Because it provides both high magnification and high resolution, transmission electron microscopy (TEM) is a powerful technique for visualizing subcellular organelles, viruses, bacteria, and large molecules. The variety of research projects to which TEM has contributed include: (1)description of the cellular distribution of viral vectors injected into rats. Adenoviruses used as vectors for gene therapy can sometimes cause toxicity. The biodistribution of intravenously-injected adenovirus was studied in rats with cirrhosis of the liver induced by bile duct ligation. In normal rats the bulk of injected adenovirus is taken up by the hepatic Kupfer cells. In cirrhotic animals vector was taken up by large numbers of macrophages within the blood vessels of the lung. The altered biodistribution of adenovirus vector in rats with chrrhosis suggests the possibility that these vectors may behave unexpectedly in patients with pre-existing liver disease. (with A Byrnes and J Smith, CBER, DCGT) (2)documentation of the cellular effects of different strains of simian foamy virus. Differences in replication and cellular pathologic effects between the lab strains of Simian Foamy Viruses (SFV) and SFVs isolated from naturally occurring infections in macaques are being investigated by TEM. (with A. Khan, CBER, DVP) (3)monitoring the production of virus-like particles from HIV gp120 and hepatitis B surface antigen gene transfections. The HIV-1 envelope glycoprotein gp120 was expressed in tandem with hepatitis B surface antigen to create a hybrid protein that self-assembles spontaneously. The goal of these studies is to enhance the immunologic response to HIV. After isolation and purification, the assembly of the hybrids into stable multimeric particles was assessed by TEM. (with I Berkower, CBER, DVP). (4)description of the morphology of influenza virus mutants. The association of influenza virus matrix protein (M) with ribonucleoproteins (RNP) may control viral growth and morphology. Results show that virus strains with stronger binding of M to RNP have higher replication efficiency and spherical morphology; whereas those with weaker M to RNP binding replicate less efficiently and have a filamentous morphology. (with Z. Ye, CBER, DVP) (5)verification of the formation of cytoplasmic viroplasm-like structures in response to NSP5 rotavirus gene transfection. Rotavirus infection causes the formation of large cytoplasmic structures called viroplasms where virus morphogenesis begins. Experiments in which the genes of rotavirus non-structural proteins (NSP) were transfected into MA104 cells showed that NSP5 alone can form cytoplasmic viroplasm-like structures when the N-terminus is blocked by fusion with a non-rotaviral protein and its C-terminus is unmodified. (with K Mohan and CD Atreya, CBER, DVP) (6)description of the morphology of subcellular melanosome fractions. 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 tandem mass spectrometry and Western immunoblotting for specific proteins. (with V. Hearing, NCI)
