Structural characterization and biological function of bivalent binding of CD2AP to intrinsically disordered domain of chikungunya virus nsP3 protein

Abstract

Alphavirus nsP3 proteins contain long, intrinsically disordered, hypervariable domains, HVD, which serve as hubs for interaction with many cellular proteins. Here, we have deciphered the mechanism and function of HVD interaction with host factors in alphavirus replication. Using NMR spectroscopy, we show that CHIKV HVD contains two SH3 domain-binding sites. Using an innovative chemical shift perturbation signature approach, we demonstrate that CD2AP interaction with HVD is mediated by its SH3-A and SH3-C domains, and this leaves the SH3-B domain available for interaction with other cellular factor(s). This cooperative interaction with two SH3 domains increases binding affinity to CD2AP and possibly induces long-range allosteric effects in HVD. Our data demonstrate that BIN1, CD2AP and SH3KBP1 play redundant roles in initiation of CHIKV replication. Point mutations in both CHIKV HVD binding sites abolish its interaction with all three proteins, CD2AP, BIN1 and SH3KBP1. This results in strong inhibition of viral replication initiation.

Keywords: Alphaviruses; BIN1; CD2AP; Chikungunya virus; Intrinsically disordered proteins; NMR; SH3 domain-containing proteins; SH3KBP1; Viral replication; nsP3.

Fig. 1.. Differential expression of CD2AP, SH3KBP1 and BIN1 in the cells susceptible to CHIKV infection.

(A) The schematic presentation of CHIKV genome. (B) The aa sequence of CHIKV 181–25 nsP3 HVD, which corresponds to aa 325–523 of the full-length nsP3 protein. The previously predicted motifs that could interact with SH3 domain-containing proteins are depicted in red. Two sites of G3BP1/2 binding are depicted in blue. (C) The schematic presentation of the domain structures of CD2AP, SH3KBP1 and BIN1. (D) Western blot analysis of CD2AP, SH3KBP1 and BIN1 expression in different cell lines. Of all isoforms, the bands, which were used for quantitative analysis of protein expression, are marked by red asterisks.

Fig. 2.. ¹H-¹⁵N-TROSY-HSQC spectrum of the CHIKV-HVD with assignment of the cross peaks that belong to the amide groups.

(A) The area between ¹H: 8.65–7.65 ppm and ¹⁵N: 130–107 ppm that covers all observed cross peaks. (B) The magnified overlapped spectrum region indicated in panel (A). The cross peaks were labeled according to the numbers of the aa in CHIKV-HVD in the sequence presented in Fig. 1B. Spectrum was acquired on a 800MHz Bruker AVANCE III-HD spectrometer at 20° C.

Fig. 3.. CHIKV HVD interaction with individual SH3 domains of CD2AP and their combination in SH3-All.

Weighted chemical shift perturbations of the amide bonds (¹H and ¹⁵N nuclei) in the ¹⁵N-labeled CHIKV HVD are presented as superimposed bars on the CHIKV HVD sequence. The lists of the titration ratios (SH3:HVD) are indicated at the right side of the panels. The amino acids that showed broadening of cross peaks during titrations are indicated by grey bars. The unassigned resonances and prolines are indicated by black and red dots, respectively. The thresholds of interacting/non-interacting aa are indicated as grey lines at the level of two standard deviations (2σ_o). Solid red lines on the tops of the panels indicate the positions of binding sites 1 and 2 and allosteric site 3. The green lines show the positions of G3BP-binding sites.

Fig. 4.. Different CHIKV HVD/SH3 complexes demonstrate specific CSP signatures.

Superposition of the expanded ¹H-¹⁵N best-TROSY spectra of the apo ¹⁵N-CHIKV HVD (black cross peaks) with the corresponding spectra of ¹⁵N-CHIKV HVD, titrated with unlabelled SH3 domains. The SH3-A-, SH3-B-, SH3-C- and SH3-All-specific cross peaks are indicated in red, blue, green and pink, respectively. (A) The N-terminal element of Site 1, represented by aa 70–74; (B) The N- and C-terminal elements of Site 2, represented by aa 101–105 and 108–111, respectively; and (C) Site 3, represented by aa 164–172. Arrows indicate the directions of the chemical shift perturbations in the presence of the SH3 domains at increasing concentrations. Several cross peaks of these sequences are not shown because they are either located in a crowed area of the spectrum or the intensity decreased below the detection limit.

Fig. 5.. Individual CD2AP SH3 domains and SH3-All have different affinities to Sites 1 and 2 in CHIKV HVD.

Experimental titration and globally fitted curves of (¹H, ¹⁵N) weighted CSPs extracted from ¹H-¹⁵N best-TROSY spectra of the ¹⁵N-CHIKV HVD in the titration experiments with different concentrations of unlabelled domains SH3-A, SH3-B, SH3-C and SH3-All.

Fig. 6.. Mutation is Site 1 and Site 2 of CHIKV HVD abolish binding of CD2AP and SH3KBP1 and strongly inhibit viral replication.

(A) The schematic presentation of CHIKV/GFP genome and mutations introduced into CD2AP-binding sites of CHIKV HVD. Dashes indicate identical aa. (B) The schematic presentation of the designed VEEV replicons, which encoded Flag-GFP alone, and wt and mutant HVDs fused with Flag-GFP. (C) Human MRC-5 and BJ-5ta cells were infected with VEEV replicons encoding Flag-GFP, or Flag-GFP fused with either wt or mutant CHIKV HVD. Protein complexes were isolated using magnetic beads with anti-Flag MAbs and further analyzed by Western blot using indicated antibodies. An asterisk marks the nonspecific staining of Flag-GFP-HVDs. (D) Equal numbers of indicated cells in the wells of 6-well plate were infected with the indicated viruses at an MOI of 0.04 PFU/cell. After incubation for 15 h at 37° C, media were harvested, and viral titers were determined by plaque assay on BHK-21 cells. Significances of differences among the values were determined by two-tailed unpaired t-test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; n=3). Data are presented as mean with SD (n=3). Numbers under asterisks indicate average fold differences between the titers.

Fig. 7.. Mutations in CD2AP-binding sites of CHIKV HVD strongly affect viral infectivity.

(A) The RNA electroporation-derived stocks of CHIKV/GFP and CHIKVmut/GFP were synchronously titrated on the indicated cell lines as described in Materials and Methods. Infectious viral titers were normalized to those determined on BHK-21 cells. (B) In the titration experiments, images of GFP-positive foci formed by viruses on both cell lines were taken at 40 h p.i. directly under agarose cover. (C) Ratio of viral concentrations in the same stocks determined in GE/ml and inf.u/ml on the indicated cell lines. Since CHIKVmut/GFP was unable to form plaques on most of cell lines, for uniform presentation, infectious titers were used as infectious units per ml (inf.u/ml) instead of PFU/ml. Significances of differences among the values were determined by two-tailed unpaired t-test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; n=3). Data are presented as mean with SD (n=3). Numbers under asterisks indicate average fold differences.

Fig. 8.. Schematic model of CHIKV HVD-CD2AP complex.

(A) CD2AP and CHIKV HVD form bivalent complex. SH3-C and SH3-A domains of CD2AP bind to Site 1 and 3 of CHIKV HVD respectively. Such bivalent interaction strongly increases affinity of CD2AP interaction with CHIKV HVD. Site 3 undergoes an allosteric modification upon interaction of any SH3 domain with Site 1. This modification may regulate binding of G3BP1 and G3BP2 to the motifs that flank Site 3. (B) Amino acid sequences of binding sites 1 and 2 and allosteric site 3 of CHIKV nsP3 HVD. The docking motifs in Sites 1 and 2 are underlined. The amino acids that were mutated in this work are depicted in red. The allosteric Site 3 is marked by bold font and flanking G3BP binding sites are depicted in green.