengue virus (DENV) infections, we decided to use affinity chromatography with domain III from the envelope (E) protein of DENV2 (DIIIE2) as a ligand to isolate virus-binding proteins from human plasma. This approach yielded serum amyloid P (SAP) and α2-macroglobulin (α2M) as novel viral interactors. After confirming the specific binding of both SAP and α2M to DIIIE2 by ELISA, the latter interaction was examined in greater detail. We obtain evidence suggesting that the binding species was actually the receptor-activated form of α2M (α2M*), that α2M* could bind monovalently to recombinant domain III from all four DENV serotypes with affinities in the micromolar range ranking as DENV4>DENV1~DENV2>DENV3 and that this interaction exhibited a strong avidity effect when multivalent binding was favoured (KD 8 × 10(-8) M for DIIIE2). We also showed that α2M* bound to DENV virions of the four serotypes, protecting the virus from temperature-induced inactivation in the absence of serum and enhancing infectivity. The latter effect exhibited an ED50 of 2.9 × 10(-8) M, also suggesting an avidity effect due to multivalent binding. These results will further contribute to the characterization of the virus-host factor interaction network during human DENV infection.

Characterization of the interaction of domain III of the envelope protein of dengue virus with putative receptors from CHO cells.

Domain III (DIII) of the envelope protein of dengue virus (DENV) contains structural determinants for the interaction with cellular receptors. In the present study a solid phase assay and recombinant fusion proteins containing DENV-DIII of serotypes 1 and 2 were used to study structural features of the interaction of the envelope protein with putative receptors present in the microsomal fraction of CHO cells. Recombinant fusion proteins showed specific interaction with proteins present in the microsomal fraction. Binding of the fusion proteins across the pH range of 5.5-8.0 resembled that of virus particles, peaking at pH 6.0. This suggests that the interaction of DIII with cell receptor(s) is strengthened at endosomal pH. The effect of reduction and carbamidomethylation of cysteine residues on the binding to the microsomal fraction and in their recognition by antibodies suggests that the region of DIII that is interacting with putative receptor(s) overlaps only partially with a dominant epitope of the antibody response. The analysis of the residue conservation profile indicates that the surface of DIII is composed typically of specific sub-complex residues with an increased representation of specific type/subtype residues found at the surface that closely correlates with the dominant neutralizing epitope.