The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition.

Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1-35 and 140-210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

Identification and characterization of heme-interacting proteins in the malaria parasite, Plasmodium falciparum.

The degradation of hemoglobin by the malaria parasite, Plasmodium falciparum, produces free ferriprotoporphyrin IX (FP) as a toxic by-product. In the presence of FP-binding drugs such as chloroquine, FP detoxification is inhibited, and the build-up of free FP is thought to be a key mechanism in parasite killing. In an effort to identify parasite proteins that might interact preferentially with FP, we have used a mass spectrometry approach. Proteins that bind to FP immobilized on agarose include P. falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), P. falciparum glutathione reductase (PfGR), and P. falciparum protein disulfide isomerase. To examine the potential consequences of FP binding, we have examined the ability of FP to inhibit the activities of GAPDH and GR from P. falciparum and other sources. FP inhibits the enzymic activity of PfGAPDH with a Ki value of 0.2 microm, whereas red blood cell GAPDH is much less sensitive. By contrast, PfGR is more resistant to FP inhibition (Ki > 25 microm) than its human counterpart. We also examined the ability of FP to inhibit the activities of the additional antioxidant enzymes, P. falciparum thioredoxin reductase, which exhibits a Ki value of 1 microm, and P. falciparum glutaredoxin, which shows more moderate sensitivity to FP. The exquisite sensitivity of PfGAPDH to FP may indicate that the glycolytic pathway of the parasite is particularly susceptible to modulation by FP stress. Inhibition of this pathway may drive flux through the pentose phosphate pathway ensuring sufficient production of reducing equivalents to counteract the oxidative stress induced by FP build-up.