Latrophilin, neurexin, and their signaling-deficient mutants facilitate α-latrotoxin insertion into membranes but are not involved in pore formation

Pure alpha -latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the alpha -latrotoxin pores are permeable to Ca2+ and small molecules including fluorescein isothiocyanate and norepinephrine. Bound alpha -latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin tin the presence of Ca2+). Interestingly, on subsequent removal of Ca2+, alpha -latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-alpha -latrotoxin antibodies. Our results indicate that (i) c alpha -latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha -latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) alpha -latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha -latrotoxin interaction with other neuronal proteins

Sequential Adaptive Mutations Enhance Efficient Vector Switching by Chikungunya Virus and Its Epidemic Emergence

The adaptation of Chikungunya virus (CHIKV) to a new vector, the Aedes albopictus mosquito, is a major factor contributing to its ongoing re-emergence in a series of large-scale epidemics of arthritic disease in many parts of the world since 2004. Although the initial step of CHIKV adaptation to A. albopictus was determined to involve an A226V amino acid substitution in the E1 envelope glycoprotein that first arose in 2005, little attention has been paid to subsequent CHIKV evolution after this adaptive mutation was convergently selected in several geographic locations. To determine whether selection of second-step adaptive mutations in CHIKV or other arthropod-borne viruses occurs in nature, we tested the effect of an additional envelope glycoprotein amino acid change identified in Kerala, India in 2009. This substitution, E2-L210Q, caused a significant increase in the ability of CHIKV to develop a disseminated infection in A. albopictus, but had no effect on CHIKV fitness in the alternative mosquito vector, A. aegypti, or in vertebrate cell lines. Using infectious viruses or virus-like replicon particles expressing the E2-210Q and E2-210L residues, we determined that E2-L210Q acts primarily at the level of infection of A. albopictus midgut epithelial cells. In addition, we observed that the initial adaptive substitution, E1-A226V, had a significantly stronger effect on CHIKV fitness in A. albopictus than E2-L210Q, thus explaining the observed time differences required for selective sweeps of these mutations in nature. These results indicate that the continuous CHIKV circulation in an A. albopictus-human cycle since 2005 has resulted in the selection of an additional, second-step mutation that may facilitate even more efficient virus circulation and persistence in endemic areas, further increasing the risk of more severe and expanded CHIK epidemics