Figure 1. Proposed rabies capsid-assembly pathway. Rabies viral proteins are translated by the ribosome (I[a]). The rabies phosphoprotein (blue) associates with unassembled nucleoprotein monomers (I[b]). One (or more) host multiprotein complex then catalyzes construction of the virus capsid through discrete assembly intermediates (I[c]) to provide the complete capsid (I[d]).

The cell-free protein synthesis (CFPS) whole-pathway screen (II) contains cellular extract, rabies nucleoprotein, matrix protein and phosphoprotein mRNAs, amino acids and an energy-regenerating system (II[a]). The proposed synthesis and assembly of rabies capsid proteins occurs as described in (I) (II[b(1)]). The CFPS translation products are transferred to a capture plate coated with anti-rabies nucleoprotein antibody. The captured rabies assembly intermediates bind a biotinylated secondary antibody (II[c(1)]), which is used to produce a fluorescent readout that indicates capsid assembly (II[d(1)]).

The CFPS system components are combined, and the drug is added <= six hours later (II[a]). A drug that blocks the formation of large capsid multimers (II[b(2)]) will lead to the capture plate being coated with monomeric units of rabies proteins (II[c(2)]) and inhibition of the fluorescent readout (II[d(2)]). (Figure based on Figures 2 and 3 from ref. 1.)