In enteroviruses, the inhibition of protein synthesis from capped host cell mRNA is catalyzed by the virally encoded 2A proteinase (2A), which cleaves eukaryotic initiation factors (eIF) 4GI and 4GII. Despite much investigation, the exact mechanism of 2A cleavage remains however unclear. Here, we identify the domains responsible for the eIF4E/HRV2 2A interaction using molecular modelling and describe mutations that impair this interaction and delay in vitro cleavage of eIF4G isoforms. Furthermore, we produced HRV1A viruses bearing the mutation L17R, Y32A or Y86A in the 2A sequence. All three viruses showed reduced yield and were appreciably delayed during infection in eIF4GI cleavage. Thus, we propose for genetic group A HRVs that the eIF4E/2A interaction is essential for successful viral replication. In contrast, HRV4 2A and coxsackievirus B4 2A failed to form complexes with eIF4E, suggesting that the mechanism of eIF4G isoform cleavage in these and related viruses is different.
New technologies are changing the way research is performed in many areas of molecular allergology. 1 Recombinant (r) allergens have influenced the development of allergy diagnosis and allergen-specific immunotherapy (AIT) for over three decades. 2 Although the gold rush of allergen discovery is over, gaps in our knowledge of structures, cross-reactivities, and conformational epitopes are being constantly filled. 2,3
Cytolytic protein (Cyt) is a member of insecticidal proteins produced by Bacillus thuringiensis. Cyt protein has activity against insect cells and mammalian cells, which differ in lipid and cholesterol composition. This study presents the lipid binding behavior of Cyt2Aa2 protein on model membranes containing different levels of cholesterol content by combining Quartz Crystal Microbalance with Dissipation (QCM-D) and Atomic Force Microscopy (AFM). QCM-D results revealed that cholesterol enhances the binding rate of Cyt2Aa2 protein onto lipid bilayers. In addition, the thicker lipid bilayer was observed for the highest cholesterol content. These results were confirmed by AFM. The analysis of protein surface coverage as a function of time showed a slower process for 5:0 and 5:0.2 (POPC:Chol) ratios than for 5:1 and 5:2 (POPC:Chol) ratios. Significantly, the Cyt2Aa2-lipid binding behavior and the protein–lipid layer were different for the 5:3 (POPC:Chol) ratio. Furthermore, AFM images revealed a transformation of Cyt2Aa2/lipid layer structure from strip pattern to ring shape structures (which showed a strong repulsion with AFM tip). In summary, cholesterol increases the binding rate and alters the lipid binding behavior of Cyt2Aa2 protein, although it is not required for Cyt2Aa2 protein binding onto lipid bilayers.
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