PROPERTIES OF THE PROTEIN ENCODED BY THE U(L)32 OPEN READING FRAME OFHERPES-SIMPLEX-VIRUS-1

The functions previously assigned to the essential herpes simplex viru s 1 U(L)32 protein were in cleavage and/or packaging of viral DNA and in maturation and/or translocation of viral glycoproteins to the plasm a membrane, The amino acid sequence predicts N-linked glycosylation si tes and sequences conserved in aspartyl proteases and in zinc-binding proteins, We report the following, (i) The 596-amino-acid U(L)32 prote in accumulated predominantly in the cytoplasm of infected cells but wa s not metabolically labeled with glucosamine and did not band with mem branes containing a known glycoprotein in flotation sucrose density gr adients, The U(L)32 protein does not, therefore, have the properties o f an intrinsic membrane protein, (ii) Experiments designed to demonstr ate aspartyl protease activity in a phage display system failed to rev eal proteolytic activity, Moreover, substitution of Asp-110 with Gly i n the sequence Asp-Thr-Gly, the hallmark of aspartyl proteases, had no effect on viral replication in Vero and SK-N-SH cell lines or in huma n foreskin fibroblasts. Therefore, if the U(L)32 protein functions as a protease, this function is not required in cells in culture, (iii) B oth the native U(L)32 protein and a histidine-tagged U(L)32 protein ma de in recombinant baculovirus-infected insect cells bound zinc, The co nsensus sequence is conserved in the U(L)32 homologs from varicella-zo ster virus and equine herpesvirus 1, U(L)32 protein is therefore a cys teine-rich, zinc-binding essential cytoplasmic protein whose function is not yet clear.