(1) The activation of latent retroviruses was studied using murine cells. Cultures of KBalb cells were treated with agents known to induce viral replication and were subsequently examined by TEM for the presence of retroviral particles. Results showed specific induction of Type C particles with IdU and Type A particles with Aza C. Treatment with Aza C and IdU together caused greatly increased activiation of Type C retroviruses, while the activation of Type A retroviruses was the same as for Aza C alone. (With A. Khan & J. Sears, CBER, DVP) (2) The EM lab is also participating in a research project related to vaccine development with Dr. I Berkower (CBER, DVP). The goal of these studies is to enhance the immunologic response to HIV by creating novel multimeric hybrid particles containing the HIV gp120 envelope molecule. After isolation and purification, the assembly of the hybrids into stable multimeric particles is assessed by TEM. (3) To study the assembly of influenza virus ribonucleoprotein (RNP), RNPs were reconstituted from nucleoprotein (NP), M1, and viral RNA synthesized in vitro and their morphology was examined by TEM. Protomers of NP self-assembled into circular oligomers. However, helical structures, similar in conformation to RNPs purified directly from influenza virus, were formed only when M1 and vRNA were also present. In the absence of vRNA, no helical structures were formed from NP and M!. (with Z. Ye, CBER, DVP) (4) Mitogenic activation of porcine peripheral blood monomuclear cells results in the activation and release of a retrovirus (PERV) capable of infecting human cells. In further studies, cultures of non-human primate cells positive for proviral DNA, genomic RNA, and viral proteins were examined by TEM. No viral particles were seen, suggesting a block to productive infection may occur at the assembly stage. (with C. Wilson, CBER, DCGT) (5) Melanin synthesis takes place in membrane-bound cytoplasmic structures termed melanosomes. The biogenesis and maturation of melanosomes is accompanied by a morphological transition from an amorphous, rounded vesicle into an elongated, fibrillar structure. However, the sequence in which melanosomal proteins are sorted to the organelle and the role(s) they play in its maturation remain largely unknown. In this study melanosome fractions were prepared by sucrose density gradient centrifugation followed by free-flow electrophoresis. They were characterized by TEM for purity and predominant melanosome stage and by Western immunoblotting for specific proteins. Our results show that all melanogenic enzymes found in early stage melanosomes are effectively cleaved and inactivated. It is only after the transition to later stages that the enzymes become resistant to proteolysis and melanin production ensues. (with V. Hearing, NCI) (6) Melanin-loaded melanosomes are transferred to neighboring keratinocytes where their presence confers to the skin its characteristic color and photoprotective properties. Using murine melanocytes and keratinocytes, alone and in co-culture, we found that melanosome transfer to keratinocytes occurs by processes of exocytosis-phagocytosis and is at least partially regulated by local signals involving UV radiation and MSH. UV and MSH were found to increase melanosome synthesis by melanocytes and to increase melanosome uptake by keratinocytes. In addition, MSH increased melanosome exocytosis and the activity of genes associated with exocytosis. Co-culture of melanocytes and keratinocytes increased melanosome exocytosis and MSH secretion. (with V. Virador, NCI)
