Initial events in bovine coronavirus infection: analysis through immunogold probes and lysosomotropic inhibitors

The early events in the infection of human rectal tumor cells by bovine coronavirus were investigated by colloidal gold-mediated immunoelectron microscopy and by analysis of the effect of lysosomotropic weak bases on virus yield. Electron microscopic studies revealed sites of fusion between the virus envelope and the plasmalemma but fusion events along intracellular membranes were not observed despite extensive searches. Virion-antibody-colloidal gold complexes were, in fact, endocytosed by synchronously infected cells. These complexes were apparently non-infectious, and they accumulated in vacuoles that resembled secondary lysosomes. Exposure of cells to ammonium chloride or to methylamine during the first hour of infection had little inhibitory effect on the production of infectious virus. Chloroquine treatments were inhibitory but this effect depended on relatively late events in the infectious process. The chloroquine inhibitory step blocked infection of virus absorbed to cells that were exposed to buffers in the pH range of 4.4 to 8.4. These findings indicate that BCV penetrates its host cell by direct fusion with the plasmalemma and does not require an acidic intracellular compartment for infectious entry.