PROTECTIVE ANTIBODIES INHIBIT REOVIRUS INTERNALIZATION AND UNCOATING BY INTRACELLULAR PROTEASES

We identified in vitro correlates of in vivo protection mediated by no nneutralizing antibodies specific for reovirus capsid proteins. We def ined mechanisms of antibody action by analyzing monoclonal antibody (M Ab) effects at sequential steps in reovirus serotype 3 strain Dearing (T3D) infection of L, cells. Two types of experiments showed that prot ective MAbs specific for the outer capsid proteins sigma 3 or mu 1 inh ibited T3D infection independent of effects on binding. First, MAbs wh ich had no effect on T3D binding inhibited T3D growth. Second, MAb-coa ted T3D attached to L cells did not replicate as efficiently as T3D wi thout bound antibody. We therefore defined sigma 3-specific MAb effect s on postbinding steps in T3D infection. T3D coated with MAb sigma 3-1 0G10 exhibited prolonged sensitivity to growth inhibition by ammonium chloride. Since ammonium chloride inhibits endosomal acidification and proteolytic processing of the T3D capsid, this suggested that MAbs in hibit early steps in T3D infection. This was confirmed by direct demon stration that several sigma 3-specific MAbs inhibited proteolytic unco ating of virions by fibroblasts. We identified two mechanisms for anti body-mediated inhibition of virion uncoating: (i) inhibition of intern alization of T3D-MAb complexes bound to the cell surface, and (ii) inh ibition of intracellular proteolysis of the T3D capsid. Studies using a cell-free system confirmed that sigma 3-specific MAbs directly block proteolytic uncoating of the T3D virion. In addition, we found that s igma 3-specific MAbs block (and therefore define) two distinct steps i n proteolytic uncoating of the reovirion. We conclude that antibodies which are protective in vivo inhibit postbinding events in reovirus in fection of permissive cells. Protective antibodies act by inhibiting i nternalization and intracellular proteolytic uncoating of the virion. Analysis of postbinding mechanisms of MAb action may identify targets for vaccine development and antiviral therapy.