Physical interaction between two varicella zoster virus gene regulatory proteins, 1E4 and IE62

Transfection assays demonstrate that the varicella tester virus (VZV) immed iate-early 62 (IE62) protein is a major transactivator of VZV gene expressi on, whereas a second immediate-early protein, IE4, can act as a major coact ivator of transactivation mediated through IE62. To test whether IE62 and I E4 interact physically, we performed several protein-protein interaction as says. Coimmunoprecipitation analyses using VZV-infected cell lysates as wel l as purified protein mixtures demonstrate that IE62 and IE4 form stable co mplexes in solution under stringent salt conditions. Enzyme-linked immunoso rbent assay protein-protein interaction assays and maltose-binding protein capture assays demonstrate that IE62 binds IE4 in a concentration- and dose -dependent manner. Far Western blot analyses show that IE4 binds to an unde rmodified form of IE62, and the use of calf intestinal phosphatase and prot ein kinases suggests that the interaction with IE4 is dependent on the phos phorylation state of IE62. An IE4 binding domain on IE62 has been mapped us ing a set of truncated IE62 fusion peptides. Collectively, these results im ply a direct and specific physical interaction between IE4 and less-phospho rylated forms of IE62. These data have implications for Virion assembly, as well as for the regulation of gene expression in VZV-infected cells. (C) 2 000 Academic Press.