The large phosphoprotein of human cytomegalovirus

A safe, protective, and cost-effective vaccine against Cytomegalovirus (CMV) infection is one of the highest priorities of the US Institute of Medicine based on the economic costs that would be avoided and the years of life and disability that would be saved by a successful vaccine. The urgent need for a CMV vaccine, however, could not be met over a period of more than 40 years. The majority of CMV vaccines developed so far incorporated the same CMV antigen, glycoprotein B (gB). The disappointing results obtained with this vaccine target may suggest the use of a different CMV antigen. The large phosphoprotein pUL32 (pp150) is a highly immunogenic tegument protein that induces both, humoral and cellular immunity. Nevertheless, we found evidence that pUL32 may be a B-cell superantigen and involved in the evolution of chronic lymphocytic leukemia (CLL). A related tegument protein (pp65) was implicated in the pathogenesis of autoimmune diseases. In addition, pUL32 has outstanding structural characteristics of undefined immunological significance extensive phosphorylation like most other immunogenic CMV tegument proteins, glycosylations like immunogenic influenzavirus proteins, and very likely no tertiary structure. Definition of the immunological significance of these properties may elucidate the role of pUL32 in immunity knowledge that may lead also to a reappraisal of the second most frequently used vaccine target, pp65. To elucidate the immunological significance of the outstanding structural properties of pUL32, we will generate and purify recombinant pUL32 proteins (r-pUL32) derived from laboratory-adapted and clinical CMV strains, modify these proteins for their posttranslational modifications by enzymatic digestion, and evaluate their immunogenicity with the use of an array of assays (immunoblots, indirect ELISA, and B-cell stimulation assays), immune sera, and monoclonal antibodies. The computer prediction that pUL32 is an intrinsically unstructured protein (IUP) will be validated experimentally by NMR Spectroscopy and testing the immunogenicity of the structured and unstructured form of pUL32 by co-expression with its natural ligand, Bicaudal D1. The proposed study will provide important insights into the immunobiology of one of the most immunogenic CMV proteins knowledge that may facilitate the study of B-cell superantigens, direct future vaccine and diagnostic strategies, and will allow a thorough evaluation of the role of CMV in CLL.

The focus of the present research project was the elucidation of the structure, post-translational modifications and immunological properties of a prominent Cytomegalovirus (CMV) protein - the large phosphoprotein pUL32 (pp150). The reasons for studying this particular CMV protein were manifold. A safe, protective, and cost-effective vaccine against Cytomegalovirus (CMV) infection is one of the highest priorities of the US Institute of Medicine based on the economic costs that would be avoided and the years of life and disability that would be saved by a successful vaccine. The urgent need for a CMV vaccine, however, could not be met so far. pUL32 is a highly immunogenic tegument protein that induces both, humoral and cellular immunity and hence may be a potential vaccine candidate. Nevertheless, we found also evidence that pUL32 may be a B-cell superantigen and involved in the evolution of chronic lymphocytic leukemia (CLL) which would disqualify the protein as vaccine candidate. A hypothetical explanation for this conflicting evidence may be the outstanding structural characteristics of pUL32 with undefined immunological significance extensive phosphorylation like most other immunogenic CMV tegument proteins, glycosylations like immunogenic influenzavirus proteins. We discovered several surprising properties of the protein: (1) extent of phosphorylation has been grossly overestimated previously by indirect assumptions on the status of phosphorylation, (2) the reason for the hypophosphorylation is the activity of a cellular phosphatase, that is packaged in the virion, (3) the protein is intrinsically unstructured and attains a secondary structure only upon ligation with other interaction partners, which (4) makes it impossible to purify this large protein as one, large molecule. These findings shed light on the biological, immunological, and biochemical properties of an important CMV antigen.