Effect of inhibitors of the intracellular exocytic pathway on glycoprotein processing and maturation of Junin virus

The effect of glycoprotein processing and transport on Junin virus (JV) maturation was studied by using brefeldin A (BFA) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), two inhibitors affecting different steps of the intracellular exocytic pathway, combined with low temperature incubation. Both compounds inhibited the multiplication of JV, strain IV4454, in Vero cells in a dose dependent manner. The addition of the compounds after several cycles of infection for a very short treatment period produced an immediate arrest on the formation of JV infectious particles, due to their effect on a late event in JV infective cycle. Extracellular and cell-associated virus yields were reduced to the same extent, suggesting that the assembly of virus particles was the blocked stage. In infected cells treated with CCCP and BFA an altered subcellular distribution of partially processed viral glycoproteins was observed, with an accumulation of JV glycoproteins at the endoplasmic reticulum and a blockade of their transport to the site of envelopment in the plasma membrane. Concomitantly, the cleavage of the precursor GPC to the mature glycoprotein GP38 was strongly inhibited when the exocytic pathway was affected. Thus, results obtained with BFA allow to conclude that the proteolytic processing of GPC probably occurs at the trans-Golgi network and this cleavage together with the glycoprotein presence at the cell surface is required for maturation of infectious JV particles.