The conditions for reactivation of latent HCMV from latency are poorly understood. The applicant has been using the following two methods to reactivate HCMV replication in MDM: (i) ConA-stimulated T cell induced MDM, (ii) allogenically stimulated T cell induced MDM. In the former, the virus is found in intracytoplasmic vacuoles and not in the extracellular fluid. In the latter, the virus replicates more efficiently and virus is found in the extracellular fluid. The two types of MDM presumably differ in the degree of cellular differentiation which effects the replication of HCMV.
Two specific aims are proposed to address these observations. In the first specific aim, the applicant proposes to investigate the growth properties of HCMV in allogenically stimulated T-cell induced MDM. Cells from asymptomatic sero positive individuals will be allogenically stimulated for the reactivation and recovery of HCMV clinical isolates. In the second specific aim, the applicant will focus on the trafficking of the viral glycoprotein gB. The applicant proposes that the phosphorylation of gB in ConA-stimulated versus allogenically stimulated MDM or in permissive human fibroblast cells is different. This difference influences the compartmentalization of gB which may influence the biological behavior of virions in the different MDM.
The specific aims will contribute towards our understanding of HCMV pathogenesis and reactivation from latency. For the first specific aim, the applicant will compare the rate of HCMV replication the two types of MDM. With the ConA-stimulated system, only 5 to 13% of the cells can be infected with HCMV. With the allo-stimulated system 30 to 60% of the cells can be infected. The two methods of generating MDM will be analyzed by depletion experiments using antibodies to CD4, CD8, B cells (CD19), NK cells (CD56) and HLA class I and class II molecules. Anti-mouse IgG attached to magnetic beads will be used to remove the cells from the system. The effect of cytokines will be determined using antibodies to IL-1, IL-2, IL-4, TNF-a, TGF-beta, GM-CSF and IFN-g. The applicant proposes that the stage and extent of cellular differentiation of the MDM influences the replication of HCMV and that the antibody depletion experiments will identify critical elements and stages of macrophage differentiation. In the second specific aim, the applicant will investigate the phosphorylation and trafficking of the viral gB molecule. Since gB is not detectable on the surface on ConA-stimulated MDM, the applicant proposes that the trafficking of gB is altered and this is related to HCMV being retained in intracytoplasmic vacuoles. To quantitate the levels of viral gB in the different MDM or control cells, the applicant will use purified His-tagged gB. Antibodies that recognize both the gB precursor or cleaved products are available. In addition, antibodies that recognize phosphorylated serines are available for these studies. Apparently, HCMV gB is phosphorylated at only one site. Dephosphorylation experiments followed by 32P-labeled phosphorylation experiments can be used to determine the extent of phosphorylation. Experiments will be designed to determine whether gB is phosphorylated on the cell surface of MDM or control cells, but not in the Golgi-derived vacuoles. As a control for investigating trafficking mechanisms, the applicant will use dynamin to block clatherin dependent retrieval mechanisms. Recombinant vaccinia viruses with and without dynamin expression are available. The applicant predicts that a dynamin mutant will block intracellular retrieval of surface localized viral gB, but will still allow for the incorporation of gB into the viral envelope. Experiments to mutagenize the viral gB molecule in different locations were also proposed. To more effectively follow the trafficking of the viral gB molecules, a green fluorescent protein-gB chimera is proposed. Lastly, the incorporation of mutant of gB molecules into HCMV for investigating the effects on gB trafficking and HCMV replication are proposed.
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