Dissecting Virus Entry: Replication-Independent Analysis of Virus Binding, Internalization, and Penetration Using Minimal Complementation of β-Galactosidase

Burkard, Christine; Bloyet, Louis-Marie; Wicht, Oliver; van Kuppeveld, Frank J.; Rottier, Peter J. M.; de Haan, Cornelis A. M.; Bosch, Berend Jan

Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli β-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide) is complementing the inactive mutant form ΔM15 of β-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).