Oncornavirus precursor particles and the microtubule organizing centers.

The relationship of intracytoplasmic type A particles, precursor particles of type B retroviruses, to microtubule organizing centers and to the mitotic apparatus have been studied electronmicroscopically in hamster and murine cell lines in various stages of mitotic arrest by Colcemid and vincristine sulfate. It was shown that the migration of these precursor particles from the cytoplasm to the kinetochore region of chromosomes, occurring in early metaphase, is a function of the inhibition of microtubule formation at the pericentriolar cytocentrum and of the attachment of spindle fibers at one sister kinetochore plate of chromosomes. The association of these type A particles with the mitotic apparatus during Colcemid arrest is reversible by removal of the inhibitor and is inversely proportional to the reattachment of spindle fibers and to the reformation of microtubules. The active participation of microtubules and cytoskeletal proteins in the transport and maturation of type B oncornaviruses is strongly suggested by these findings. Their implications, as to a possible epigenetic mode of transmission of these viruses as well as to the induction of the transformed state are discussed.

Quantitative electron microscopy of intracytoplasmic type A particles at kinetochores of metaphase chromosomes isolated from Chinese hamster and murine cell lines.

We have successfully isolated and spread individual chromosomes of CHO-KI cells for electron microscopic karyotyping. Controlled preparation permitted a quantitative evaluation of the association between endogenous intracytoplasmic type A virus precursor particles and the centromeric region (kinetochores) of isolated chromosomes at prophase and metaphase. Our results suggest the transfer of type A particles from the cytoplasmic to the centromeric regions during early metaphase in conjunction with microtubule assembly at a time when the kinetochores are structurally mature and capable of binding microtubules. Preliminary comparable studies of the endogenous M432 virus propagated in murine cells support these findings. Our results are discussed with respect to mechanisms of intracellular movement of virus precursor particles and the interference with components of both the cytoskeleton and the mitotic apparatus.

Enhanced proliferation of endogenous virus in Chinese hamster cells associated with microtubules and the mitotic apparatus of the host cell.

Chinese hamster ovary cells harbour intracytoplasmic virus-like particles of type A which are closely associated with sites of microtubule formation. We report here the enhanced proliferation of these particles and their release at the cell membrane by using either 5-bromodeoxyuridine or dibutyryl cyclic AMP. The extracellular mature particles are similar in morphology to retroviruses of type B. Close association of the type A virus precursors with microtubule organizing centres, i.e. kinetochores, centrioles and basal bodies, and with microtubules per se, is confirmed by studying the effects of the microtubule inhibitors Colcemid and vincristine sulphate. The role of microtubules in the activation and transport of the intracytoplasmic type A particles is discussed.

A quantitative autoradiographic study of nucleolus-associated RNA and DNA synthesis during the eclipse phase in Rous sarcoma virus-infected chicken fibroblasts.

Functional and morphologic differences between the sensitivity of nucleoli of Rous sarcoma virus-transformed cells and that of newly infected cells to the action of actinomycin D (AD) have been demonstrated by quantitative light and electron microscope autoradiography and utilized to investigate the function of the nucleolus in the early stages of infection. After a pulse exposure to low doses of AD, increased RNA synthesis is induced within 80 minutes in the fibrillar portion of the nucleolus by infection. A concomitant increase in the retention of tritiated AD in the nucleolus and a quantitative redistribution of intranuclear and cytoplasmic DNA label are interpreted as evidence for a virus-induced amplification of the binding sites of AD in nucleolar chromatin.

Effect of temperature of aldehyde fixation on the radioautographic localization of ribonucleoprotein in nucleoli of HeLa cells. Inhibition by puromycin and actinomycin D.

In efforts to clarify the role of the nucleolus and substructures thereof in the assembly or synthesis of protein associated with formation of the complete ribosome, the effect of variation of some conditions of aldehyde fixation on the intranuclear distribution of lysine-(3)H, arginine-(3)H, and uridine-(3)H was studied by differential grain count in radioautographs of PPLO-free HeLa cells. It was found that the nucleolus is a site of rapid assembly or synthesis of a protein, the synthesis of which is inhibited equally by puromycin (200 microg/ml) and by actinomycin D under conditions inhibitory for ribosomal precursor RNA synthesis (P < 0.01). This protein is fixed by phosphate-buffered formalin or glutaraldehyde at pH 7.3, but the label is diminished by fixation in customarily employed acetic ethanol or in formalin at acid pH. Elevation of temperature of formalin or glutaraldehyde fixatives to 37 degrees C consistently reduces the nucleolar protein label, but not the RNA label, by a proportion identical with that incurred by puromycin or actinomycin inhibition. This proportional reduction of nucleolar protein label occurs without evident loss of total grain count and is independent of length of fixation between 30 min and 4 hr, but it is not observed at 23 degrees C. The data support the interpretation that the proportion of nucleolar protein not fixed at 37 degrees C is associated with nucleolar ribosomal RNA but that it is dissociated at 37 degrees C in formalin or glutaraldehyde fixatives, probably on the basis of ionic dissociation of a conjugated ribonucleoprotein.