ALPHAVIRUS BUDDING IS DEPENDENT ON THE INTERACTION BETWEEN THE NUCLEOCAPSID AND HYDROPHOBIC AMINO-ACIDS ON THE CYTOPLASMIC DOMAIN OF THE E2ENVELOPE GLYCOPROTEIN

The interaction between the nucleocapsid core and the glycoprotein spi kes is a critical component in the budding process of alphaviruses. A molecular model was previously proposed which suggested that this inte raction was mediated by the binding of the cytoplasmic domain of glyco protein E2 into a hydrophobic pocket found on the surface of the nucle ocapsid protein [S. Lee, K.E. Owen, H.-K. Choi, H. Lee, G. Lu, G. Weng ler, D.T. Brown, M.G. Rossmann, and R.J. Kuhn (1996) Structure 4, 531- 541; U. Skoging, M. Vihinen, L. Nilsson, and P. Liljestrom (1996) Stru cture 4, 519-529]. Two hydrophobic amino acids in the cytoplasmic doma in of E2 were predicted to be important in the contact between the pro teins. One of the residues, Y400 (Sindbis virus numbering), had previo usly been shown by mutational studies to be important in the budding o f Semliki Forest Virus [H. Zhao, B. Lindqvist, H. Garoff, C. H. von Bo nsdorf, and P. Liljestrom (1994) EMBO J. 13, 4204-4211]. The role of t he second residue, L402, had not been examined. By creating a panel of amino acid substitutions at this residue, followed by phenotypic anal ysis of rescued mutant viruses, we now show that L402 is critical for the production of Sindbis virus. Substitutions at this amino acid inhi bit budding, and the data suggest that L402 plays an important role in the interaction between the glycoprotein and the nucleocapsid core. T hese data support the model and suggest that the proposed molecular in teractions are important for the budding of alphaviruses from the cell . (C) 1997 Academic Press.