Viral envelope glycoproteins expressed on cellular membranes and/or incorporated into the envelope, mediate critical steps in the lifecycle such as entry, egress and cell to cell spread of infection. A hallmark and defining feature of human cytomegalovirus (HCMV) infection, is its capacity to enter and spread cell to cell between cell types of distinct and divergent developmental lineages. Since few cell surface molecules are distributed on all of these cell types, it is likely that multiple viral glycoproteins participate in HCMV pathogenesis. Sequence analysis predicts 67 possible glycoproteins reflecting the potential to utilize multiple viral ligand/cellular interactions. Biochemical analysis of HCMV envelope revealed the presense of at least 10 glycoproteins, many of which are organized into large disulfide-linked complexes. However, only 5 of these proteins are mapped to the viral genome. Before the mechanism of viral entry, egress and spread can be delineated, the composition of the envelope must be defined and the genes encoding the envelope proteins identified. This outlines an experimental plan that will define the composition of two major HCMV envelop complexes, gCII and gCIII. Both of these complexes are implicated in virus-cell interactions and both contain identified (gM and gH/gL respectively) as well as unidentified components. The availability of monoclonal antibodies to the known constituents of these complexes will allow purification of gCII and gCIII. Further purification and separation of individual complex proteins will precede microsequence analysis and genetic identification of the unmapped glycoproteins. The feasibility of this approach is blostered by significant preliminary data regarding the identification of a previously unmapped component of gCIII. Once reagents to the newly mapped proteins are produced, the composition of the complexes will be verified. Biophysical parameters such as the molecular mass and stoichiometry of gCII and gCIII will be determined using a variety of biochemical techniques. The biosynthetic pathway and cellular localization within infected cells will be characterized. These studies will facilitate our understanding of virion maturation and egress, an area of the HCMV lifecycle with enormous gaps in knowledge. In studies aimed at future functional analysis of the gCII and gCIII, the complexes will be reconstituted by co-expression of the individual components. The information learned in the course of the studies is critical for future advances in the understanding of the HCMV lifecycle and pathogenesis and may also serve as the basis for future antiviral drug design.
| Lopper, Matthew; Compton, Teresa (2004) Coiled-coil domains in glycoproteins B and H are involved in human cytomegalovirus membrane fusion. J Virol 78:8333-41 |
