VIRAL-INFECTION .2. HEMIN INDUCES OVEREXPRESSION OF P67 AS IT PARTIALLY PREVENTS APPEARANCE OF AN ACTIVE P67-DEGLYCOSYLASE IN BACULOVIRUS-INFECTED INSECT CELLS

The roles of p67-deglycosylase (p67-DG) in the regulation of protein s ynthesis in baculovirus-infected insect cells were studied, Like vacci nia viral infection, baculovirus infection of insect cells also induce d the appearance of a p67-DG. However, p67-DG activity could not be de tected because these cells do not contain a detectable level of p67. T he baculovirus expression vector system (BEVS), however, promotes sign ificant expression of cloned p67-cDNA, The expression of p67 was signi ficantly enhanced by the addition of hemin to the growth medium. Maxim um enhancement was observed at 5 mu M hemin. Data suggest that hemin p revents the activation of latent p67-DG inside the cell and does not h ave any effect on p67 gene transcription, To gain a better understandi ng of the mechanism of p67-DG; activation and hemin stimulation of p67 synthesis, we have now purified p67-DG; from baculovirus-infected ins ect cells, We prepared antibodies against this protein, These antibodi es reacted with a 105-kDa protein in cell extracts from the uninfected insect cells (Sf9), KRC-7, and L929 (animal cells), In addition, thes e antibodies reacted with an additional 60-kDa protein in the cell ext racts of baculovirus-infected Sf9 cells and vaccinia virus-infected KR C-7 and L929 cells, Data are also presented to show that the antibodie s against p67-DG reacted more efficiently (40%) with the 60-kDa protei n in both hemin-deficient reticulocyte lysate and hemin-deficient bacu lovirus-infected cells, We suggest that hemin prevents the conversion of an inactive p67-DG into an active form possibly by covalent modific ation such as protein phosphorylation or protein glycosylation. The ac tive form is more efficiently recognized by the p67-DG antibodies sinc e these antibodies were prepared against the active form of p67-DG. (C ) 1997 Academic Press.