Intracellular type A retrovirus movement associated with an intact microtubule system.

Intracytoplasmic type A particles known to be precursors to type B retroviruses in murine, hamster and marsupial cells are closely associated with microtubules and microtubule organizing centres. In this publication, the active participation of microtubules in the intracellular transport of the particles to the cell surface has been examined in NIH 3T3 cells infected with M432 virus using vincristine sulphate (VCR) as inhibitor of microtubule polymerization. The release of virus at different times after exposure to VCR was quantified by reverse transcriptase determinations of cell supernatants and by electron microscopic quantification of the number of virions at the cell surface using freeze-dried whole cell replicas. These studies indicate that VCR inhibits both microtubule polymerization and virus release, and thus suggest that intact cytoplasmic microtubules are necessary for intracellular transport and release of virus.

Interferon-treated cells release vesicular stomatitis virus particles lacking glycoprotein spikes: correlation with biochemical data.

Earlier we reported a reduction to 1/30th-1/100th of the original number of infectious particles in the infectious vesicular stomatitis virus (VSV) released from L cells treated with 10 or 30 reference units of interferon per ml. However, in these cultures virus particle production, as measured by VSV particle-associated viral RNA, virus nucleocapsid protein, and viral transcriptase, was inhibited by less than 10%. Data reported in this paper show that there was a significant reduction in glycoprotein and membrane protein of VSV particles released from interferon-treated cells. Evidence supporting the deficiency of glycoprotein in VSV released from interferon-treated cells was derived from electron microscopic studies. Under conditions where glycoprotein spikes or projections were clearly detectable on the surface of VSV released from cells not treated with interferon, very few spikes were observed on VSV released from interferon-treated cells. These results suggested that interferon-treated cells produced VSV particles with low infectivity and that this low infectivity may be related to the reduced amount of glycoprotein and membrane protein incorporated into such particles.

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.