SINDBIS VIRUS ATTACHMENT - ISOLATION AND CHARACTERIZATION OF MUTANTS WITH IMPAIRED BINDING TO VERTEBRATE CELLS

Sindbis virus can infect a broad range of insect and vertebrate cell t ypes. The ability to restrict tissue tropism and target virus infectio n to specific cell types would expand the usefulness of engineered alp haviruses as gene expression vectors. In this study, virus pools deriv ed from libraries of full-length Sindbis virus cDNA clones containing random insertion mutations in the PE2 or E1 virion glycoprotein gene w ere screened for mutants defective for binding to vertebrate cells. Bi nding-competent mutants were depleted by serial adsorption to chicken embryo fibroblast (CEF) monolayers at 4-degrees-C, and the remaining p opulation was amplified by immune-enhanced infection of P388D1 cells. From the PE2 libraries, 12 candidate mutants showing reduced cytopathi c effects on CEF monolayers were isolated and three representative mut ants, NB1, NB2, and NB12, were characterized in detail. Insertion muta tions for NB1 and NB12 were found near the PE2 cleavage site, whereas the insertion in NB2 occurred between residues 69 and 74 of E2. Althou gh virion assembly and release occurred normally for all three mutants , PE2 cleavage was completely (NB1) or partially (NB12) blocked for th e mutants with insertions near the PE2 cleavage site. Both NB1 and NB2 were defective for binding to CEF and BHK-21 cells. Mild trypsin dige stion of isolated NB1 virions resulted in PE2 cleavage and partially r estored binding to CEF. Besides defective binding, NB1 also exhibited slower CEF penetration kinetics. Consistent with previous work, these results implicate PE2 cleavage and domains in the N-terminal portion o f E2 as important determinants of alphavirus binding and penetration. Binding-defective mutants such as NB2, which exhibit normal particle a ssembly, release, and penetration, may be useful for future efforts to target Sindbis virus infection.